One-month old horsegram (Macrotyloma uniflorum (Lam.) Verdc. cv VZM1) and bengalgram (Cicer arietinum L. cv Annogiri) were exposed to different regimes of lead stress as Pb(NO<em>3</em>)2 at 0, 200, 500 and 800 ppm concentrations. The extent of oxidative damage as the rate of lipid peroxidation, antioxidative response and the accumulation of lead in roots and shoots of both plants were evaluated after 12 days of lead stress. Lead (Pb) treated plants showed increased levels of lipid peroxidation as evidenced from the increased malondialdehyde content coupled with the increase in the activities of superoxide dismutase (<em>SOD</em>), catalase (CAT), peroxidase (POD), glutathione reductase (GR), glutathione S-transferase (GST) compared to control (untreated) plants. Lead stress caused significant changes in the activity of antioxidative enzymes. The effect of lead was found to be concentration dependent. Higher concentration of lead (800 ppm) resulted 2- to <em>3</em>-fold increase in <em>SOD</em>, catalase and peroxidase activities, <em>3</em>- to 5-fold increase in GR activity and <em>3</em>- to 4-fold increase in GST activity in roots and leaves of both horsegram and bengalgram plants. Lead stress caused a significant increase in the rate of peroxidation as showed in the levels of malondialdehyde content in roots and leaves of both plant species. Horsegram registered lower Pb accumulation than bengalgram, however localization of Pb was greater in roots than leaves in both plants. In general, lipid peroxide levels and antioxidative enzyme activities were higher in horsegram than bengalgram and also more in roots than leaves which best concordance with the lead contents of both the plants and organs. These results suggest that Pb toxicity causes oxidative stress in plants and the antioxidative enzymes <em>SOD</em>, CAT, POD, GR, GST could play a pivotal role against oxidative injury.

Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene <em>sod</em>-<em>3</em>; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools.

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (Mn<em>SOD</em>) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of Mn<em>SOD</em> mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat Mn<em>SOD</em> promoter. <em>SOD</em> and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state Mn<em>SOD</em> mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated Mn<em>SOD</em> mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the Mn<em>SOD</em> promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine <em>3</em>',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus <em>SOD</em> plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of Mn<em>SOD</em> gene expression is demonstrated.

BACKGROUND

Anticancer drugs act by increasing intracellular hydrogen peroxide levels. Mangafodipir, a superoxide dismutase (SOD) mimic with catalase and glutathione reductase activities, protects normal cells from apoptosis induced by H2O2. We investigated its and other oxidative stress modulators' effects on anticancer drug activity in vitro and in vivo.

METHODS

Cell lysis and intracellular reactive oxygen species levels were assessed in vitro in human leukocytes from healthy subjects and in murine CT26 colon cancer cells. Cells were exposed to the chemotherapeutic agents paclitaxel, oxaliplatin, or 5-fluorouracil, either in the presence or absence of mangafodipir and other oxidative stress modulators. Cell viability was evaluated by the methylthiazoletetrazolium assay. The effects of mangafodipir and other oxidative stress modulators on peripheral blood counts and on tumor growth were studied in BALB/c mice that were implanted with CT26 tumors and treated with 20 mg/kg paclitaxel. Survival of BALB/c mice infected with Staphylococcus aureus was also examined by treatment group. Statistical tests were two-sided.

RESULTS

In vitro lysis of leukocytes exposed to paclitaxel, oxaliplatin, or 5-fluorouracil in combination with mangafodipir was decreased by 46% (95% confidence interval [CI] = 44% to 48%), 30.5% (95% CI = 29% to 32%), and 15% (95% CI = 10% to 20%), compared with lysis of cells treated with anticancer agent alone. Mangafodipir also statistically significantly enhanced in vitro anticancer drug cytotoxicity toward CT26 cancer cells. In vivo, mangafodipir protected mice against paclitaxel-induced leukopenia. Moreover, the survival rate of mice infected with S. aureus and treated with paclitaxel was higher when mangafodipir was also administered (survival: 3 of 17 versus 14 of 17, P < .001). In addition, mangafodipir amplified the inhibitory effect of paclitaxel on CT26 tumor growth in mice.

CONCLUSIONS

Mangafodipir decreased hematotoxicity and enhanced cytotoxicity of anticancer agents.

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5 μm (PM2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM2.5 exposure and adverse effects has not fully elucidated, PM2.5-induced oxidative stress has been considered as an important molecular mechanism of PM2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (<em>SOD</em>) and catalase (CAT) activities compared to the unexposed control cells. PM2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-<em>3</em> (LC<em>3</em>) puncta, and accumulation of LC<em>3</em> in both time- and concentration-dependent manner. PM2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM2.5-induced impairment of pulmonary function.

Epigallocatechin gallate (EGCG), a main active ingredient of green tea, is believed to be beneficial in association with anticarcinogenesis, antiobesity, and blood pressure reduction. Here we report that EGCG extended Caenorhabditis elegans longevity under stress. Under heat stress (<em>3</em>5 degrees C), EGCG improved the mean longevity by 1<em>3</em>.1% at 0.1 microg/ml, 8.0% at 1.0 microg/ml, and 11.8% at 10.0 microg/ml. Under oxidative stress, EGCG could improve the mean longevity of C. elegans by 172.9% at 0.1 microg/ml, 177.7% at 1.0 microg/ml, and 88.5% at 10.0 microg/ml. However, EGCG could not extend the life span of C. elegans under normal culture conditions. Further studies demonstrated that the significant longevity-extending effects of EGCG on C. elegans could be attributed to its in vitro and in vivo free radical-scavenging effects and its up-regulating effects on stress-resistance-related proteins, including superoxide dismutase-<em>3</em> (<em>SOD</em>-<em>3</em>) and heat shock protein-16.2 (HSP-16.2), in transgenic C. elegans with <em>SOD</em>-<em>3</em>::green fluorescent protein (GFP) and HSP-16.::GFP expression. Quantitative real-time PCR results showed that the up-regulation of aging-associated genes such as daf-16, <em>sod</em>-<em>3</em>, and skn-1 could also contribute to the stress resistance attributed to EGCG. As the death rate of a population is closely related to the mortality caused by external stress, it could be concluded that the survival-enhancing effects of EGCG on C. elegans under stress are very important for antiaging research.

Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca(2+) sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca(2+) spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca(2+) sparks were detected with fluo-<em>3</em> and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca(2+) load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca(2+) transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca(2+) spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca(2+) sparks did not correlate with the SR Ca(2+) content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (<em>SOD</em>) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 microm H(2)O(2) sped up the development of Ca(2+) sparks and increased their frequency. These results indicate that the appearance of Ca(2+) sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight oxidative stress is likely to be responsible for unmasking Ca(2+) sparks in skinned preparations. They also suggest that under physiological and pathophysiological conditions the appearance of Ca(2+) sparks may be, at least in part, limited by the fine-tuned equilibrium between mitochondrial ROS production and cellular ROS scavenging mechanisms.

Norepinephrine (NE) causes hypertrophic growth of cardiac myocytes via stimulation of alpha1-adrenergic receptors (alpha1-AR). Reactive oxygen species (ROS) can act as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate alpha1-AR-stimulated hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the superoxide dismutase (<em>SOD</em>)-mimetic MnTMPyP. NE also caused the induction of Mn<em>SOD</em> mRNA. alpha1-AR stimulation with NE (1 microM) in the presence of propranolol (2 microM) for 48-96 h caused a hypertrophic growth phenotype characterized by a <em>3</em>6+/-<em>3</em>% increase in <em>3</em>H-leucine incorporation, a 49+/-14% increase in protein accumulation, a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of Mn<em>SOD</em> mRNA. These responses were all prevented by pretreatment with the alpha1-AR-selective antagonist prazosin (100 n M) or the <em>SOD</em>-mimetics MnTMPyP (50 microM) and Euk-8 (100 microM). MnTMPyP had no effect on alpha1-AR-stimulated <em>3</em>H-inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-myristate-1<em>3</em>-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to alpha1-AR-stimulation in ARVM.

Three new Mn(III) porphyrin catalysts of O2.-dismutation (superoxide dismutase mimics), bearing ether oxygen atoms within their side chains, were synthesized and characterized: Mn(III) 5,10,15,20-tetrakis[N-(2-methoxyethyl)pyridinium-2-yl]porphyrin (MnTMOE-2-PyP(5+)), Mn(III)5,10,15,20-tetrakis[N-methyl-N'-(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTM,MOE-2-ImP(5+)) and Mn(III) 5,10,15,20-tetrakis[N,N'-di(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTDMOE-2-ImP(5+)). Their catalytic rate constants for O2.-dismutation (disproportionation) and the related metal-centered redox potentials vs. NHE are: log k(cat)= 8.04 (E(1/2)=+251 mV) for MnTMOE-2-PyP(5+), log k(cat)= 7.98 (E(1/2)=+<em>3</em>56 mV) for MnTM,MOE-2-ImP(5+) and log k(cat)= 7.59 (E(1/2)=+<em>3</em>65 mV) for MnTDMOE-2-ImP(5+). The new porphyrins were compared to the previously described <em>SOD</em> mimics Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)), Mn(III) 5,10,15,20-tetrakis(N-n-butylpyridinium-2-yl)porphyrin (MnTnBu-2-PyP(5+)) and Mn(III) 5,10,15,20-tetrakis(N,N'-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP(5+)). MnTMOE-2-PyP(5+) has side chains of the same length and the same E(1/2), as MnTnBu-2-PyP(5+)(k(cat)= 7.25, E(1/2)=+ 254 mV), yet it is 6-fold more potent a catalyst of O2.-dismutation , presumably due to the presence of the ether oxygen. The log k(cat)vs. E(1/2) relationship for all Mn porphyrin-based <em>SOD</em> mimics thus far studied is discussed. None of the new compounds were toxic to Escherichia coli in the concentration range studied (up to <em>3</em>0 microM), and protected <em>SOD</em>-deficient E. coli in a concentration-dependent manner. At <em>3</em> microM levels, the MnTDMOE-2-ImP(5+), bearing an oxygen atom within each of the eight side chains, was the most effective and offered much higher protection than MnTE-2-PyP(5+), while MnTDE-2-ImP(5+) was of very low efficacy.

OBJECTIVE

To evaluate if using a soft-tipped guidewire to cannulate the common bile duct may ameliorate development of post-ERCP pancreatitis and facilitate cannulation of the CBD.

METHODS

A single-center, blinded, randomized trial of conventional cannulation technique using sphinctertome and contrast injection versus guidewire cannulation technique.

METHODS

We prospectively randomized <em>3</em>00 patients to conventional cannulation (group I) or guidewire cannulation (group II) technique.

METHODS

Primary outcome measure was incidence of acute pancreatitis and secondary outcome measures were ease of cannulation of common bile duct (assessed by attempts required for common bile duct cannulation & rates of precut sphincterotomy) and overall complication rates.

RESULTS

Guidewire cannulation was associated with significantly lower likelihood of post-ERCP pancreatitis (adjusted OR 0.4<em>3</em>, 95% CI 0.21-0.89, P= 0.02). Twenty-five patients (16.6%) in group I and thirteen patients (8.6%) in group II developed acute pancreatitis, P= 0.0<em>3</em>7. All instances of pancreatitis were mild. There were more women in group II; 41 in group I and 59 in group II, P= 0.028. Otherwise the two groups were comparable for age, age under <em>3</em>5 yr, indication for ERCP, diagnosis, and number of patients with <em>SOD</em>. The number of patients requiring 0-<em>3</em>, 4-6, and 7-10 attempts for successful cannulation of the common bile duct were 87, 48, and 15 in group I and 117, 24, and 9 in group II, respectively, P= 0.001. A total of <em>3</em><em>3</em> patients in group I and 1<em>3</em> patients in group II required precut sphincterotomy, P= 0.007. Rates of accidental pancreatic duct cannulation were 21 in group I and 27 in group II, P= 0.<em>3</em>4. Rates of overall complication were not significantly different in the two groups.

CONCLUSIONS

Guidewire technique for bile duct cannulation lowers likelihood of post-ERCP pancreatitis by facilitating cannulation and reducing need for precut sphincterotomy.

OBJECTIVE

The aim of this study is to investigate the status of oxidative stress and nitric oxide related parameters in type II diabetes mellitus (DM) patients in which heart disease, atherosclerosis, retinopathy, and nephropathy commonly occur, and also to determine the effect of glycemic control on these parameters.

METHODS

Erythrocyte copper zinc-superoxide dismutase (CuZn-<em>SOD</em>), erythrocyte and plasma selenium dependent glutathione peroxidase (Se-GPx), erythrocyte catalase (CAT) activities, erythrocyte and plasma thiobarbituric acid reactive substances (TBARS) levels; nitrite/nitrate (NO(2)(-)/NO(<em>3</em>)(-)), cyclic guanosine monophosphate (cGMP) and nitrotyrosine levels in plasma of type II DM patients were measured.

RESULTS

Erythrocyte CuZn-<em>SOD</em> activities in type II DM were significantly higher than those of the control subjects (p < 0.05). TBARS levels in type II DM were significantly higher than the control subjects (p < 0.001). Plasma NO(2)(-)/NO(<em>3</em>)(-) levels in type II DM patients both during poor glycemic control and after three months of oral antidiabetic treatment were significantly higher than those of the control subjects (p < 0.001). Plasma cGMP levels in type II DM patients during poor glycemic control were significantly lower than those of control subjects (p < 0.001).

CONCLUSIONS

These results indicate that oxidative status and nitric oxide metabolism are affected in type II DM patients. We found high CuZn-SOD activity in type II DM patients. This increased activity could not protect the patients against the reactive oxygen species (ROS), since lipid peroxidation (defined by erythrocyte and plasma TBARS levels) still occurs in DM patients. After the therapy with oral antidiabetic agents for three months, erythrocyte SE-GPx and CAT activities were found to be decreased below the control values. Our results suggested that the low cGMP levels in the study may be a good marker of endothelium dysfunction in DM.

The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, <em>SOD</em>-1, has been fused with an efficient rbcS promoter fragment and <em>3</em>' flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of <em>SOD</em>-1 which occur in wild-type plants. However, tobacco plants with <em>3</em>0- to 50-fold the normal <em>SOD</em>-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2- to 4-fold increases in <em>SOD</em>-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO2 concentrations). Our data indicate that increasing the chloroplastic <em>SOD</em> level in a plant cell is not sufficient to reduce the toxicity of superoxide.

OBJECTIVE

Sustained beta-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown.

RESULTS

Adult male Wistar rats were implanted with osmotic minipumps to infuse either l-isoproterenol (ISO, 25 microg/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and <em>3</em>) augmentation of oxidative stress, with a approximately <em>3</em>-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (<em>SOD</em>) enzyme activity (<em>3</em>0%), protein (40%), and mRNA (60%), without changes in Mn-<em>SOD</em>, catalase, or glutathione peroxidase. Elk-1 and YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn-<em>SOD</em> expression, respectively. ISO-treated hearts exhibited a <em>3</em>-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn-<em>SOD</em> gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a beta1-AR antagonist, but not by ICI118,551, a beta2-AR antagonist. CuZn-<em>SOD</em> downregulation paralleled the increase in ROS, and were similarly blocked by beta1- but not beta2-AR blockade. There were no changes in CuZn-<em>SOD</em> mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn-<em>SOD</em> mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn-<em>SOD</em> siRNA transfection of H9c2 cardiomyocytes to suppress CuZn-<em>SOD</em> protein by approximately 40-50% (analogous to the in vivo changes) induced cellular apoptosis.

CONCLUSIONS

Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta1-AR, thereby contributing to beta-AR-mediated oxidative stress.

To test the hypothesis that the antioxidant enzyme superoxide dismutase (<em>SOD</em>) mimetic TEMPOL improves arterial aging, young (Y, 4-6 months) and old (O, 26-28 months) male C57BL6 mice received regular or TEMPOL-supplemented (1mM) drinking water for <em>3</em> weeks (n = 8 per group). Aortic superoxide was 65% greater in O (P < 0.05 vs. Y), which was normalized by TEMPOL. O had large elastic artery stiffening, as indicated by greater aortic pulse wave velocity (aPWV, 508 ± 22 vs. 418 ± 22 AU), which was associated with increased adventitial collagen I expression (P < 0.05 vs. Y). TEMPOL reversed the age-associated increases in aPWV (4<em>3</em>4 ± 21 AU) and collagen in vivo, and <em>SOD</em> reversed the increases in collagen I in adventitial fibroblasts from older rats in vitro. Isolated carotid arteries of O had impaired endothelial function as indicated by reduced acetylcholine-stimulated endothelium-dependent dilation (EDD) (75.6 ± <em>3</em>.2 vs. 94.5 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (L-NAME) associated with decreased endothelial NO synthase (eNOS) expression (P < 0.05 vs. Y). TEMPOL restored EDD (94.5 ± 1.4%), NO bioavailability and eNOS in O. Nitrotyrosine and expression of NADPH oxidase were ~100-200% greater, and Mn<em>SOD</em> was ~75% lower in O (P < 0.05 vs. Y). TEMPOL normalized nitrotyrosine and NADPH oxidase in O, without affecting Mn<em>SOD</em>. Aortic pro-inflammatory cytokines were greater in O (P < 0.05 vs. Y) and normalized by TEMPOL. Short-term treatment of excessive superoxide with TEMPOL ameliorates large elastic artery stiffening and endothelial dysfunction with aging, and this is associated with normalization of arterial collagen I, eNOS, oxidative stress, and inflammation.

Ozone is a major gaseous pollutant that is known to have detrimental effects on plant growth and metabolism. We have investigated the effects of ozone on Arabidopsis thaliana growth and the pattern of expression of several stress-related genes. A. thaliana plants treated with either 150 or <em>3</em>00 parts per billion (ppb) ozone daily for 6 h exhibited reduced growth and leaf curling. Fresh and dry weights of ozone-treated plants were reduced <em>3</em>0 to 48% compared to ambient air controls. RNA blot analyses demonstrated that mRNA levels for glutathione S-transferase (GST), phenylalanine ammonia-lyase (PAL), a neutral peroxidase, and a cytosolic Cu/Zn superoxide dismutase (<em>SOD</em>) were higher in plants treated with <em>3</em>00 ppb ozone than in ambient air-treated controls. The mRNA levels of lipoxygenase and a catalase were not affected by ozone treatment. Of the transcripts examined, GST mRNA levels increased the most, showing a 26-fold induction <em>3</em> h after the initiation of ozone treatment. PAL mRNA was also rapidly induced, reaching <em>3</em>-fold higher levels than controls within <em>3</em> h of ozone treatment. The neutral peroxidase and <em>SOD</em> mRNA levels rose more slowly, with both reaching maximum levels corresponding to 5-fold and <em>3</em>-fold induction, respectively, approximately 12 h after ozone treatment. These studies indicate that ozone-induced expression of stress-related genes in A. thaliana provides an excellent model system for investigating the molecular and genetic basis of ozone-induced responses in plants.

When seedlings of rice (Oryza sativa L.) cultivar Pant-12 were raised in sand cultures containing 80 and 160 muM Al(<em>3</em>+) in the medium for 5-20 days, a regular increase in Al(<em>3</em>+) uptake with a concomitant decrease in the length of roots as well as shoots was observed. Al(<em>3</em>+) treatment of 160 muM resulted in increased generation of superoxide anion (O(2) (-)) and hydrogen peroxide (H(2)O(2)), elevated amount of malondialdehyde, soluble protein and oxidized glutathione and decline in the concentrations of thiols (-SH) and ascorbic acid. Among antioxidative enzymes, activities of superoxide dismutase (<em>SOD</em> EC 1.15.1.1), guaiacol peroxidase (Guaiacol POX EC 1.11.1.7), ascorbate peroxidase (APX EC 1.11.1.11), monodehydroascorbate reductase (MDHAR EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) increased significantly, whereas the activities of catalase (EC EC 1.11.1.6) and chloroplastic APX declined in 160 muM Al(<em>3</em>+ )stressed seedlings as compared to control seedlings. The results suggest that Al(<em>3</em>+) toxicity is associated with induction of oxidative stress in rice plants and among antioxidative enzymes <em>SOD</em>, Guaiacol POX and cytosolic APX appear to serve as important components of an antioxidative defense mechanism under Al(<em>3</em>+) toxicity. PAGE analysis confirmed the increased activity as well as appearance of new isoenzymes of APX in Al(<em>3</em>+) stressed seedlings. Immunoblot analysis revealed that changes in the activities of APX are due to changes in the amounts of enzyme protein. Similar findings were obtained when the experiments were repeated using another popular rice cv. Malviya-<em>3</em>6.

The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known [maximum lifespan potential (MLSP): >28 yr] and is a unique model of successful aging showing attenuated declines in most physiological function. This study addresses age-related changes in endothelial function and production of reactive oxygen species in NMR arteries and vessels of shorter-living Fischer <em>3</em>44 rats (MLSP: approximately <em>3</em> yr). Rats exhibit a significant age-dependent decline in acetylcholine-induced responses in carotid arteries over a 2-yr age range. In contrast, over a 10-yr age range nitric oxide (NO)-mediated relaxation responses to acetylcholine and to the NO donor S-nitrosopencillamine (SNAP) were unaltered in NMRs. Cellular superoxide anion (O(2)(*-)) and H(2)O(2) production significantly increased with age in rat arteries, whereas they did not change substantially with age in NMR vessels. Indicators of apoptotic cell death (DNA fragmentation rate, caspase <em>3</em>/7 activity) were significantly enhanced ( approximately 250-<em>3</em>00%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a approximately 50% increase in apoptotic cell death. In the hearts of NMRs (2 to 26 yr old), expression of endothelial NO synthase, antioxidant enzymes (Cu,Zn-<em>SOD</em>, Mn-<em>SOD</em>, catalase, and glutathione peroxidase), the NAD(P)H oxidase subunit gp91(phox), and mitochondrial proteins (COX-IV, ATP synthase, and porin, an indicator of mitochondrial mass) did not change significantly with age. Thus long-living NMRs can maintain a youthful vascular function and cellular oxidant-antioxidant phenotype relatively longer and are better protected against aging-induced oxidative stress than shorter-living rats.

Melatonin confers cardioprotective effect against myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress. Activation of silent information regulator 1 (SIRT1) signaling also reduces MI/R injury. We hypothesize that melatonin may protect against MI/R injury by activating SIRT1 signaling. This study investigated the protective effect of melatonin treatment on MI/R heart and elucidated its potential mechanisms. Rats were exposed to melatonin treatment in the presence or the absence of the melatonin receptor antagonist luzindole or SIRT1 inhibitor EX527 and then subjected to MI/R operation. Melatonin conferred a cardioprotective effect by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase release, upregulating SIRT1, Bcl-2 expression and downregulating Bax, caspase-<em>3</em> and cleaved caspase-<em>3</em> expression. Melatonin treatment also resulted in reduced myocardium superoxide generation, gp91(phox) expression, malondialdehyde level, and increased myocardium superoxide dismutase (<em>SOD</em>) level, which indicate that the MI/R-induced oxidative stress was significantly attenuated. However, these protective effects were blocked by EX527 or luzindole, indicating that SIRT1 signaling and melatonin receptor may be specifically involved in these effects. In summary, our results demonstrate that melatonin treatment attenuates MI/R injury by reducing oxidative stress damage via activation of SIRT1 signaling in a receptor-dependent manner.

Oxidative stress plays an important role in diabetic vascular dysfunction. The sources and regulation of reactive oxygen species production in diabetic vasculature continue to be defined. Because peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduced superoxide anion (O(2)(-.)) generation in vascular endothelial cells in vitro by reducing NADPH oxidase and increasing Cu/Zn superoxide dismutase (<em>SOD</em>) expression, the current study examined the effect of PPARgamma ligands on vascular NADPH oxidase and O(2)(-.) generation in vivo. Lean control (db(+)/db(-)) and obese, diabetic, leptin receptor-deficient (db(-)/db(-)) mice were treated with either vehicle or rosiglitazone (<em>3</em> mg/kg/day) by gavage for 7-days. Compared to controls, db(-)/db(-) mice weighed more and had metabolic derangements that were not corrected by treatment with rosiglitazone for 1-week. Aortic O(2)(-.) generation and mRNA levels of the NADPH oxidase subunits, Nox-1, Nox-2, and Nox-4 as well as Nox-4 protein expression were elevated in db(-)/db(-) compared to db(+)/db(-) mice, whereas aortic Cu/Zn <em>SOD</em> protein and PPARgamma mRNA levels were reduced in db(-)/db(-) mice. Treatment with rosiglitazone for 1-week significantly reduced aortic O(2)(-.) production and the expression of Nox-1, 2, and 4 but failed to increase Cu/Zn <em>SOD</em> or PPARgamma in aortic tissue from db(-)/db(-) mice. These data demonstrate that the vascular expression of Nox-1, 2, and 4 subunits of NADPH oxidase is increased in db(-)/db(-) mice and that short-term treatment with the PPARgamma agonist, rosiglitazone, has the potential to rapidly suppress vascular NADPH oxidase expression and O(2)(-.) production through mechanisms that do not appear to depend on correction of diabetic metabolic derangements.

A common property of aging in all animals is that chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for Caenorhabditis elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with the superoxide dismutase gene <em>sod</em>-<em>3</em> expression being the best single predictor of remaining lifespan. Correlation with remaining lifespan became stronger if expression from two aging markers was monitored simultaneously, accounting for up to 49% of the variation in individual lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability is different pathogenicity from individual to individual and that the mechanism involves variable activation of the insulin-signaling pathway.

1. Oxygen free radicals have been suggested to be a contributory factor in complications of diabetes mellitus. There are many reports indicating the changes in parameters of oxidative stress in diabetes mellitus. In this study we aimed to identify whether oxidative stress occurs in the liver and pancreas in the initial stages of development of diabetes. 2. We therefore investigated the lipid peroxide level (thiobarbituric acid-reactive substances, TBARS) and activities of antioxidant enzymes [superoxide dismutase (<em>SOD</em>), catalase and glutathione peroxidase] in liver and pancreas of control and streptozotocin-induced diabetic rats at various stages of development of diabetes. <em>3</em>. Male Sprague-Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups consisting of six rats each. Rats in these subgroups were studied at weekly intervals (0 to 6 weeks). Plasma glucose levels, TBARS levels and activities of antioxidant enzymes were measured in liver and pancreas at various time intervals. 4. There was a significant (P < 0.05) and progressive increase in TBARS levels of liver and pancreas in the diabetic group. Total <em>SOD</em> and Cu-Zn-<em>SOD</em> activity increased (P < 0.05) with progression of diabetes while Mn-<em>SOD</em> activity showed no significant change in either tissue. Catalase and glutathione peroxidase activities increased significantly (P < 0.05) in liver and pancreas. 5. Immunohistochemical study of pancreatic islet revealed a decrease in the expression of insulin with progression of diabetes. However, glucagon and somatostatin showed an increase in immunoreactivity and a difference in their distribution pattern. 6. The findings of the present study suggest that oxidative stress starts at early onset of diabetes mellitus and increases progressively. In conclusion, the structural damage to these tissues or complications of diabetes mellitus may be due to oxidative stress.

A low dose (0.5 mg/kg) of lipopolysaccharide (LPS), administered 72 hours before 60-minute middle cerebral artery occlusion, induced a delayed neuroprotection proven by the significant decrease (-<em>3</em>5%) of brain infarct volume in comparison with control, whereas infarct volumes remained unchanged in rats treated 12, 24, or 168 hours before ischemia. This delayed neuroprotective effect of LPS was induced only with low doses (0.25 to 1 mg/kg), whereas this effect disappeared with a higher dose (2 mg/kg). The delayed neuroprotection of LPS was induced in the cortical part of the infarcted zone, not in the subcortical part. The beneficial effect of LPS on consequences of middle cerebral artery occlusion was suppressed by dexamethasone (<em>3</em> mg/kg) and indomethacin (<em>3</em> mg/ kg) administered 1 hour before LPS, whereas both drugs had no direct effect on infarct volume by themselves, suggesting that activation of inflammatory pathway is involved in the development of LPS-induced brain ischemic tolerance. Preadministration of cycloheximide, an inhibitor of protein synthesis, also blocked LPS-induced brain ischemic tolerance suggesting that a protein synthesis is also necessary as a mediating mechanism. Superoxide dismutase (<em>SOD</em>) could be one of the synthesized proteins because lipopolysaccharide increased <em>SOD</em> brain activity 72 hours, but not 12 hours, after its administration, which paralleled the development of brain ischemic tolerance. In contrast, catalase brain activity remained unchanged after LPS administration. The LPS-induced delayed increase in <em>SOD</em> brain content was suppressed by a previous administration of indomethacin. These data suggest that the delayed neuroprotective effect of low doses of LPS is mediated by an increased synthesis of brain <em>SOD</em> that could be triggered by activation of inflammatory pathway.

Oxidative stress plays an important role in the pathogenesis of lung inflammation. Respiratory syncytial virus (RSV) infection induces reactive oxygen species (ROS) production in vitro and oxidative injury in lungs in vivo; however, the mechanism of RSV-induced cellular oxidative stress has not been investigated. Therefore, we determined whether RSV infection of airway epithelial cells modified the expression and/or activities of antioxidant enzymes (AOE). A549 cells, a human alveolar type II-like epithelial cell line, and small airway epithelial (SAE) cells, normal human cells derived from terminal bronchioli, were infected with RSV and harvested at various time points to measure F(2)-8 isoprostanes by enzyme-linked immunosorbent assay and total and reduced glutathione (GSH and GSSG) by colorimetric assay. Superoxide dismutase (<em>SOD</em>) 1, 2, and <em>3</em>, catalase, glutathione peroxidase (GPx), and glutathione S-transferase (GST) expression was determined by quantitative real-time PCR and Western blot, and their activity was measured by colorimetric assays. RSV infection induced a significant increase of lipid peroxidation products as well as a significant decrease in the GSH/GSSG ratio. There was a significant decrease in <em>SOD</em> 1, <em>SOD</em> <em>3</em>, catalase, and GST expression with a concomitant increase of <em>SOD</em> 2 in RSV-infected cells, compared with uninfected cells. Total <em>SOD</em> activity was increased, but catalase, GPx, and GST activities were decreased, after RSV infection. Our findings suggest that RSV-induced cellular oxidative damage is the result of an imbalance between ROS production and antioxidant cellular defenses. Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation.

The use of methamphetamine (METH) leads to neurotoxic effects in mammals. These neurotoxic effects appear to be related to the production of free radicals. To assess the role of peroxynitrite in METH-induced dopaminergic, we investigated the production of <em>3</em>-nitrotyrosine (<em>3</em>-NT) in the mouse striatum. The levels of <em>3</em>-NT increased in the striatum of wild-type mice treated with multiple doses of METH (4 x 10 mg/kg, 2 h interval) as compared with the controls. However, no significant production of <em>3</em>-NT was observed either in the striata of neuronal nitric oxide synthase knockout mice (nNOS -/-) or copper-zinc superoxide dismutase overexpressed transgenic mice (<em>SOD</em>-Tg) treated with similar doses of METH. The dopaminergic damage induced by METH treatment was also attenuated in nNOS-/- or <em>SOD</em>-Tg mice. These data further confirm that METH causes its neurotoxic effects via the production of peroxynitrite.