Glutathione peroxidase (GPX), superoxide dismutase (<em>SOD</em>) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, <em>SOD</em> and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (<em>3</em>0 min, <em>3</em>7 degrees C) for hydrogen peroxide, <em>3</em> x 10(-4) M (15 min, <em>3</em>7 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, <em>3</em>7 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. <em>SOD</em> was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, <em>3</em>7 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

OBJECTIVE

Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-<em>3</em> and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells.

METHODS

Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-<em>3</em> activity and ELISA assay.

RESULTS

Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis.

CONCLUSIONS

Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with <em>3</em>H-1,2-dithiole-<em>3</em>-thione (D<em>3</em>T) led to a significant induction of superoxide dismutase (<em>SOD</em>), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of <em>SOD</em>, catalase, GSH, GR, GST, and NQO1, but not GPx by D<em>3</em>T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D<em>3</em>T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D<em>3</em>T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.

The genomic distribution of actin-related sequences in tomato was investigated using a cloned actin gene from soybean. Ten actin loci account for most of the hybridizing fragments observed with Southern analysis. Single loci were found on chromosomes 1, <em>3</em> and 10 and two loci on chromosome 4. One locus is linked to an unmapped isozyme marker, <em>Sod</em>-1. The four remaining actin loci are independent of each other and of any of the other markers tested. The number of actin loci in tomato (10) is greater than that estimated for soybean (8). As soybean is apparently a tetraploid and tomato a diploid, these results suggest that the number of actin loci has not been stable during the evolution of dicots. A number of these mapped loci lie in regions of the genome previously devoid of molecular markers and thus may be useful in basic and applied genetic research.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-<em>3</em> cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, <em>3</em>-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable <em>SOD</em> but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Several recently identified intracellular proteins associate with the tumor necrosis factor (TNF) receptor and activate nuclear transcription factor (NF)-kappaB, c-Jun kinase, and apoptosis. However, the mechanism is not understood. In the present report, we investigated the role of reactive oxygen intermediates in TNF-induced signaling. Overexpression of manganese superoxide dismutase (Mn-<em>SOD</em>) in human breast cancer MCF-7 cells completely abolished TNF-mediated NF-kappaB activation, IkappaB alpha degradation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Besides TNF, phorbol ester-, okadaic acid-, ceramide-, and lipopolysaccharide-induced activation of NF-kappaB was blocked by Mn-<em>SOD</em>, indicating a common pathway of activation. H2O2-induced NF-kappaB activation, however, was potentiated. In addition, Mn-<em>SOD</em> blocked the TNF-mediated activation of activated protein-1, stress-activated c-Jun protein kinase, and mitogen-activated protein kinase kinase. TNF-induced antiproliferative effects and caspase-<em>3</em> activation, indicators of apoptosis, were also completely suppressed by transfection of cells with Mn-<em>SOD</em>. Suppression of apoptosis induced by okadaic acid, H2O2, and taxol was also inhibited by Mn-<em>SOD</em> but not that induced by vincristine, vinblastine, or daunomycin. Overall, these results demonstrate that, in addition to several recently identified signaling molecules, reactive oxygen intermediates play a critical role in activation of NF-kappaB, activated protein-1, c-Jun kinase, and apoptosis induced by TNF and other agents.

The aim of the present study was the evaluation of possible protective effects of quercetin (QE) against beta-cell damage in experimental streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg kg(-1) for diabetes induction. QE (15 mg kg(-1) day, intraperitoneal (i.p.) injection) was injected for <em>3</em> days prior to STZ administration; these injections were continued to the end of the study (for 4 weeks). It has been believed that oxidative stress plays a role in the pathogenesis of diabetes mellitus (DM). In order to determine the changes of cellular antioxidant defense system, antioxidant enzymes such as glutathione peroxidase (GSHPx), superoxide dismutase (<em>SOD</em>) and catalase (CAT) activities were measured in pancreatic homogenates. Moreover we also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, if there is an imbalance between oxidant and antioxidant status. Pancreatic beta-cells were examined by immunohistochemical methods. STZ induced a significant increase lipid peroxidation, serum NO concentrations and decreased the antioxidant enzyme activity. Erythrocyte MDA, serum NO and pancreatic tissue MDA significantly increased (P < 0.05) and also the antioxidant levels significantly decreased (P < 0.05) in diabetic group. QE treatment significantly decreased the elevated MDA and NO (P < 0.05), and also increased the antioxidant enzyme activities (P < 0.05). QE treatment has shown protective effect possibly through decreasing lipid peroxidation, NO production and increasing antioxidant enzyme activity. Islet cells degeneration and weak insulin immunohistochemical staining was observed in STZ induced diabetic rats. Increased staining of insulin and preservation of islet cells were apparent in the QE-treated diabetic rats. These findings suggest that QE treatment has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic beta-cell integrity.

Sympathetic neurons in culture die by apoptosis when deprived of nerve growth factor (NGF). We used this model of programmed cell death to study the mechanisms that mediate neuronal apoptosis. Cultured sympathetic neurons were injected with copper/zinc superoxide dismutase protein (<em>SOD</em>) or with an expression vector containing an <em>SOD</em> cDNA. In both cases apoptosis was delayed when the neurons were deprived of NGF. The delay was similar to that seen when a bcl-2 expression vector was injected. <em>SOD</em>, injected 8 hr after NGF deprivation, provided no protection, indicating that superoxide production may occur early in response to trophic factor deprivation. We have demonstrated, with a redox-sensitive dye, an increase in reactive oxygen species (ROS) that peaked at <em>3</em> hr after sympathetic neurons were deprived of NGF. If NGF was added back to the culture medium after the period of peak ROS generation, apoptosis was completely prevented, suggesting that ROS production serves as an early signal, rather than a toxic agent, to mediate apoptosis.

A systematic study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with HT-1080 and A4<em>3</em>1 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. Upon exposure to SNP (up to 6.25 microg/mL), morphology of both the cell types remained unaltered. However, at higher concentrations (6.25-50 microg/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC(50) values for HT-1080 and A4<em>3</em>1 as revealed by XTT assay were 10.6 and 11.6 microg/mL, respectively. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (6.25 microg/mL), clear signs of oxidative stress, i.e. decreased GSH ( approximately 2.5-folds in HT-1080, approximately 2-folds in A4<em>3</em>1) and <em>SOD</em> ( approximately 1.6-folds in HT-1080, <em>3</em>-folds in A4<em>3</em>1) as well as increased lipid peroxidation ( approximately 2.5-folds in HT-1080, approximately 2-folds in A4<em>3</em>1) were seen. Changes in the levels of catalase and GPx in A4<em>3</em>1 cells were statistically insignificant in both cell types. DNA fragmentation in SNP-exposed cells suggested apoptosis. When the apoptotic thresholds of SNP were monitored with caspase-<em>3</em> assay the concentrations required for the onset of apoptosis were found to be much lower (0.78 microg/mL in HT-1080, 1.56 microg/mL in A4<em>3</em>1) than the necrotic concentration (12.5 microg/mL in both cell types). These results can be used to define a safe range of SNP for the intended application as a topical antimicrobial agent after appropriate in vivo studies.

OBJECTIVE

The aim of this study was to determine whether oxidative stress is increased in calcified, stenotic aortic valves and to examine mechanisms that might contribute to increased oxidative stress.

BACKGROUND

Oxidative stress is increased in atherosclerotic lesions and might play an important role in plaque progression and calcification. The role of oxidative stress in valve disease is not clear.

METHODS

Superoxide (dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence), hydrogen peroxide H2O2 (dichlorofluorescein fluorescence), and expression and activity of pro- and anti-oxidant enzymes were measured in normal valves from hearts not suitable for transplantation and stenotic aortic valves that were removed during surgical replacement of the valve.

RESULTS

In normal valves, superoxide levels were relatively low and distributed homogeneously throughout the valve. In stenotic valves, superoxide levels were increased 2-fold near the calcified regions of the valve (p < 0.05); noncalcified regions did not differ significantly from normal valves. Hydrogen peroxide levels were also markedly elevated in calcified regions of stenotic valves. Nicotinamide adenine dinucleotide phosphate oxidase activity was not increased in calcified regions of stenotic valves. Superoxide levels in stenotic valves were significantly reduced by inhibition of nitric oxide synthases (NOS), which suggests uncoupling of the enzyme. Antioxidant mechanisms were reduced in calcified regions of the aortic valve, because total superoxide dismutase (<em>SOD</em>) activity and expression of all <em>3</em> <em>SOD</em> isoforms was significantly decreased. Catalase expression also was reduced in pericalcific regions.

CONCLUSIONS

This study provides the first evidence that oxidative stress is increased in calcified regions of stenotic aortic valves from humans. Increased oxidative stress is due at least in part to reduction in expression and activity of antioxidant enzymes and perhaps to uncoupled NOS activity. Thus, mechanisms of oxidative stress differ greatly between stenotic aortic valves and atherosclerotic arteries.

OBJECTIVE

We have demonstrated in a previous study that superoxide radicals play a role in the pathogenesis of cerebral infarction, using a transgenic mouse model of distal middle cerebral artery occlusion, permanent ipsilateral cerebral carotid artery occlusion, and 1-hour contralateral cerebral carotid artery occlusion that produced infarction only in the cortex. However, the role of superoxide radicals in reperfusion injury in transgenic mice overexpressing superoxide dismutase (SOD) is unknown. Using a mouse model of intraluminal blockade of middle cerebral artery that produced both cortical and striatal infarction, we now further examined the role of superoxide radicals in ischemic cerebral infarction after reperfusion in transgenic mice overexpressing human CuZn-SOD activity.

METHODS

Transgenic mice of strain Tg HS/SF-218, carrying human SOD-1 genes, and nontransgenic littermates were anesthetized with chloral hydrate (350 mg/kg IP) and xylazine (4 mg/kg IP). Physiological parameters were maintained at a normal range using a 30% O2/70% N2O gas mixture inserted via an inhalation mask. Body temperature was maintained at 37 +/- 0.5 degrees C by using a heating pad throughout the studies. The middle cerebral artery occlusion was achieved with a 5-0 rounded nylon suture placed within the internal cerebral artery for 3 hours followed by the removal of the suture to allow reperfusion for another 3 hours. Cerebral infarct size in brain slices and infarct volume, neurological deficit, cortical blood flow, and glutathione levels were measured in both transgenic and nontransgenic mice.

RESULTS

Compared with the nontransgenic mice, the infarcted areas were significantly decreased in coronal slices from transgenic mice. The infarct volume (in cubic millimeters) was reduced by 26% in transgenic mice after ischemia and reperfusion. This decrease in the infarct volume in transgenic mice closely paralleled the reduced neurological deficits. Introduction of the suture to block blood supply to the middle cerebral artery territory produced a rapid decrease in the relative surface blood flow in the ipsilateral core and the peri-ischemic (penumbra) areas. There were no significant differences in the local cerebral blood flow in the ischemic core or the penumbra areas between the transgenic and nontransgenic groups. However, the level of reduced glutathione in the penumbra area was significantly higher in transgenic mice than in nontransgenic mice, whereas there was no difference in the reduced glutathione levels in the ischemic core between these two groups.

CONCLUSIONS

Our study demonstrated that superoxide radicals play a major role in the pathogenesis of cerebral infarction in reperfusion injury after a focal stroke. The reduction in infarct volume and neurological deficits is not dependent on the changes in cerebral blood flow but rather correlate with reduced oxidative stress in the ischemic brain tissue, which was indicated by the relatively high levels of endogenous reduced glutathione in transgenic mice.

Three <em>3</em>-hydroxy-<em>3</em>-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O(2)(-)) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 micromol/L). The HMG-CoA reductase product mevalonic acid (400 micromol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C<em>3</em>). A mevalonate-sensitive inhibition of phorbol ester-stimulated O(2)(-) formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (<em>SOD</em>) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced approximately 2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of <em>SOD</em>. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O(2)(-) formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O(2)(-) in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.

Electron paramagnetic resonance (EPR) spectroscopy was used to investigate whether (i) the free radicals produced in the "stunned" myocardium (myocardium with postischemic contractile dysfunction) are derived from O2, (ii) inhibition of radical reactions improves function, and (iii) i.v. spin traps are effective. Open-chest dogs undergoing a 15-min coronary occlusion received an i.v. infusion of the spin trap, alpha-phenyl N-tert-butylnitrone (PBN) (50 mg/kg). In group I (n = 6), EPR signals characteristic of radical adducts of PBN appeared in the coronary venous blood during ischemia and increased dramatically after reperfusion. In group II (n = 6), which received PBN and i.v. superoxide dismutase (<em>SOD</em>; 16,000 units/kg) plus catalase (12,000 units/kg), myocardial production of PBN adducts was undetectable during ischemia (delta = -100%, P less than 0.01 vs. group I) and markedly inhibited after reperfusion (delta = -86%, P less than 0.001). This effect was seen at all levels of ischemic zone flow but was relatively greater in the low-flow range. In group III (n = 8), the same dosages of <em>SOD</em> and catalase without PBN markedly enhanced contractile recovery (measured as systolic wall thickening) after reperfusion [P less than 0.01 at <em>3</em> hr vs. controls (group IV, n = 7)]. Systemic plasma activity of <em>SOD</em> and catalase averaged 127 +/- 24 and 12<em>3</em> +/- 82 units/ml, respectively, 2 min after reperfusion. PBN produced no apparent adverse effects and actually improved postischemic contractile recovery in group I (P less than 0.05 at <em>3</em> hr vs. controls). This study shows that (i) <em>SOD</em> and catalase are highly effective in blocking free radical reactions in vivo, (ii) the radicals generated in the "stunned" myocardium are derived from univalent reduction of O2, and (iii) inhibition of radical reactions improves functional recovery. The results provide direct, in vivo evidence to support the hypothesis that reactive oxygen metabolites play a causal role in the myocardial "stunning" seen after brief ischemia.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+<em>SOD</em> cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.<em>3</em>, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in Mn<em>SOD</em> activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+<em>SOD</em> cells was reduced following expression of antisense Mn<em>SOD</em>. DNA microarray analysis and immunoblotting identified p21, Myc, 14-<em>3</em>-<em>3</em> zeta, cyclin A, cyclin B1, and GADD15<em>3</em> as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+<em>SOD</em> cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+<em>SOD</em> cells expressing antisense Mn<em>SOD</em>. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of Mn<em>SOD</em>, 14-<em>3</em>-<em>3</em> zeta, GADD15<em>3</em>, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-<em>3</em>-<em>3</em> zeta, GADD15<em>3</em>, cyclin A, and cyclin B1 mRNAs in MCF+<em>SOD</em> cells, where Mn<em>SOD</em> overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of Mn<em>SOD</em>, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

Cerium oxide nanoparticles (nanoceria) have recently been shown to protect cells against oxidative stress in both cell culture and animal models. Nanoceria has been shown to exhibit superoxide dismutase (<em>SOD</em>) activity using a ferricytochrome C assay, and this mimetic activity that has been postulated to be responsible for cellular protection by nanoceria. The nature of nanoceria's antioxidant properties, specifically what physical characteristics make nanoceria effective at scavenging superoxide anion, is poorly understood. In this study electron paramagnetic resonance (EPR) analysis confirms the reactivity of nanoceria as an <em>SOD</em> mimetic. X-ray photoelectron spectroscopy (XPS) and UV-visible analyses of nanoceria treated with hydrogen peroxide demonstrate that a decrease in the Ce <em>3</em>(+)/4(+) ratio correlates directly with a loss of <em>SOD</em> mimetic activity. These results strongly suggest that the surface oxidation state of nanoceria plays an integral role in the <em>SOD</em> mimetic activity of nanoceria and that ability of nanoceria to scavenge superoxide is directly related to cerium(III) concentrations at the surface of the particle.

A simple and reproducible microtiter plate assay for measuring superoxide dismutase (<em>SOD</em>) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[<em>3</em>-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,<em>3</em>-be nzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized <em>SOD</em> solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform-ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4+/-0.1 ng of Cu, Zn<em>SOD</em> per well with the coefficient of variation 4.2%.

OBJECTIVE

Many of the effects of resveratrol are consistent with the activation of AMP-activated protein kinase (AMPK), silent information regulator T1 (SIRT1) and peroxisome proliferator-activated receptor (PPAR)γ co-activator 1α (PGC-1α), which play key roles in the regulation of lipid and glucose homeostasis, and in the control of oxidative stress. We investigated whether resveratrol has protective effects on the kidney in type 2 diabetes.

METHODS

Four groups of male C57BLKS/J db/m and db/db mice were used in this study. Resveratrol was administered via gavage to diabetic and non-diabetic mice, starting at 8 weeks of age, for 12 weeks.

RESULTS

The db/db mice treated with resveratrol had decreased albuminuria. Resveratrol ameliorated glomerular matrix expansion and inflammation. Resveratrol also lowered the NEFA and triacylglycerol content of the kidney, and this action was related to increases in the phosphorylation of AMPK and the activation of SIRT1-PGC-1α signalling and of the key downstream effectors, the PPARα-oestrogen-related receptor (ERR)-1α-sterol regulatory element-binding protein 1 (SREBP1). Furthermore, resveratrol decreased the activity of phosphatidylinositol-<em>3</em> kinase (PI<em>3</em>K)-Akt phosphorylation and class O forkhead box (FOXO)<em>3</em>a phosphorylation, which resulted in a decrease in B cell leukaemia/lymphoma 2 (BCL-2)-associated X protein (BAX) and increases in BCL-2, superoxide dismutase (<em>SOD</em>)1 and <em>SOD</em>2 production. Consequently, resveratrol reversed the increase in renal apoptotic cells and oxidative stress, as reflected by renal 8-hydroxy-deoxyguanosine (8-OH-dG), urinary 8-OH-dG and isoprostane concentrations. Resveratrol prevented high-glucose-induced oxidative stress and apoptosis in cultured mesangial cells through the phosphorylation of AMPK and activation of SIRT1-PGC-1α signalling and the downstream effectors, PPARα-ERR-1α-SREBP1.

CONCLUSIONS

The results suggest that resveratrol prevents diabetic nephropathy in db/db mice by the phosphorylation of AMPK and activation of SIRT1-PGC-1α signalling, which appear to prevent lipotoxicity-related apoptosis and oxidative stress in the kidney.

Stroke is a severe and prevalent syndrome for which there is a great need for treatment, including agents to block the cascade of brain injury that occurs in the hours after the onset of ischemia. Reactive oxygen species (ROS) have been implicated in this destructive process, but antioxidant enzymes such as superoxide dismutase (<em>SOD</em>) have been unsatisfactory in experimental stroke models. This study is an evaluation of the effectiveness of salen-manganese complexes, a class of synthetic <em>SOD</em>/catalase mimetics, in a rat focal ischemia model involving middle cerebral artery occlusion. We focus on EUK-1<em>3</em>4, a newly reported salen-manganese complex demonstrated here to have greater catalase and cytoprotective activities and equivalent <em>SOD</em> activity compared with the previously described prototype EUK-8. The administration of EUK-1<em>3</em>4 at <em>3</em> hr after middle cerebral artery occlusion significantly reduced brain infarct size, with the highest dose apparently preventing further infarct growth. EUK-8 was also protective but substantially less effective. These findings support a key role for ROS in the cascade of brain injury after stroke, even well after the onset of ischemia. The enhanced activity of EUK-1<em>3</em>4 suggests that, in particular, hydrogen peroxide contributes significantly to this injury. Overall, this study suggests that synthetic <em>SOD</em>/catalase mimetics might serve as novel, multifunctional therapeutic agents for stroke.

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (<em>SOD</em>) sensitive reaction at a 5-10-fold higher rate than membranes containing <em>3</em> other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by <em>SOD</em>. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,1<em>3</em>-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.

Paclitaxel, one of the most commonly prescribed chemotherapeutic agents, is active against a wide spectrum of human cancer. The mechanism of its cytotoxicity, however, remains controversial. Our results indicate that paclitaxel treatment increases levels of superoxide, hydrogen peroxide, nitric oxide (NO), oxidative DNA adducts, G2-M arrest, and cells with fragmented nuclei. Antioxidants pyruvate and selenium, the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester, and the NO scavenger manganese (III) 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-<em>3</em>-oxide all decreased paclitaxel-mediated DNA damage and sub-G1 cells. In contrast, the glutamylcysteine synthase inhibitor buthionine sulfoximine (BSO) and the superoxide dismutase (<em>SOD</em>) inhibitor 2-methoxyestradiol (2-ME) increased the sub-G1 fraction in paclitaxel-treated cells. These results suggest that reactive oxygen and nitrogen species are involved in paclitaxel cytotoxicity. This notion is further supported with the observation that concentrations of paclitaxel required to inhibit cell growth by 50% correlate with total antioxidant capacity. Moreover, agents such as arsenic trioxide (As2O<em>3</em>), BSO, 2-ME, PD98059, U0126 [mitogen-activated protein/extracellular signal-regulated kinase inhibitors], and LY294002 (phosphatidylinositol <em>3</em>-kinase/Akt inhibitor), all of which decrease clonogenic survival, also decrease the total antioxidant capacity of paclitaxel-treated cells, regardless whether they are paclitaxel sensitive or paclitaxel resistant. These results suggest that paclitaxel chemosensitivity may be predicted by taking total antioxidant capacity measurements from clinical tumor samples. This, in turn, may then improve treatment outcomes by selecting out potentially responsive patients.

Antioxidants are emerging as prophylactic and therapeutic agents. These are the agents, which scavenge free radicals otherwise reactive oxygen species and prevent the damage caused by them. Free radicals have been associated with pathogenesis of various disorders like cancer, diabetes, cardiovascular diseases, autoimmune diseases, neurodegenerative disorders and are implicated in aging. Several antioxidants like <em>SOD</em>, CAT, epigallocatechin-<em>3</em>-O-gallate, lycopene, ellagic acid, coenzyme Q10, indole-<em>3</em>-carbinol, genistein, quercetin, vitamin C and vitamin E have been found to be pharmacologically active as prophylactic and therapeutic agents for above mentioned diseases. Antioxidants are part of diet but their bioavailability through dietary supplementation depends on several factors. This major drawback of dietary agents may be due to one or many of the several factors like poor solubility, inefficient permeability, instability due to storage of food, first pass effect and GI degradation. Conventional dosage forms may not result in efficient formulation owing to their poor biopharmaceutical properties. Principles of novel drug delivery systems need to be applied to significantly improve the performance of antioxidants. Novel drug delivery systems (NDDS) would also help in delivery of these antioxidants by oral route, as this route is of prime importance when antioxidants are intended for prophylactic purpose. Implication of NDDS for the delivery of antioxidants is largely governed by physicochemical characteristics, biopharmaceutical properties and pharmacokinetic parameters of the antioxidant to be formulated. Recently, chemical modifications, coupling agents, liposomes, microparticles, nanoparticles and gel-based systems have been explored for the delivery of these difficult to deliver molecules. Results from several studies conducted across the globe are positive and provided us with new anticipation for the improvement of human healthcare.

This review offers a systematic understanding about how polyphenols target multiple inflammatory components and lead to anti-inflammatory mechanisms. It provides a clear understanding of the molecular mechanisms of action of phenolic compounds. Polyphenols regulate immunity by interfering with immune cell regulation, proinflammatory cytokines' synthesis, and gene expression. They inactivate NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and modulate mitogen-activated protein Kinase (MAPk) and arachidonic acids pathways. Polyphenolic compounds inhibit phosphatidylinositide <em>3</em>-kinases/protein kinase B (PI<em>3</em>K/AkT), inhibitor of kappa kinase/c-Jun amino-terminal kinases (IKK/JNK), mammalian target of rapamycin complex 1 (mTORC1) which is a protein complex that controls protein synthesis, and JAK/STAT. They can suppress toll-like receptor (TLR) and pro-inflammatory genes' expression. Their antioxidant activity and ability to inhibit enzymes involved in the production of eicosanoids contribute as well to their anti-inflammation properties. They inhibit certain enzymes involved in reactive oxygen species ROS production like xanthine oxidase and NADPH oxidase (NOX) while they upregulate other endogenous antioxidant enzymes like superoxide dismutase (<em>SOD</em>), catalase, and glutathione (GSH) peroxidase (Px). Furthermore, they inhibit phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) leading to a reduction in the production of prostaglandins (PGs) and leukotrienes (LTs) and inflammation antagonism. The effects of these biologically active compounds on the immune system are associated with extended health benefits for different chronic inflammatory diseases. Studies of plant extracts and compounds show that polyphenols can play a beneficial role in the prevention and the progress of chronic diseases related to inflammation such as diabetes, obesity, neurodegeneration, cancers, and cardiovascular diseases, among other conditions.

BACKGROUND

It has been previously shown that hyperglycemia enhances free radical production, inducing oxidative damage, which in its turn activates the death pathways implicated in cell apoptosis and necrosis. But the possible involvement of this pathway in the hyperglycemia-induced apoptosis of endothelial cells has not yet been reported.

METHODS

To verify a possible connection between mitochondrial ROS production and apoptosis induced by both stable and oscillating high glucose, <em>SOD</em>, MnTBAP and TTFA was added to HUVEC cell culture medium. We measured nitrotyrosine and 8OHdG as oxidative stress parameters and Bcl-2 expression and Caspase-<em>3</em> expression and activity as apoptosis indicators.

RESULTS

Our results show that hyperglycemia, both stable or oscillating, increases oxidative stress and endothelial cell apoptosis through ROS overproduction at the mitochondrial transport chain level.

CONCLUSIONS

The prevention of mitochondrial oxidative damage seems to be a future important therapeutic strategy in diabetes.

Neonatal and adult animals of five species were exposed to 95+% O2. Survival time and changes in lung antioxidant enzyme activity (superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GP)) in response to hyperoxia were determined. Adult animals succumbed to O2 lung toxicity in <em>3</em>--5 days. Neonatal rats, mice and rabbits showed minimal lung changes after 7 days of hyperoxic exposure and these same neonatal animals showed rapid and significant increases in lung antioxidant enzyme activities. In contrast, neonatal guinea pigs and hamsters had no lung antioxidant enzyme response to hyperoxia and these neonates died in 95+% O2 as readily as their respective parent animals. Results from an in vitro hyperoxic exposure system suggest that the lack of enzymic response of the guinea pig (and hamster) neonates to O2 challenge is due to an inherent pulmonary biochemical unresponsiveness rather than to a deficiency of a necessary "serum factor." The results of this species and age study support the important role of the lung antioxidant enzyme defense system in protection of the lung from O2-induced injury.