BACKGROUND

The aim of this study was to determine glutathione levels and antioxidant enzyme activities in the drug-naive first-episode patients with schizophrenia in comparison with healthy control subjects.

METHODS

It was a case-controlled study carried on twenty-three patients (20 men and <em>3</em> women, mean age = 29.<em>3</em> ± 7.5 years) recruited in their first-episode of schizophrenia and 40 healthy control subjects (<em>3</em>6 men and 9 women, mean age = 29.6 ± 6.2 years). In patients, the blood samples were obtained prior to the initiation of neuroleptic treatments. Glutathione levels: total glutathione (GSHt), reduced glutathione (GSHr) and oxidized glutathione (GSSG) and antioxidant enzyme activities: superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPx), catalase (CAT) were determined by spectrophotometry.

RESULTS

GSHt and reduced GSHr were significantly lower in patients than in controls, whereas GSSG was significantly higher in patients. GPx activity was significantly higher in patients compared to control subjects. CAT activity was significantly lower in patients, whereas the SOD activity was comparable to that of controls.

CONCLUSIONS

This is a report of decreased plasma levels of GSHt and GSHr, and impaired antioxidant enzyme activities in drug-naive first-episode patients with schizophrenia. The GSH deficit seems to be implicated in psychosis, and may be an important indirect biomarker of oxidative stress in schizophrenia early in the course of illness. Finally, our results provide support for further studies of the possible role of antioxidants as neuroprotective therapeutic strategies for schizophrenia from early stages.

The aim of this study was to characterise biomarker responses in three-spined sticklebacks exposed to copper. For this purpose, adult sticklebacks were exposed for <em>3</em> weeks to copper sulphate at 0, 25, 100 and 200μgL(-1) as Cu. At days 4, 8, 12 and 21, several parameters were measured including liver, gonad and spleen somatic indexes, hepatic biomarkers (catalase (CAT), superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPx), glutathione (GSH), glutathione-S-transferase (GST) and 7-ethoxyresorufin-O-deethylase (EROD)) and hepatic copper and zinc concentrations. Copper induced a rapid and transient increase of antioxidant enzymes and a depletion of glutathione content during the first 8 days of exposure. Significant copper and zinc accumulation in fish liver were observed for the two higher exposure concentrations after 8 and 12 days, respectively. This study showed that copper induced an oxidative stress in fish liver before significant metal accumulation in the liver could be detected, suggesting the involvement of differential mechanisms in copper uptake and metabolism. Three-spined stickleback appears to be a sensitive model to study oxidative stress induced by metals.

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-<em>SOD</em>) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-<em>SOD</em> newborn mice at postnatal days <em>3</em>, 5, and 7 (P<em>3</em>-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-<em>SOD</em> ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P<em>3</em>, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-<em>SOD</em> expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P<em>3</em>, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P<em>3</em>, WT>> TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P<em>3</em> and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P<em>3</em>.

The aim of this study was to examine the effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation (LPO) and the activities of antioxidant enzymes such as superoxide dismutase (<em>SOD</em>), catalase (CAT), and glutathione peroxidase (GSH-Px) in the liver of streptozotocin (STZ)-induced diabetic rats. Twenty-seven rats were randomly divided into three groups: group I, control non-diabetic rats (n = 9); group II, STZ-induced, untreated diabetic rats (n = 8); group III, STZ-induced, CAPE-treated diabetic rats (n = 10), which were intraperitoneally injected with CAPE (10 microM kg(-1) day(-1)) after <em>3</em> days followed by STZ treatment. The liver was excised after 8 weeks of CAPE treatment, the levels of malondialdehyde (MDA) and the activities of <em>SOD</em>, CAT, and GSH-Px in the hepatic tissues of all groups were analyzed. In the untreated diabetic rats, MDA markedly increased in the hepatic tissue compared with the control rats (p < 0.0001). However, MDA levels were reduced to the control level by CAPE. The activities of <em>SOD</em>, CAT, and GSH-Px in the untreated diabetic group were higher than that in the control group (p < 0.0001). The activities of <em>SOD</em> and GSH-Px in the CAPE-treated diabetic group were higher than that in the control group (respectively, p < 0.0001, p < 0.0<em>3</em>5). There were no significant differences in the activity of CAT between the rats of CAPE-treated diabetic and control groups. Rats in the CAPE-treated diabetic group had reduced activities of <em>SOD</em> and CAT in comparison with the rats of untreated diabetic group (p < 0.0001). There were no significant differences in the activity of GSH-Px between the rats of untreated diabetic and CAPE-treated groups. It is likely that STZ-induced diabetes caused liver damage. In addition, LPO may be one of the molecular mechanisms involved in STZ-induced diabetic damage. CAPE can reduce LPO caused by STZ-induced diabetes.

1. We studied the effects of <em>3</em>-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-<em>3</em>':5' cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters. 2. YC-1 elicited a direct concentration-dependent activation of sGC (EC50 18.6 +/- 2.0 microM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing KM and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,<em>3</em>-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound. <em>3</em>. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive. 4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 microM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (<em>3</em> microM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (<em>SOD</em>). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O-2 in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O-2 at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). <em>SOD</em> restored the vasodilator responsiveness of SHR rings to YC-1. 6. In conclusion, these results indicate that YC-1 is an NO-independent, O-2-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.

Manganese-salen complexes (Mn-Salen), including EUK-8 [manganese N,N'-bis(salicylidene)ethylenediamine chloride] and EUK-1<em>3</em>4 [manganese <em>3</em>-methoxy N,N'-bis(salicylidene)ethylenediamine chloride], have been reported to possess combined superoxide dismutase (<em>SOD</em>) and catalase mimetic functions. Because of this <em>SOD</em>/catalase mimicry, EUK-8 and EUK-1<em>3</em>4 have been investigated as possible therapeutic agents in neurological disorders resulting from oxidative stress, including Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. These actions have been explained by the ability of the Mn-Salen to remove deleterious superoxide (O(2)(-)) and H(2)O(2). However, in addition to oxidative stress, cells in models for neurodegenerative diseases may also be subjected to damage from reactive nitrogen oxides (nitrosative stress), resulting from elevated levels of NO and sister compounds, including peroxynitrite (ONOO(-)). We have been examining the interaction of EUK-8 and EUK-1<em>3</em>4 with NO and ONOO(-). We find that in the presence of a per-species (H(2)O(2), ONOO(-), peracetate and persulphate), the Mn-Salen complexes are oxidized to the corresponding oxo-species (oxoMn-Salen). OxoMn-Salens are potent oxidants, and we demonstrate that they can rapidly oxidize NO to NO(2) and also oxidize nitrite (NO(2)(-) to nitrate (NO(2)(-)). Thus these Mn-Salens have the potential to ameliorate cellular damage caused by both oxidative and nitrosative stresses, by the catalytic breakdown of O(2)(-), H(2)O(2), ONOO(-) and NO to benign species: O(2), H(2)O, NO(2)(-) and NO(<em>3</em>)(-).

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-<em>SOD</em> activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-<em>3</em> activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.

OBJECTIVE

To investigate whether activation of adenosine A1 receptor (A1R) through limb remote ischemic preconditioning (RIPC) by a noninvasive tourniquet contribute neuroprotective effects against rat focal cerebral ischemic injury induced by transient middle cerebral artery occlusion (MCAO).

METHODS

One hundred twenty-eight Sprague-Dawley (SD) rats were randomly assigned into eight groups (n=16 each): MCAO, Control, 8-cyclopentyl-1,<em>3</em>-dipropulxanthine (DPCPX, Adenosine A1 receptor antagonist), RIPC, DPCPX+RIPC, Vehicle+RIPC, 2-chloro-N(6)-cyclopentyladenosine (CCPA, Adenosine A1 receptor agonist) and CCPA+DPCPX groups. All animals underwent right middle cerebral artery occlusion (MCAO) for 2 h. Limb RIPC consisted of three cycles of 5-minute ischemia followed by 5-minute reperfusion in right hind-limb by tourniquet application. Neurological deficit scores were evaluated 24 h after reperfusion, and then the infarct volume was assessed with diffusion weighted imaging (DWI) and 2, <em>3</em>, 5-triphenyltetrazolium chloride (TTC) staining. Inflammation was assessed by serum tumor necrosis factor α (TNF(α)) and NO; oxidative stress was estimated by malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAD), superoxide dismutase (<em>SOD</em>) activity and GSH.

RESULTS

Animals in the RIPC, Vehicle+RIPC and CCPA groups developed lower neurological deficit scores and smaller brain infarct volumes than the Control group (P<0.01). Animals in the DPCPX, DPCPX+RIPC and CCPA+DPCPX groups developed higher neurological deficit scores and larger brain infarct volumes than the RIPC, Vehicle+RIPC and CCPA groups (P<0.01). DPCPX abolished the protective effects of RIPC and CCPA. RIPC or CCPA induced a significant increase in brain MnSOD (manganese SOD) activity and NO generation, and this activity was abolished by DPCPX pretreatment. RIPC or CCPA induced a significant reduction (P<0.05) in the GSH and MDA+4HDA concentration and an accumulation in the GSSG concentration in both compartments (serum and tissue) as compared with the MCAO group.

CONCLUSIONS

The present study demonstrates that limb RIPC induced by noninvasive tourniquet reduces cerebral ischemic injury in rats, and the effect of neuroprotection may depend on the activation of adenosine A1 receptors. CCPA pretreatment can induce delayed ischemic tolerance against cerebral ischemia/reperfusion injury. These protective effects are associated with a reduction in oxidative stress, inflammation and endogenous antioxidant preservation.

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-<em>SOD</em>), catalase, heme oxygenase-1 (HO-1) and <em>3</em>-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-1<em>3</em>0 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-<em>SOD</em>, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and <em>3</em>0 mg L(-1)) for 24h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (<em>SOD</em>), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-<em>3</em>, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes.

1. Oxidative stress is believed to be a pathogenetic factor in the development of diabetic complications. In the present study, we investigated the ethanolic extract of the aerial parts of Andrographis paniculata for antihyperglycaemic and anti-oxidant effects in normal and streptozotocin-induced type I diabetic rats. 2. Normal and diabetic rats were randomly divided into groups and treated orally by gavage with vehicle (distilled water), metformin (500 mg/kg bodyweight) or the extract (400 mg/kg bodyweight), twice a day for 14 days. <em>3</em>. At the end of the 14 day period, the extract, like metformin, significantly increased bodyweight (P < 0.01) and reduced fasting serum glucose in diabetic rats (P < 0.001) when compared with vehicle, but had no effect on bodyweight and serum glucose in normal rats. Levels of liver and kidney thiobarbituric acid-reactive substances (TBARS) were significantly increased (P < 0.0001, P < 0.01, respectively), while liver glutathione (GSH) concentrations were significantly decreased (P < 0.005) in vehicle-treated diabetic rats. Liver and kidney TBARS levels were significantly lower (P < 0.0001, P < 0.005, respectively), whereas liver GSH concentrations were significantly higher (P < 0.05) in extract- and metformin-treated diabetic rats compared with vehicle-treated diabetic rats. Andrographis paniculata significantly decreased kidney TBARS level (P < 0.005) in normal rats. Hepatic superoxide dismutase (<em>SOD</em>), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were significantly lower in vehicle-treated diabetic rats compared with vehicle-treated normal rats. The extract, as well as metformin, significantly increased the activity of <em>SOD</em> and CAT, but had no significant effect on GSH-Px activity in diabetic rats. The extract and metformin did not produce significant changes in the activity of these anti-oxidant enzymes in normal rats. 4. Our results show that oxidative stress is evident in streptozotocin-diabetic rats and indicate that the ethanolic extract of A. paniculata not only possesses an antihyperglycaemic property, but may also reduce oxidative stress in diabetic rats.

In order to examine antioxidant status and lipid peroxidation in schizophrenia patients, activities of three free radical scavenging enzymes (superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px) and catalase (CAT)), and the level of thiobarbituric acid-reactive substances (TBARS) as an index of lipid peroxidation have been studied in red blood cells. Schizophrenic patients were divided into three groups (disorganized (n = 21), paranoid (n = 26) and residual types (n = 18)) to determine differences between subgroups. <em>SOD</em>, CAT and GSH-Px activities in the control group were found to be 1461.0 +/- 248.6 U g(-1) Hb, 148.2 +/- 59.<em>3</em> k g(-1) Hb and 25.87 +/- 4.25 U g(-1) Hb, respectively. We found no significant differences in <em>SOD</em> activities between study and control groups. There was a significant increase in <em>SOD</em> activity in the residual group compared to the paranoid group (P < 0.005). CAT activity was found to be increased in disorganized (148%), paranoid (147%), and residual (165%) groups compared to the control group. GSH-Px activity was markedly increased in the study groups except the paranoid group. Statistically significant (<em>3</em>-4 fold) increases in TBARS levels of red blood cells were found in all the study groups. It is proposed that antioxidant status may be changed in schizophrenia and thus may induce lipid peroxidation. Therefore, oxidative stress may have a pathophysiological role in all the subtypes of schizophrenia.

BACKGROUND

Acute RT-induced damage to the lung is characterized by inflammatory changes, which proceed to the development of fibrotic lesions in the late phase of injury. Ultimately, complete structural ablation will ensue, if the source of inflammatory/fibrogenic mediators and oxidative stress is not removed or attenuated. Therefore, the purpose of this study is to determine whether overexpression of extracellular superoxide dismutase (EC-<em>SOD</em>) in mice ameliorates acute radiation induced injury by inhibiting activation of TGFbeta1 and downregulating the Smad <em>3</em> arm of its signal transduction pathway.

METHODS

Whole thorax radiation (single dose, 15 Gy) was delivered to EC-<em>SOD</em> overexpressing transgenic (XRT-TG) and wild-type (XRT-WT) animals. Mice were sacrificed at 1 day, 1 week, <em>3</em>, 6, 10 and 14 weeks. Breathing rates, right lung weights, total/differential leukocyte count, activated TGFbeta1 and components of its signal transduction pathway (Smad <em>3</em> and p-Smad 2/<em>3</em>) were assessed to determine lung injury.

RESULTS

Irradiated wild-type (XRT-WT) animals exhibited time dependent increase in breathing rates and right lung weights, whereas these parameters were significantly less increased (p < 0.05) at <em>3</em>, 6, 10 and 14 weeks in irradiated transgenic (XRT-TG) mice. An inflammatory response characterized predominantly by macrophage infiltration was pronounced in XRT-WT mice. This acute inflammation was significantly attenuated (p < 0.05) in XRT-TG animals at 1, <em>3</em>, 6 and 14 weeks. Expression of activated TGFbeta1 and components of its signal transduction pathway were significantly reduced (p < 0.05) at later time-points in XRT-TG vs. XRT-WT.

CONCLUSIONS

This study shows that overexpression of EC-SOD confers protection against RT-induced acute lung injury. EC-SOD appears to work, in part, via an attenuation of the macrophage response and also decreases TGFbeta1 activation with a subsequent downregulation of the profibrotic TGFbeta pathway.

AMP-activated protein kinase influences cellular metabolism, glucose-regulated gene expression, and insulin secretion of pancreatic beta cells. Its sustained activation by culture at low glucose concentrations or in the presence of 5-aminoimidazole-4-carboxamide riboside (AICAR) was shown to trigger apoptosis in beta cells. This study shows that both low glucose- and AICAR-induced apoptosis are associated with increased formation of mitochondrial superoxide-derived radicals and decreased mitochondrial activity. Mitochondrial dysfunction was reflected by an increased oxidized state of the mitochondrial flavins (FMN/FAD) but not of NAD(P)H. It was accompanied by suppression of glucose oxidation and glucose-induced insulin secretion, while palmitate oxidation appeared unaffected. When the cellular accumulation of superoxide-derived radicals was quenched by the ROS scavengers vitamin E, N-acetylcysteine, or the <em>SOD</em>-mimetic compound MnTBAP, apoptosis was significantly inhibited. Both low glucose and AICAR also elevated the expression of BH<em>3</em>-domain-only Bcl-2 antagonists, and induced caspase-<em>3</em> activation, causing caspase-dependent truncation of Bcl-2. Overexpression of recombinant human Bcl-2 prevented caspase-<em>3</em> activation, endogenous Bcl-2 processing, and apoptosis, but did not attenuate oxygen radical formation, AMPK activation, or JNK phosphorylation. We conclude that apoptosis by prolonged AMPK activation in beta cells results from enhanced production of mitochondria-derived oxygen radicals and onset of the intrinsic mitochondrial apoptosis pathway, followed by caspase activation and Bcl-2 cleavage which may amplify the death signal.

CSF from patients with motor neurone disease (MND) has been reported to be toxic to cultured primary neurones. We found that CSF from MND patients homozygous for the D90A CuZn-superoxide dismutase (CuZn-<em>SOD</em>) mutation, patients with sporadic MND and patients with familial MND without CuZn-<em>SOD</em> mutations significantly increased apoptosis and reduced phosphorylation of neurofilaments in cultured spinal cord neurones when compared with the effects of CSF from patients with other neurological diseases. Exposure of spinal cord cultures to MND CSF also triggered microglial activation. The toxicity of MND CSF was independent of the presence of the CuZn-<em>SOD</em> mutation, and it did not correlate with gelatinase activity or the presence of immunoglobulin G autoantibodies in the CSF. The concentrations of glutamate, aspartate and glycine in MND CSF were not elevated. Antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-<em>3</em>-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors prevented the toxic CSF-induced neuronal death but not microglial activation, whereas minocycline, a tetracycline derivative with anti-inflammatory potential independent of antimicrobial activity, reduced both the apoptotic neuronal death and microglial activation. We conclude that the cytotoxic action of CSF is prevalent in all MND cases and that microglia may mediate the toxicity of CSF by releasing excitotoxicity-enhancing factors.

Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 microg/kg, subsequent dose 0.07 microg.kg(-1).day(-1) im, <em>3</em>0 days) or vehicle. Blood levels at the end of treatment were 7.97 +/- 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; <em>3</em>) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM). Superoxide dismutase (<em>SOD</em>, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.<em>3</em> mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, <em>SOD</em> did not affect phenylephrine responses; however, when coincubated with l-NAME, the l-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and <em>SOD</em> restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2(-) production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease.

To obtain insight into the morphological and molecular correlates of motoneuron degeneration in amyotrophic lateral sclerosis (ALS) mice that express G9<em>3</em>A mutant superoxide dismutase (<em>SOD</em>)1 (G9<em>3</em>A mice), we have mapped and characterized 'sick' motoneurons labelled by the 'stress transcription factors' ATF<em>3</em> and phospho-c-Jun. Immunocytochemistry and in situ hybridization showed that a subset of motoneurons express ATF<em>3</em> from a relatively early phase of disease before the onset of active caspase <em>3</em> expression and motoneuron loss. The highest number of ATF<em>3</em>-expressing motoneurons occurred at symptom onset. The onset of ATF<em>3</em> expression correlated with the appearance of ubiquitinated neurites. Confocal double-labelling immunofluorescence showed that all ATF<em>3</em>-positive motoneurons were immunoreactive for phosphorylated c-Jun. Furthermore, the majority of ATF<em>3</em> and phospho-c-Jun-positive motoneurons were also immunoreactive for CHOP (GADD15<em>3</em>) and showed Golgi fragmentation. A subset of ATF<em>3</em> and phosphorylated c-Jun-immunoreactive motoneurons showed an abnormal appearance characterized by a number of distinctive features, including an eccentric flattened nucleus, perikaryal accumulation of ubiquitin immunoreactivity, juxta-nuclear accumulation of the Golgi apparatus and the endoplasmic reticulum, and intense Hsp70 immunoreactivity. These abnormal cells were not immunoreactive for active caspase <em>3</em>. We conclude that motoneurons in ALS-<em>SOD</em>1 mice prior to their death and disappearance experience a prolonged sick phase, characterized by the gradual accumulation of ubiquitinated material first in the neurites and subsequently the cell body.

The importance of superoxide dismutase (<em>SOD</em>) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial <em>SOD</em> and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial <em>SOD</em> antibody inhibited the effect of nocardial <em>SOD</em> on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial <em>SOD</em>, another monoclonal antinocardial antibody (not reactive with <em>SOD</em>), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at <em>3</em>, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-<em>SOD</em> antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-<em>SOD</em> antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for <em>SOD</em>. The data demonstrate that surface-associated <em>SOD</em> protects N. asteroides for oxidative killing in vivo during all stages of infection.

Experimental evidences suggest that hyperglycaemia-induced overproduction of reactive oxygen species and subsequent damage to proteins, lipids and DNA may play a key role in the development of distal symmetric polyneuropathy (DSPN)-the most common complication of diabetes mellitus. The study population consisted of 51 individuals aged 52-82 years classified into <em>3</em> groups: 16 patients diagnosed with type 2 diabetes mellitus (T2DM) with DSPN, 16 T2DM patients without DSPN and 19 control subjects without diabetes and neuropathy. The study was conducted to determine the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPX) and total antioxidant status (TAS) in the examined groups. An alkaline comet assay was used to determine the extent of DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites. A significant decrease of <em>SOD</em> (P < 0.05), GPX (P < 0.05) and nonsignificant decrease of CAT (P>> 0.05), and TAS status (P>> 0.05) were seen in T2DM patients with neuropathy compared to T2DM patients as well as controls. T2DM patients with or without neuropathy revealed significantly lower (P < 0.05) plasma concentration of nitrous oxide compared to the control subjects. Endogenous level of oxidative DNA damage in T2DM patients with DSPN was significantly higher compared both to the controls and T2DM patients without DSPN (P < 0.001). Moreover, lymphocytes isolated from T2DM patients with DSPN were more susceptible to oxidative DNA lesions induced by hydrogen peroxide than from T2DM patients without DSPN (P < 0.001). Our results confirm hypothesis that oxidative stress may play a substantial role in the development and progression of diabetic distal symmetric polyneuropathy.

Radix Angelica sinensis, known as Danggui in Chinese, has been used to treat cardiovascular and cerebrovascular diseases in Traditional Chinese Medicine for a long time. Modern phytochemical studies showed that Z-ligustilide (LIG) is the main lipophilic component of Danggui. In this study, we examined whether LIG could protect ischemia/reperfusion-induced brain injury by minimizing oxidative stress and anti-apoptosis. Transient forebrain cerebral ischemia (FCI) was induced by the bilateral common carotid arteries occlusion for <em>3</em>0 min. LIG was intraperitoneally injected to ICR mice at the beginning of reperfusion. As determined via 2,<em>3</em>,5-triphenyl tetrazolium chloride (TTC) staining at 24 h following ischemia, the infarction volume in the FCI mice treated without LIG (22.1 +/- 2.6%) was significantly higher than that in the FCI mice treated with 5 mg/kg (11.8 +/- 5.2%) and 20 mg/kg (2.60 +/- 1.5%) LIG (P < 0.05 or P < 0.01). LIG treatment significantly decreased the level of malondialdehyde (MDA) and increased the activities of the antioxidant enzyme glutathione peroxidase (GSH-PX) and superoxide dismutase (<em>SOD</em>) in the ischemic brain tissues (P < 0.05 or P < 0.01 vs. FCI group). In addition, LIG provided a great increase in Bcl-2 expression as well as a significant decrease in Bax and caspase-<em>3</em> immunoreactivities in the ischemic cortex. The findings demonstrated that LIG could significantly protect the brain from damage induced by transient forebrain cerebral ischemia. The antioxidant and anti-apoptotic properties of LIG may contribute to the neuroprotective potential of LIG in cerebral ischemic damage.

We examined antioxidant activity in the pre-conditioned canine myocardium with four 5-min episodes of regional ischemia and reperfusion. Immediately after repetitive brief ischemia, mitochondrial Mn-superoxide dismutase (<em>SOD</em>) activity in the ischemic myocardium significantly increased compared with that in the nonischemic myocardium (18.7 +/- 2.1 vs. 14.9 +/- 1.0 U/mg protein, P < 0.05). Although no difference was seen in the activity between these regions after <em>3</em> h of the sublethal ischemia, a significant increase in the activity of the ischemic myocardium reappeared after 24 h compared with that of the nonischemic myocardium (26.7 +/- 0.9 vs. 20.8 +/- 0.9 U/mg protein, P < 0.05). Mn-<em>SOD</em> content increased gradually in the ischemic myocardium after sublethal ischemia, with a peak after 24 h (2.8 +/- 0.1 vs. 2.1 +/- 0.1 microgram/mg protein, P < 0.05). There were no differences in the activity and content of Cu, Zn-<em>SOD</em> between these regions after sublethal ischemia. Activities of glutathione peroxidase and reductase were significantly higher and lower, respectively, in the ischemic myocardium than those of the nonischemic myocardium immediately after repetitive brief ischemia, but no differences between these regions were seen in activities after <em>3</em> or 24 h. These results indicate that a brief ischemic insult alters myocardial antioxidant activity not only immediately after but also 24 h after sublethal ischemia.

Mutations of copper,zinc-superoxide dismutase (cu,zn <em>SOD</em>) are found in patients with a familial form of amyotrophic lateral sclerosis. When expressed in transgenic mice, mutant human cu,zn <em>SOD</em> causes progressive loss of motor neurons with consequent paralysis and death. Expression profiling of gene expression in <em>SOD</em>1-G9<em>3</em>A transgenic mouse spinal cords indicates extensive glial activation coincident with the onset of paralysis at <em>3</em> months of age. This is followed by activation of genes involved in metal ion regulation (metallothionein-I, metallothionein-III, ferritin-H, and ferritin-L) at 4 months of age just prior to end-stage disease, perhaps as an adaptive response to the mitochondrial destruction caused by the mutant protein. Induction of ferritin-H and -L gene expression may also limit iron catalyzed hydroxyl radical formation and consequent oxidative damage to lipids, proteins, and nucleic acids. Thus, glial activation and adaptive responses to metal ion dysregulation are features of disease in this transgenic model of familial amyotrophic lateral sclerosis.

We tested two hypotheses, first that exercise training reverses age-related decrements in endothelium-dependent dilation in soleus muscle feed arteries and second that this improved endothelium-dependent dilation is the result of increased nitric oxide (NO) bioavailability due to increased content and phosphorylation of endothelial NO synthase (eNOS) and/or increased antioxidant enzyme content. Young (2 mo) and old (22 mo) male Fischer <em>3</em>44 rats were exercise trained (Ex) or remained sedentary (Sed) for 10-12 wk, yielding four groups of rats: 1) young Sed (4-5 mo), 2) young Ex (4-5 mo), <em>3</em>) old Sed (24-25 mo), and 4) old Ex (24-25 mo). Soleus muscle feed arteries (SFA) were isolated and cannulated with two glass micropipettes for examination of endothelium-dependent (ACh) and endothelium-independent [sodium nitroprusside (SNP)] vasodilator function. To determine the mechanism(s) by which exercise affected dilator responses, ACh-induced dilation was assessed in the presence of N(omega)-nitro-l-arginine (l-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and l-NNA + Indo. Results indicated that ACh-induced dilation was blunted in old Sed SFA relative to young Sed SFA. Exercise training improved ACh-induced dilation in old SFA such that vasodilator responses in old Ex SFA were similar to young Sed and young Ex SFA. Addition of l-NNA, or l-NNA + Indo, abolished the exercise effect. Immunoblot analysis revealed that extracellular superoxide dismutase (<em>SOD</em>) protein content was increased by training in old SFA, whereas eNOS and <em>SOD</em>-1 protein content were not altered. Addition of exogenous <em>SOD</em>, or <em>SOD</em> + catalase, improved ACh-induced dilation in old Sed SFA such that vasodilator responses were similar to young Sed SFA. Addition of l-NNA abolished the effect of exogenous <em>SOD</em> in old Sed arteries. Collectively, these results indicate that exercise training reverses age-induced endothelial dysfunction in SFA by increasing NO bioavailability and that increases in vascular antioxidant capacity may play an integral role in the improvement in endothelial function.

UV radiation from the sun is a potent environmental risk factor in the pathogenesis of skin damage. Much of the skin damage caused by ultraviolet A (UVA) irradiation from the sun is associated with oxidative stress. The aim of this study was to investigate the protective role of ellagic acid (25-75 μM), a natural antioxidant, against UVA (5-20 J/cm(2))-induced oxidative stress and apoptosis in human keratinocyte (HaCaT) cells and to reveal the possible mechanisms underlying this protective efficacy. Ellagic acid pre-treatment markedly increased HaCaT cell viability and suppressed UVA-induced ROS generation and MDA formation. Moreover, ellagic acid pre-treatment prevented UVA-induced DNA damage as evaluated by the comet assay. Ellagic acid treatment also significantly inhibited the UVA-induced apoptosis of HaCaT cells, as measured by a reduction of DNA fragmentation, mitochondria dysfunction, ER stress, caspase-<em>3</em> activation, and Bcl-2/Bax deregulation. Notably, the antioxidant potential of ellagic acid was directly correlated with the increased expression of HO-1 and <em>SOD</em>, which was followed by the downregulation of Keap1 and the augmented nuclear translocation and transcriptional activation of Nrf2 with or without UVA irradiation. Nrf2 knockdown diminished the protective effects of ellagic acid. Therefore, ellagic acid may be useful for the treatment of UVA-induced skin damage.