These experiments tested the hypothesis that high intensity (interval) training is superior to moderate intensity (continuous) exercise training in the upregulation of antioxidant enzyme activity in skeletal muscle. To test this postulate, we examined changes in oxidative and antioxidant enzyme activities in rat skeletal muscle following 12 wk of either interval (6 x approximately 5-min intervals at approximately 80-95% VO2max) or continuous (45 min at approximately 70% VO2max) exercise training. Both continuous and interval training resulted in significantly elevated (P < 0.05) succinate dehydrogenase (SDH) and <em>3</em>-hydroxyacyl-CoA-dehydrogenase (HADH) activities in the gastrocnemius (G) and soleus (S) muscles compared with controls. SDH and HADH activities in the G and S muscles did not differ between the two exercise groups. Glutathione peroxidase (GPX) activity exceeded controls (P < 0.05) in only the interval trained S muscle. Soleus superoxide dismutase (<em>SOD</em>) activity was higher (P < 0.05) in both exercise groups compared with controls. No differences in <em>SOD</em> activity existed between interval and continuous trained animals. We conclude that when matched for oxygen cost, interval and continuous exercise training result in similar increases in <em>SOD</em> activity. However, high intensity interval exercise is superior to moderate intensity continuous exercise in the promotion of GPX activity in the S.

Pre-eclampsia is a multi-system disorder unique to human pregnancy. Although the aetiology of pre-eclampsia is still unknown, increased placental oxidative stress contributes to the pathophysiology of this pregnancy disorder. The goal of this study was to determine if placental trophoblast cells generate superoxide, and if there was a difference in superoxide generation and superoxide dismutase (<em>SOD</em>) activity between trophoblast cells isolated from pre-eclamptic placentae versus normal placentae. Placentae were obtained from nine normal and 10 pre-eclamptic pregnancies immediately after delivery. Trophoblast cells were isolated and purified by Percoll density gradient centrifugation. Superoxide generation and <em>SOD</em> activity were determined by spectrophotometric assays. Localization of CuZn-<em>SOD</em> protein within the placenta was examined by immunohistochemical staining. mRNA expression of CuZn-<em>SOD</em> was determined in trophoblast cells isolated from five normal and five pre-eclamptic pregnancies by Northern blot analysis. 18S ribosomal mRNA expression was used as an internal standard. We found: (1) trophoblast cells from pre-eclamptic placentae generated significantly more superoxide than trophoblast cells from normal placentae: 17.62+/-<em>3</em>.19 versus 4.70+/-0.76 nmol/5x10(6) cells (mean+/-s.e.), P< 0.01; (2) protein and mRNA expression for CuZn-<em>SOD</em> was mainly localized in the trophoblast cells within the placenta; and (<em>3</em>) <em>SOD</em> activity and relative mRNA expression for CuZn-<em>SOD</em> were significantly decreased in trophoblast cells from pre-eclamptic placentae as compared to trophoblast cells from normal placentae, <em>SOD</em> activity: 6.46+/-1.76 versus 1<em>3</em>.01+/-1.67 units/mg protein, P< 0.05; relative mRNA expression for CuZn-<em>SOD</em>: 0.25+/-0.09 versus 0.7<em>3</em>+/-0.07, P< 0.01. We conclude that increased superoxide generation was associated with decreased <em>SOD</em> activity and mRNA expression for CuZn-<em>SOD</em> in trophoblast cells isolated from pre-eclamptic placentae. These findings support the notion of increased oxidative stress in the pre-eclamptic placenta, which may contribute to the pathophysiology of this pregnancy disorder.

The objective of this experiment was to investigate oxidative injury and the development of an antioxidant system after early weaning in piglets. A total of 40 piglets (Landrace× Large White, weaned at 14 d after birth) were randomly slaughtered 0 (w0d), 1 (w1d), <em>3</em> (w<em>3</em>d), 5 (w5d), or 7 d (w7d; n = 8) after weaning. Concentrations of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), and protein carbonyl and the activities of superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px), and catalase were measured in plasma. Gene expressions of antioxidant enzymes were determined by quantitative reverse transcription PCR analysis. The mediation of transcription factor 65 (p65) and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways by oxidative stress was determined by Western blot analysis. Results showed that the plasma MDA level was significantly higher at <em>3</em> d (P < 0.05) and that the protein carbonyl level increased at 1, <em>3</em>, and 5 d (P < 0.05) compared with w0d. In addition, early weaning suppressed the plasma activity of <em>SOD</em> at 1 d (P < 0.05) and reduced the GSH-Px activity at <em>3</em> d (P < 0.05). The expression results in the jejunum indicate that the genes related to antioxidant enzymes were downregulated (P < 0.05) at <em>3</em> and 5 d after weaning. Uncoupling protein 2 (Ucp2), which is considered to be a feedback regulation on reactive oxygen species generation, tended to decrease in the ileum (P < 0.05) after weaning. Tumor protein 5<em>3</em> (p5<em>3</em>), which regulates reactive oxygen species generation, was enhanced (P < 0.05) in the jejunum after weaning. Meanwhile, early weaning suppressed p65 (at <em>3</em>, 5, and 7 d; P < 0.05) and Nrf2 (at 5 and 7 d; P < 0.05) signals in the jejunum, which might feedback-regulate antioxidant gene expression and promote the development of the antioxidant system. Therefore, we speculate that weaning disrupted oxidative balance and caused oxidative injury in piglets, and this imbalance can recover with the development of an antioxidant system via feedback regulation.

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer <em>3</em>44 rats. In Fischer rats at 4, 14, and 2<em>3</em> mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- <em>3</em>% (P < 0.05) at 2<em>3</em> mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (<em>3</em>6 +/- 2 or <em>3</em>4 +/- <em>3</em>%) and 14 mo (29 +/- 1 or 25 +/- <em>3</em>%) but markedly (P < 0.05) reduced in 2<em>3</em>-mo-old Fischer rats (15 +/- <em>3</em> or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (<em>3</em>5%, <em>3</em>5%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 2<em>3</em> mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- <em>3</em>%) or enalaprilat (28 +/- <em>3</em>%) in 2<em>3</em>-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn <em>SOD</em>, Cu/Zn <em>SOD</em>, extracellular <em>SOD</em>, and p67phox, however, did not differ between 4- and 2<em>3</em>-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 2<em>3</em> mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 2<em>3</em>-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.

Thgoal of this study was to evaluate the efficacy of the antioxidant olive constituent, oleuropein, on infarct size, oxidative damage, and the metabolic profile in rabbits subjected to ischemia. Oleuropein, 10 or 20 mg/(kg x d), was administered to 8 groups that consumed a normal or hypercholesterolemic diet for 6 wk or only the higher dose for <em>3</em> wk. Circulating levels of malondialdehyde, protein carbonyl, nitrite+nitrate, cholesterol, triglycerides, <em>SOD</em> activity, and the metabolic profile were measured using 1H NMR spectra. In rabbits that consumed the normal diet, the infarct size (percentage of infarct to risk areas) was reduced by the administration of 10 mg oleuropein/(kg x d) (16.1 +/- 2.9%) or 20 mg oleuropein/(kg x d) for <em>3</em> wk (21.7 +/- 2.2%) or for 6 wk (24.<em>3</em> +/- 1.<em>3</em>%) compared with the control group (48.05 +/- 2.0%, P < 0.05). Only the higher dose of 20 mg/(kg x d) reduced the infarct size in hypercholesterolemic rabbits (<em>3</em>4.7 +/- 4.4% for 6 wk and <em>3</em>4.8 +/- 6.1% for <em>3</em> wk) compared with the cholesterol-fed control group (52.8 +/- 2.4%, P < 0.05). Oleuropein decreased the plasma lipid peroxidation product and protein carbonyl concentrations compared with the control groups, in which these factors increased relative to baseline due to ischemia and reperfusion. Furthermore, in rabbits administered oleuropein, RBC superoxide dismutase activity did not change during ischemia and reperfusion. This activity was significantly higher than in both control groups in which it was reduced by ischemia and reperfusion compared with baseline. Treatment for 6 wk with both doses of oleuropein reduced total cholesterol and triglyceride concentrations. 1H NMR spectra revealed a different profile of glycolysis metabolites in the oleuropein-treated groups compared with the controls. Oleuropein, for <em>3</em> or 6 wk, reduced the infarct size, conferred strong antioxidant protection and reduced the circulating lipids. This is the first experimental study in vivo that suggests the possibility of using an olive constituent in the treatment of ischemia.

Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-<em>SOD</em>) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-<em>SOD</em> (Ad.<em>SOD</em>2) or beta-galactosidase (Ad.lacZ) and then fed alcohol enterally for 4 weeks. Mn-<em>SOD</em> expression and activity of Ad.<em>SOD</em>2 in liver mitochondria of infected animals was increased nearly <em>3</em>-fold compared with Ad.lacZ-infected controls. Mitochondrial glutathione levels in Ad.lacZ-infected animals were decreased after 4 weeks of chronic ethanol, as expected, but were unchanged in Ad.<em>SOD</em>2-infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.<em>SOD</em>2. Moreover, pathology (e.g. the sum of steatosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ-treated rats. This effect was also blunted in animals infected with Ad.<em>SOD</em>2. Neutrophil infiltration was increased about <em>3</em>-fold in livers from both Ad.lacZ- and Ad.<em>SOD</em>2-infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using (1<em>3</em>)C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the alpha-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.<em>SOD</em>2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.<em>SOD</em>2. Interestingly, tumor necrosis factor-alpha mRNA was elevated to the same extent in both Ad.lacZ- and Ad.<em>SOD</em>2-infected animals follows ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production, and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol.

Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta <em>3</em>1K (n = 4), S19 delta <em>SOD</em> (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta <em>3</em>1K and S19 delta <em>SOD</em>, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta <em>3</em>1K and S19 delta <em>SOD</em> strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2<em>3</em>08 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; <em>3</em> aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

Low rates of angiotensin II (Ang II) infusion raise blood pressure, renal vascular resistance (RVR), NADPH oxidase activity, and superoxide. We tested the hypothesis that these effects are ameliorated by extracellular superoxide dismutase (EC-<em>SOD</em>). EC-<em>SOD</em> knockout (-/-) and wild type (+/+) mice were equipped with blood pressure telemeters and infused subcutaneously with Ang II (400 ng/kg per minute) or vehicle for 2 weeks. During vehicle infusion, EC-<em>SOD</em> -/- mice had significantly (P<0.05) higher MAP (+/+: 107+/-<em>3</em> mm Hg versus -/-: 114+/-2 mm Hg; n=11 to 14), RVR, lipid peroxidation, renal cortical p22(phox) expression, and NADPH oxidase activity. Ang II infusion in EC-<em>SOD</em> +/+ mice significantly (P<0.05) increased MAP, RVR, p22(phox), NADPH oxidase activity, and lipid peroxidation. Ang II reduced <em>SOD</em> activity in plasma, aorta, and kidney accompanied by reduced renal EC-<em>SOD</em> expression. During Ang II infusion, both groups had similar values for MAP (+/+ Ang II: 125+/-<em>3</em> versus -/- Ang II: 124+/-<em>3</em> mmHg; P value not significant), RVR, NADPH oxidase activity, and lipid peroxidation. <em>SOD</em> activity in the kidneys of Ang II-infused mice was paradoxically higher in EC-<em>SOD</em> -/- mice (+/+: 8.8+/-1.2 U/mg protein(-1) versus -/-: 1<em>3</em>.7+/-1.6 U/mg protein(-1); P<0.05) accompanied by a significant upregulation of mRNA and protein for Cu/Zn-<em>SOD</em>. In conclusion, EC-<em>SOD</em> protects normal mice against oxidative stress by attenuating renal p22(phox) expression, NADPH oxidase activation, and the accompanying renal vasoconstriction and hypertension. However, during an Ang II slow pressor response, renal EC-<em>SOD</em> expression is reduced and, in its absence, renal Cu/Zn-<em>SOD</em> is upregulated and may prevent excessive Ang II-induced renal oxidative stress, renal vasoconstriction, and hypertension.

Histologic alterations of ischemia- and reperfusion-induced retinal damage are critically dependent on the duration of the period of ischemia. Male Sprague Dawley rats were anesthetized, and a suture was placed behind the globe including the central retinal artery. Because it was desirable that untreated eyes show a great histologic change due to reperfusion-induced damage (in order that maximum scope would exist for demonstration of any protective effect of a drug treatment), a preliminary series of studies established the time-induced characteristics for the retina with transient regional ischemia. Eyes (n = 6-12 in each group) were subjected to <em>3</em>0, 60, or 90 min of ischemia followed by 0.5, 1, 2, 4, and 24 hr of reperfusion, respectively. The <em>3</em>0-min ischemia followed by reperfusion did not result in any histologic changes; 60-min ischemia followed by reperfusion induced a moderate retinal edema which returned to the preischemic value after 24 hr of reperfusion. The 90-min ischemia followed by reperfusion further aggravated retinal edema and increased the migration of neutrophil leukocytes. Even after 24 hr of reperfusion, the retinal edema had not disappeared although an attenuation was observed. In this study, the rats were treated with superoxide dismutase (<em>SOD</em>-PEG, 15 x 10(<em>3</em>) U/kg) or EGB 761 (100 mg/kg) for 10 days (chronic treatment). The <em>SOD</em> and EGB 761 significantly reduced the development of reperfusion-induced retinal edema and significantly prevented the neutrophil leukocyte infiltration. Both also had a protective effect against reperfusion-induced injury when these agents were administered just before reperfusion ("late" administration).(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the effects of daily repetitive brief (15 min) myocardial ischemia and reperfusion (I/R) in WT C57BL6 and extracellular superoxide dismutase (EC-<em>SOD</em>)-overexpressing mice. In the absence of myocardial necrosis, I/R resulted in persistent fibrosis in ischemic areas of C57/BL6 mice associated with persistent global and segmental anterior wall dysfunction. The I/R protocol induced chemokines (peak <em>3</em> days) followed sequentially by infiltration of macrophages and myofibroblasts (5 days). Fibrosis peaked at 7 days and was stable at 28 days despite regression of the chemokine and cellular response. Discontinuation of I/R at 7 or 28 days led to regression of fibrosis and ventricular dysfunction. In contrast, the EC-<em>SOD</em> mice developed markedly less chemokine induction, cell response, and fibrosis, with no ventricular dysfunction. Reversible fibrosis and ventricular dysfunction are features of human hibernating myocardium. The reduction of the cellular and functional response in EC-<em>SOD</em> mice suggests a role for reactive O(2) in the pathogenesis of ischemic cardiomyopathy.

Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 microg/mL and 100 microg/mL SNP, respectively, although with minor decrease in confluence. IC(50) values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 microg/mL and 449 microg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC(50) SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (i.e. <em>3</em>0 microg/mL and 225 microg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (approximately 1.2 fold) and depletion of lipid peroxidation (approximately 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of <em>SOD</em> ( approximately 1.4 fold) and GSH ( approximately 1.1 fold) as compared to unexposed cells were observed. Caspase-<em>3</em> activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (<em>3</em>.12 microg/mL in primary fibroblasts, 12.5 microg/mL in primary liver cells) than the necrotic concentration (100 microg/mL in primary fibroblasts, 500 microg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC(50) SNP (resulting in apoptosis) and 2 x IC(50)) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (approximately 20 microg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

Male mice receiving vitamin E (5.0 g alpha-tocopherol acetate/kg of food) from 28 wk of age showed a 40% increased median life span, from 61 +/- 4 wk to 85 +/- 4 wk, and 17% increased maximal life span, whereas female mice equally supplemented exhibited only 14% increased median life span. The alpha-tocopherol content of brain and liver was 2.5-times and 7-times increased in male mice, respectively. Vitamin E-supplemented male mice showed a better performance in the tight-rope (neuromuscular function) and the T-maze (exploratory activity) tests with improvements of 9-24% at 52 wk and of 28-45% at 78 wk. The rates of electron transfer in brain mitochondria, determined as state <em>3</em> oxygen uptake and as NADH-cytochrome c reductase and cytochrome oxidase activities, were 16-25% and <em>3</em>5-<em>3</em>8% diminished at 52-78 wk. These losses of mitochondrial function were ameliorated by vitamin E supplementation by <em>3</em>7-56% and by 60-66% at the two time points considered. The activities of mitochondrial nitric oxide synthase and Mn-<em>SOD</em> decreased 28-67% upon aging and these effects were partially (41-68%) prevented by vitamin E treatment. Liver mitochondrial activities showed similar effects of aging and of vitamin E supplementation, although less marked. Brain mitochondrial enzymatic activities correlated negatively with the mitochondrial content of protein and lipid oxidation products (r2 = 0.58-0.99, P < 0.01), and the rates of respiration and of complex I and IV activities correlated positively (r2 = 0.74-0.80, P < 0.01) with success in the behavioral tests and with maximal life span.

It is known that a high-cholesterol diet induces oxidative stress, inflammatory response, and beta-amyloid (Abeta) accumulation in mouse brain, resulting in neurodegenerative changes. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that quercetin protects mouse brain against D-galactose-induced oxidative damage. Against this background, we evaluated the effect of quercetin on high-cholesterol-induced neurotoxicity in old mice and explored its potential mechanism. Our results showed that oral administration of quercetin significantly improved the behavioural performance of high-cholesterol-fed old mice in both a step-through test and the Morris water maze task. This is at least in part caused by decreasing ROS and protein carbonyl levels and restoring Cu--Zn superoxide dismutase (Cu, Zn-<em>SOD</em>) activity. Furthermore, quercetin also significantly activated the AMP-activated protein kinase (AMPK) via down-regulation of protein phosphatase 2C (PP2C), which reduced the integral optical density (IOD) of activated microglia cells and CD11b expression, down-regulated iNOS and cyclooxygenase-2 (COX-2) expression, and decreased IL-1beta, IL-6, and TNF-alpha expression in the brains of high-cholesterol-fed old mice through the suppression of NF-kappaB p65 nuclear translocation. Moreover, AMPK activation significantly increased <em>3</em>-hydroxy-<em>3</em>-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase (ACC) phosphorylation and reduced fatty acid synthase (FAS) expression in the brains of high-cholesterol-fed old mice, which reduced cholesterol levels, down-regulated cholesterol 24-hydroxylase (CYP46A1) and beta-amyloid converting enzyme 1 (BACE1) expression, decreased eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation, and lowered Abeta deposits. However, the neuroprotective effect of quercetin was weakened by intraperitoneal injection of compound C, an AMPK inhibitor. These results suggest that AMPK activated by quercetin may be a potential target to enhance the resistance of neurons to age-related diseases.

Bone marrow stromal cells (BMSCs) and other cell populations derived from mesenchymal precursors are developed for cell-based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen species (ROS) of cellular and environmental origin are involved in redox signaling, cumulative cell damage, senescence, and tumor development. Selenium-dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium-independent (superoxide dismutases [<em>SODs</em>] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. <em>SODs</em> process superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT; TrxR neutralizes other ROS, such as peroxinitrite. Primary BMSCs and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) express GPx1-<em>3</em>, TrxR1, TrxR2, SOD1, SOD2, and CAT. We show here that in standard cell cultures (5%-10% fetal calf serum, 5-10 nM selenite), the activity of antioxidative selenoenzymes is impaired in hMSC-TERT and BMSCs. Under these conditions, the superoxide anion processing enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSCs. Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS-stimulated SOD1 mRNA expression and activity, and reduces ROS accumulation in hMSC-TERT and micronuclei generation in BMSCs. In conclusion, BMSCs in routine cell culture have low antioxidative capacity and are subjected to oxidative stress, as indicated by the generation of micronuclei. Selenite supplementation of BMSC cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures.

1. This study was conducted to determine the effects of heat stress on fearfulness, leucocyte components, oxidative stress and lipid peroxidation in two commercial broiler strains, Cobb (C) and Ross (R). 2. At <em>3</em>6 and <em>3</em>7 d of age birds were exposed to <em>3</em>8 +/- 1 degree C for <em>3</em> h. Rectal temperatures, duration of tonic immobility (TI), haematocrit values, proportions of leucocyte components (heterophil, lymphocyte, basophil, eosinophil, monocyte), malondialdehyde (MDA) concentrations and antioxidant enzyme activities (CAT, <em>SOD</em>, GPx) of all the birds were determined, before and after heat treatment. <em>3</em>. Rectal temperatures increased and haematocrit values decreased in birds exposed to heat stress. Heat stress caused a significant increase in heterophil/lymphocyte and in basophil ratios. 4. Exposing birds to heat stress increased duration of TI, suggesting heat-stressed birds tended to be more fearful. 5. Heat stress resulted in a significant Genotype x Treatment interaction for MDA concentration. CAT, <em>SOD</em> and GPx activities; MDA concentrations in heat-stressed R strain birds were greater than in heat-stressed C strain birds.

With the premise that oxygen free radicals may be responsible for the severity and complications of diabetes, the level of antioxidant enzymes catalase (CAT), superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GPx) as well as the oxidative damage were examined in the tissues of control, diabetic and treated rats. After <em>3</em> weeks of diabetes, the activity of CAT was significantly increased in heart in diabetes (about 6-fold) but decreased in liver. The <em>SOD</em> activity decreased significantly in liver but increased in brain. The activity of GPx decreased significantly in liver and increased in kidney. A significant increase was observed in oxidative damage in heart and kidney and a small increase in brain with decrease in liver and muscle. Vanadate and fenugreek (Trigonella foenum graecum) administration to diabetic animals showed a reversal of the disturbed antioxidant levels and peroxidative damage. Results suggest that oxidative stress play a key role in the complications of diabetes. Vanadate and fenugreek seeds showed an encouraging antioxidant property and can be valuable candidates in the treatment of the reversal of the complications of diabetes.

OBJECTIVE

Placental trophoblast cells (TCs) produce soluble Flt-1 (sFlt-1). Hypoxia induces placental oxidative stress and modulates trophoblast function. The aim of this study was to investigate whether hypoxia mediates TC sFlt-1 production and whether increased sFlt-1 production correlates with increased oxidative stress in placental TCs.

METHODS

Placentas were obtained immediately after delivery from normal pregnant women (n = 8). Placental TCs were isolated by Dispase digestion of villous tissue and purified by Percoll gradient centrifugation. Isolated TCs were cultured under normoxia (21% O2: 5% CO2/95% air) and hypoxia (2% O2/5% CO2/9<em>3</em>% N2) conditions for <em>3</em> days in vitro. TC productions of sFlt-1, VEGF, and PlGF were measured by enzyme-linked immunosorbent assay (ELISA). Lipid peroxide production and superoxide dismutase (CuZn-<em>SOD</em>) levels were evaluated. Messenger RNA expressions of Flt-1, VEGF and PlGF were determined by RT-PCR. Messenger RNA expressions for superoxide dismutase (CuZn-<em>SOD</em>) and heme oxygenase-1 (HO-1) were also determined. Data are expressed as mean +/- SE. A p level less than 0.05 was considered statistically different.

RESULTS

Our results show that sFlt-1 production was significantly increased by TCs cultured under hypoxia condition that correlates with increased lipid peroxide production. We also found that under hypoxia condition: (1) the ratio of PlGF/VEGF production was reversed; (2) the ratio of lipid peroxides to superoxide dismutase production was increased. The increased mRNA expressions for Flt-1 and VEGF and the decreased mRNA expression for PlGF in TCs were consistent with the protein productions under hypoxia condition.

CONCLUSIONS

We concluded that upregulation of sFlt-1 and unbalanced PlGF/VEGF production associated with increased oxidative stress are consequences of hypoxia in placental TCs. Our results suggest that placental TCs are major sources of sFlt-1 and VEGF levels in the maternal circulation in women with preeclampsia.

BACKGROUND

Previously, we assessed selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) on survival and prevention of bacteraemia in patients in intensive-care units. In this analysis, we aimed to assess effectiveness of these interventions for prevention of respiratory tract colonisation and bacteraemia with highly resistant microorganisms acquired in intensive-care units.

METHODS

We did an open-label, clustered group-randomised, crossover study in 13 intensive-care units in the Netherlands between May, 2004, and July, 2006. Participants admitted to intensive-care units with an expected duration of mechanical ventilation of more than 48 h or an expected stay of more than 72 h received SOD (topical tobramycin, colistin, and amphotericin B in the oropharynx), SDD (SOD antibiotics in the oropharynx and stomach plus 4 days' intravenous cefotaxime), or standard care. The computer-randomised order of study regimens was applied by an independent clinical pharmacist who was masked to intensive-care-unit identity. We calculated crude odds ratios (95% CI) for rates of bacteraemia or respiratory tract colonisation with highly resistant microorganisms in patients who stayed in intensive-care units for more than 3 days (ie, acquired infection). This trial is registered at http://isrctn.org, number ISRCTN35176830.

RESULTS

Data were available for 5927 (>99%) of 5939 patients, of whom 5463 (92%) were in intensive-care units for more than 3 days. 239 (13%) of 1837 patients in standard care acquired bacteraemia after 3 days, compared with 158 (9%) of 1758 in SOD (odds ratio 0·66, 95% CI 0·53-0·82), and 124 (7%) of 1868 in SDD (0·48, 0·38-0·60). Eight patients acquired bacteraemia with highly resistant microorganisms during SDD, compared with 18 patients (with 19 episodes) during standard care (0·41, 0·18-0·94; rate reduction [RR] 59%, absolute risk reduction [ARR] 0·6%) and 20 during SOD (0·37, 0·16-0·85; RR 63%, ARR 0·7%). Of the patients staying in intensive-care units for more than 3 days, we obtained endotracheal aspirate cultures for 881 (49%) patients receiving standard care, 886 (50%) receiving SOD, and 828 (44%) receiving SDD. 128 (15%) patients acquired respiratory tract colonisation with highly resistant microorganisms during standard care, compared with 74 (8%) during SDD (0·58, 0·43-0·78; RR 38%, ARR 5·5%) and 88 (10%) during SOD (0·65, 0·49-0·87; RR 32%, ARR 4·6%). Acquired respiratory tract colonisation with Gram-negative bacteria or cefotaxime-resistant and colistin-resistant pathogens was lowest during SDD.

CONCLUSIONS

Widespread use of SDD and SOD in intensive-care units with low levels of antibiotic resistance is justified.

BACKGROUND

None.

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (<em>SOD</em>) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1beta (IL-1beta) alone or a combination of IL-1beta, tumor necrosis factor-alpha, and gamma-interferon resulted in a time- and concentration-dependent decrease of cell viability in the <em>3</em>-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1beta alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn <em>SOD</em> protected against toxicity of the cytokine mixture but not against that of IL-1beta alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn <em>SOD</em> overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

OBJECTIVE

To investigate the mechanism of human prostate cancer cell growth inhibition by plumbagin, a constituent of the widely used medicinal herb Plumbago zeylanica L.

METHODS

Cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Cell cycle distribution and generation of reactive oxygen species (ROS) were determined by flow cytometry. The effect of plumbagin treatment on cellular redox status was determined by analysis of intracellular glutathione (GSH) levels and expression of genes involved in ROS metabolism.

RESULTS

Plumbagin treatment decreased viability of human prostate cancer cells (PC-<em>3</em>, LNCaP, and C4-2) irrespective of their androgen responsiveness or p5<em>3</em> status. Plumbagin-mediated decrease in cell viability correlated with apoptosis induction, which was accompanied by ROS generation and depletion of intracellular GSH levels. Pretreatment of cells with the antioxidant N-acetylcysteine inhibited plumbagin-mediated ROS generation and apoptosis. Plumbagin treatment also resulted in altered expression of genes responsible for ROS metabolism, including superoxide dismutase 2 (Mn-<em>SOD</em>).

CONCLUSIONS

The present study points towards an important role of ROS in plumbagin-induced apoptosis in human prostate cancer cells.

OBJECTIVE

Reactive oxygen species (ROS) have been implicated in the pathophysiology of ARDS. We investigated the pattern of antioxidants in plasma and ROS production by neutrophils in patients with ARDS over 6 days.

METHODS

Observational study. Blood samples were taken when the diagnosis was made (D0) and after <em>3</em> (D<em>3</em>) and 6 days (D6) during therapy.

METHODS

Intensive care units at a University Hospital.

METHODS

Eight patients with ARDS were investigated, 17 healthy volunteers served as controls.

RESULTS

Plasma levels of ascorbate, alpha-tocopherol, retinol, beta-carotene, selenium and lipid peroxidation products (MDA) were determined and the activities of the antioxidative enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-peroxidase (GSH-PX) in erythrocytes were measured. In addition, ROS production (superoxide anion and hydrogen peroxide) in activated neutrophils was assessed. Plasma levels of alpha-tocopherol, ascorbate, beta-carotene and selenium were reduced from the onset of illness. MDA plasma levels were increased throughout the illness. ROS generation from neutrophils was normal on D0 and decreased to D6 in ARDS patients.

CONCLUSIONS

The antioxidative system is severely compromised in patients with ARDS. Plasma levels of alpha-tocopherol, ascorbate, beta-carotene and selenium are decreased. Elevated MDA levels provide further evidence of massive oxidative stress. The routine replacement of micronutrients according to recommended daily allowances was inadequate to compensate for the increased requirements.

1. Endothelium-dependent and -independent regulation of vascular tone in small mesenteric arteries (SMA) from control (db/db +/?) and diabetic (db/db -/-) mice was compared. 2. Phenylephrine-induced maximum contraction, but not sensitivity, of SMA in db/db -/- compared to db/db +/? was enhanced. <em>3</em>. Acetylcholine (ACh), but not sodium nitroprusside (SNP), -induced relaxation was reduced in SMA from db/db -/- compared to db/db +/?. 4. ACh-induced relaxation of SMA was inhibited by a combination of N(omega)-nitro-L-arginine and indomethacin in db/db +/?, but not in db/db -/-. 5. Acute incubation of SMA with tetrahydrobiopterin (BH(4), 10 microM) and sepiapterin (100 microM) enhanced ACh-induced relaxation in SMA from db/db -/-, but not from db/db +/? 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, (10 mM), impaired the sensitivity of SMA from db/db +/? to ACh, which was restored by co-incubation with BH(4) (10 microM). 6. BH(4) and superoxide dismutase (<em>SOD</em>, 150 u ml(-1)), either alone or in combination, had no effect on either ACh or SNP-induced relaxation in SMA from eNOS -/- mice. 7. Incubation of SMA with <em>SOD</em> (150 iu ml(-1)), catalase (200 iu ml(-1)) and L-arginine (1 mM) had no effect on ACh-induced relaxation of SMA. However, the combination of polyethylene glycol-<em>SOD</em> (200 iu ml(-1)) and catalase (80 u ml(-1)) improved the sensitivity of ACh-induced relaxation in db/db -/-, but not in db/db +/?. 8. These data suggest that increased production of superoxide anions and decreased availability of BH(4) result in an 'uncoupling' of nitric oxide synthase and endothelial dysfunction in SMA from db/db -/- mice.

Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. We developed a heavy chain (H) ferritin null mutant in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. We examined heterozygous and wild-type (wt) mice between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, we hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (<em>SOD</em>) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-<em>3</em> were detected in neurons of the +/- mice but not in the wt. Thus, we have developed a mouse model that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies.

To circumvent the early lethality of manganese superoxide dismutase (<em>SOD</em>2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon <em>3</em> of the <em>SOD</em>2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a <em>SOD</em>2 "floxed" male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for <em>SOD</em>2. In sharp contrast to initial publications on <em>SOD</em>2(+/-) mice, the herein reported mice on a mixed genetic background (C57BL/6 x 129/Ola) in their heterozygous state (<em>SOD</em>(+/-)) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (<em>SOD</em>1) in the heart tissue of <em>SOD</em>2(+/-) mice, suggesting that the breakdown of mitochondrial membranes in the heart of <em>SOD</em>2(+/-) mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic <em>SOD</em>1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.