To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H2O2) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H2O2 treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor αplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

Use of Melatonin Derivatives as Spin Traps for Hydroxyl Radicals: A Computational Studies. Aaron Teye Caesar and Dr. Scott Jeffery Kirkby, Department of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN. Free radicals, especially reactive oxygen species, have been implicated in several deleterious processes which result in degenerative and cardiovascular diseases. Melatonin (N-acetyl-5-methoxytryptamin, MLT) is a naturally occurring antioxidant which has shown some potential for use as a spin trap. Spin traps react with short lived radicals such as hydroxy (.OH) or superoxide (O2-) to produce more stable products called spin adducts which may be characterized by electron paramagnetic resonance spectroscopy. This work examines whether MLT derivatives show improved spin adduct stability which may enhance their spin trapping characteristics. Electronic structure calculations of MLT, selected derivatives and 2-OH radical products were performed at the HF/6-31G(d), cc-pVDZ and DFT/B3LYP/6-31G(d) and cc-pVDZ levels of theory using NWChem. The stabilization energy was calculated using; ∆Estabilization = Espin adduct – (Espin trap + Ehydroxy radical). In units of hartrees, the results of 2-OHMLT, 2-OHMLT-Me and 2-OHMLT-CN are -0.43738, -1.60054, -1.60380 for HF/6-31G(d); -1.46071, -1.44788 and -1.46173 for DFT/6-31G(d) respectively. Also, HF/cc-pVDZ and DFTB3LYP/cc-pVDZ respectively gave -1.61268, -1.60233, -1.61409 and -1.44929, -0.26318, -1.45521.

Melatonin (N-acetyl-5-methoxytryptamin, MLT) is a naturally occurring antioxidant which has shown some potential for use as a spin trap. Spin traps react with short lived hydroxyl radicals (HO·) to produce more stable products called spin adducts which may be characterized by electron paramagnetic resonance spectroscopy. However, the relative stability of hydroxyl spin adducts of melatonin derivatives (MLTD) compared to 2-hydroxymelatonin (HO-MLT) has not been examined computationally. Computational studies have been done on four selected MLTD; methylmelatonin (Me-MLT), chloromelatonin (Cl-MLT), cyanomelatonin (CN-MLT), and nitromelatonin (NO2-MLT). Geometry of the structures were optimized at the HF/6-31G(d), cc-pVXZ, (X=D and T) and DFT/B3LYP/6-31G(d), cc-pVDZ and cc-pVTZ levels of theory and extrapolated to the complete basis set limit using cc-pVXZ (X=D, T) basis sets. The lowest relative energy was found to be a mix of results for 2-OH-MLT-Me at HF and 2-OH-MLT-NO2 at DFT.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice. Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration. Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

Cerium is the most abundant of the rare earth metals, found on average at a level of 66 parts per million in the earth's crust. The unique redox properties of cerium and cerium oxide nanoparticles have led to its use in a wide variety of industrial and commercial uses such as oxygen sensors, fertilizers and as a catalyst to remove toxic gases in automobile exhaust. The use of cerium has also garnered interest in the nanotechnology field. Nanoceria has been generated in its oxide form as nanoparticles and nanorods. Recently, nanoceria has been shown to protect against oxidative stress in both animal and cell culture models. Although not fully understood, this observed protective effect of nanoceria is believed to be the result of recently identified SOD mimetic activity. Currently there is little understanding as to how nanoceria is capable of scavenging radicals or what properties makes nanoceria an effective SOD mimetic. Our data shows strong evidence that the oxidation state of nanoceria is directly related to its reported SOD mimetic activity. As such, future studies of nanoceria should be mindful of the oxidation state of nanoceria preparations as only nanoceria with a high concentration of cerium (III) have shown effective SOD mimetic activity. In addition to the characterization of nanoceria and its SOD mimetic activity, we have evidence that free cerium is capable of generating radicals and damaging DNA in vitro in the presence of hydrogen peroxide. These data strongly suggests that the rare earth inner-transition metal cerium is capable of generating hydroxyl radicals via a Fenton-like reaction. Based on these results the use of free cerium salts should be monitored to limit environmental exposure to cerium. Altogether our data would suggest that cerium by virtue of its unique redox chemistry is quite capable of accepting and donating electrons from its surroundings. In its free form cerium is able to redox cycle easily and can generate radicals. However, paradoxically nanoceria may not easily redox cycle due to the bound lattice structure of the particle. The unique nature of nanoceria and cerium leads to a unique circumstance where nanoceria is a radical scavenger while free cerium generates radicals. As such, further investigation is needed to insure that leeching or cerium from nanoceria does not abrogate any potential benefit nanoceria may provide.

No description available.

No description available.

In this study, low density polyethylene (LDPE) and polyethylene terephalate (PETE) resin beads were ground to a coarse powder and exposed to sterilization treatments applied to the food contact surface of packaging materials used in aseptically processed and packaged food. Electron paranlagnetic resonance (EPR) analyzed free radical (-CH2CHCH=CHCHz-) production on the surface of LDPE exposed to heat (107°C) and treatments of heat (107°C) + 30% hydrogen peroxide solution (H20 2). As the temperature was raised from 100° to 200°C, peak intensity of carbon radicals produced gradually increased. The sensitivity of EPR prevented detection of free radicals on LDPE, exposed to H20 2 treatment, due to residual peroxide and H20 condensation on the surface of LDPE. D-limonene was placed in 12ml sealed glass vials containing a sodium citrate buffer solution (pH=3.7), under atmospheric O₂ (21%) conditions. Oxidation of d-limonene, placed in intimate contact with untreated, HzOz treated, and ultraviolet (VV) light (650mW/cm2) treated LDPE for 15 weeks, was measured to determine the capacity of an oxidized polymer to initiate autoxidation. The oxidation of d-limonene in vials containing no polymer was also measured. Production of carvone and carveol were used as an index for oxidation. No polymer and UV treated samples showed significantly (P<0.05) higher levels of calVone and calVeol than samples containing untreated and HzOz treated LDPE. Samples containing no polymer oxidized d-limonene at the highest rate, but not significantly faster than solutions containing UV treated LDPE. Accumulation of carvone and carveol was zero order. / Ph. D.

Oxidative stress plays a vital role in the pathogenesis of many chronic and acute inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two key mediators that are known to induce cellular and tissue oxidative stress. The generation of ROS and RNS is mediated by innate immune signaling processes. Lipopolysaccharide (LPS), a major inflammatory signal, is known to be a potent inducer of ROS/RNS. Thus, strategies that may block LPS-mediated generation of free radicals may hold promise in treating various inflammatory disease processes. However, the molecular mechanisms underlying LPS-mediated ROS/RNS production are not fully defined. Interleukin-1 Receptor associated kinase (IRAK-1), an intracellular kinase downstream of Toll-like Receptor 4 (TLR4) has been shown to contribute to the inflammatory cascade associated with LPS-TLR4 signaling pathway. However, its role in ROS production has not been defined. Therefore, we tested the hypothesis that IRAK-1 plays an important role in regulating ROS/RNS production. Both in vitro and in vivo studies were conducted to investigate the role of IRAK-1 in modulating free radicals as well as oxidative stress. In vitro studies demonstrate that IRAK-1 is a critical molecule involved in the induction of ROS/RNS. IRAK-1 deletion ablated free radical production following LPS challenge in a variety of cell types including macrophages, fibroblasts and microglia. Mechanistically, we observed that IRAK-1 is required for optimal expression and activity of NADPH oxidase subunits and iNOS. IRAK-1 deletion reduced LPS-triggered p47phox membrane translocation, suppressed NOX-1 expression and protein levels as well as hampered Rac1 activation. On the other hand, IRAK-1 deletion sustained antioxidative enzyme activity and levels in IRAK-1-/- macrophages and fibroblasts. In terms of the in vivo physiological consequences, IRAK-1-/- mice exhibited attenuated lipid peroxidation in vital organs, attenuated histopathological lesions in liver and kidney, and reduced endotoxemia-associated mortality. Taken together, IRAK-1 may, at least in part, serve as an important therapeutic target in the treatment of various inflammatory disease processes. / Ph. D.