Plasma Superoxide Dismutase-1 as a Surrogate Marker of Vivax Malaria Severity

Background: Severe outcomes have been described for both Plasmodium falciparum and P. vivax infections. The identification of sensitive and reliable markers of disease severity is fundamental to improving patient care. An intense pro-inflammatory response with oxidative stress and production of reactive oxygen species is present in malaria. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and antioxidant agents such as superoxide dismutase-1 (SOD-1) are likely candidate biomarkers for disease severity. Here we tested whether plasma levels of SOD-1 could serve as a biomarker of severe vivax malaria. Methodology/Principal Findings: Plasma samples were obtained from residents of the Brazilian Amazon with a high risk for P. vivax transmission. Malaria diagnosis was made by both microscopy and nested PCR. A total of 219 individuals were enrolled: non-infected volunteers (n = 90) and individuals with vivax malaria: asymptomatic (n = 60), mild (n = 50) and severe infection (n = 19). SOD-1 was directly associated with parasitaemia, plasma creatinine and alanine amino-transaminase levels, while TNF-alpha correlated only with the later enzyme. The predictive power of SOD-1 and TNF-alpha levels was compared. SOD-1 protein levels were more effective at predicting vivax malaria severity than TNF-alpha. For discrimination of mild infection, elevated SOD-1 levels showed greater sensitivity than TNF-alpha (76% vs. 30% respectively; p < 0.0001), with higher specificity (100% vs. 97%; p < 0.0001). In predicting severe vivax malaria, SOD-1 levels exhibited higher sensitivity than TNF-alpha (80% vs. 56%, respectively; p < 0.0001; likelihood ratio: 7.45 vs. 3.14; p, 0.0001). Neither SOD-1 nor TNF-alpha could discriminate P. vivax infections from those caused by P. falciparum. Conclusion: SOD-1 is a powerful predictor of disease severity in individuals with different clinical presentations of vivax malaria.

DNA oxidation, strand-breaks and etheno-adducts formation promoted by Cu, Zn-superoxide dismutase-H(2)O(2) in the presence and absence of bicarbonate

Biomolecule oxidation promoted by Cu, Zn-superoxide dismutase (SOD1) has been studied because of its potential role in neurodegenerative diseases. We studied the mechanism of DNA damage promoted by the SOD1-H(2)O(2) system. The system promoted the formation of strand breaks in plasmid DNA and the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA. We were also able to detect, for the. first time, 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilon dGuo) in calf thymus DNA exposed to SOD1-H(2)O(2). The addition of a copper chelator caused a decrease in the frequency of 8-oxodGuo and 1,N(2)-epsilon dGuo, indicating the participation of copper ions lost from SOD1 active sites. The addition of bicarbonate increased the levels of both DNA lesions. We conclude that copper liberated from SOD1 active sites has a central role in the mechanism of DNA damage promoted by SOD1 in the presence of H(2)O(2), and that bicarbonate can modulate the reactivity of released copper.

Moderate exercise training decreases aortic superoxide production in myocardial infarcted rats

Myocardial infarction (MI) has been associated with increases in reactive oxygen species (ROS). Exercise training (ET) has been shown to exert positive modulations on vascular function and the purpose of the present study was to investigate the effect of moderate ET on the aortic superoxide production index, NAD(P)H oxidase activity, superoxide dismutase activity and vasomotor response in MI rats. Aerobic ET was performed during 11 weeks. Myocardial infarction significantly diminished maximal exercise capacity, and increased vasoconstrictory response to norepinephrine, which was related to the increased activity of NAD(P)H oxidase and basal superoxide production. On the other hand, ET normalized the superoxide production mostly due to decreased NAD(P)H oxidase activity, although a minor SOD effect may also be present. These adaptations were paralleled by normalization in the vasoconstrictory response to norepinephrine. Thus, diminished ROS production seems to be an important mechanism by which ET mediates its beneficial vascular effects in the MI condition.

Photochemical damage and comparative performance of superoxide dismutase and ascorbate peroxidase in sugarcane leaves exposed to paraquat-induced oxidative stress

The physiological responses of sugarcane (Succharion officinarum L.) to oxidative stress induced by methyl viologen (paraquat) were examined with respect to photochemical activity, chlorophyll content, lipid peroxidation and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. Thirty-day-old sugarcane plants were sprayed with 0, 2, 4, 6 and 8 mM methyl viologen (MV). Chlorophyll fluorescence was measured after 18 It and biochemical analyses were performed after 24 and 48 h. Concentrations of MV above 2 mM caused significant damage to photosystem II (PSII) activity. Potential and effective quantum efficiency of PSII and apparent electron transport rate were greatly reduced or practically abolished. Both chlorophyll and soluble protein contents steadily decreased with MV concentrations above 2 mM after 24 It of exposure, which became more pronounced after 48 It, achieving a 3-fold decrease. Insoluble protein contents were little affected by MV. Oxidative stress induced by MV was evidenced by increases in lipid peroxidation. Specific activity of SOD increased, even after 48 h of exposure to the highest concentrations of MV, but total activity on a fresh weight basis did not change significantly. Nondenaturing YAGE assayed with H2O2 and KCN showed that treatment with MV did not change Cu/Zn-SOD and MnSOD isoform activities. In contrast, APX specific activity increased at 2 mM MV but then dropped at higher doses. Oxidative damage induced by MV was inversely related to APX activity. It is suggested that the major MV-induced oxidative damages in sugarcane leaves were related to excess H2O2, probably in chloroplasts, caused by an imbalance between SOD and APX activities, in which APX was a limiting step. Reduced photochemical activity allowed the early detection of the ensuing oxidative stress. (c) 2007 Elsevier Inc. All rights reserved.

Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia

Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.

Effect of academic psychological stress in post-graduate students: The modulatory role of cortisol on superoxide release by neutrophils

Experimental and clinical evidence shows that neutrophils play an important role in the mechanism of tissue injury in immune complex diseases through the generation of reactive oxygen species. In this study, we examined the influence of academic psychological stress in post-graduate students on the capacity of their blood neutrophils to release superoxide when stimulated by immune complexes bound to nonphagocytosable surfaces and investigated the modulatory effect of cortisol on this immune function. The tests were performed on the day before the final examination. The state-trait anxiety inventory questionnaire was used to examine whether this stressful event caused emotional distress. In our study, the psychological stress not only increased plasma cortisol concentration, but it also provoked a reduction in superoxide release by neutrophils. This decrease in superoxide release was accompanied by diminished mRNA expression for subunit p47(phox) of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase. These inhibitory effects were also observed by in vitro exposure of neutrophils from control volunteers to 10(-7) M hydrocortisone, and could be prevented by the glucocorticoid receptor antagonist RU-486. These results show that in a situation of psychological stress, the increased levels of cortisol could inhibit superoxide release by neutrophils stimulated by IgG immune complexes bound to nonphagocytosable surfaces, which could attenuate the inflammatory state.

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus ( SLE), the superoxide anion (O(2)(-)) production by neutrophils, mediated by Fc gamma R and Fc gamma R/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: ( 1) manifestations associated with the occurrence of cytotoxic antibodies ( SLE-I group); ( 2) manifestations associated with circulating immune complexes ( IC; SLE-II group), and ( 3) manifestations associated with IC and cytotoxic antibodies ( SLE-III group). O(2)(-) production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with ICIgG opsonized or not with complement. No difference in O(2)(-) production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: ( 1) SLE-I neutrophils showed lower O(2)(-) production mediated by the IgG receptor ( Fc gamma R) with the cooperation of complement receptors ( Fc gamma R/ CR) than observed in the SLE-II, SLE-III, and healthy groups; ( 2) neutrophils from the SLE-II group showed a decreased O(2)(-) production mediated by Fc gamma R/ CR compared to the SLE-III group, ( 3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and ( 4) CR showed inefficiency in mediating the O(2)(-) production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence ( CL; T(max) and CL(max)) disclosed differences only for the SLE- I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by Fc gamma R/ CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Therapeutic potential of selective superoxide dismutase mimetics

Selective superoxide dismutase (SOD) mimetics are potentially useful in pathological conditions in which there is an overproduction of the superoxide anion O-2.(-). These pathological conditions include inflammation, ischemia/reperfusion, shock, various cardiovascular disorders, amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. A major step forward in this field was the development of small-molecule selective SOD mimetics that penetrate cell membranes, These selective SOD mimetics catalytically remove O-2.(-) without interfering with nitric oxide (NO), peroxynitrite (ONOO-) or other radicals such as hydroxyl radical or hydrogen peroxide (H2O2). These selective SOD mimetics (SC-52608, SC-55858, M-40403 and M-40401) have been shown to have benefits in animal models of inflammation, ischemia/reperfusion, shock, thrombosis and diabetes. The next challenge with selective SOD mimetics is to develop therapeutic potential into therapeutic agents.

Electrochemical DNA-sensor for evaluation of total antioxidant capacity of flavours and flavoured waters using superoxide radical damage

In this paper, a biosensor based on a glassy carbon electrode (GCE) was used for the evaluation of the total antioxidant capacity (TAC) of flavours and flavoured waters. This biosensor was constructed by immobilising purine bases, guanine and adenine, on a GCE. Square wave voltammetry (SWV) was selected for the development of this methodology. Damage caused by the reactive oxygen species (ROS), superoxide radical (O2·−), generated by the xanthine/xanthine oxidase (XOD) system on the DNA-biosensor was evaluated. DNA-biosensor encountered with oxidative lesion when it was in contact with the O2·−. There was less oxidative damage when reactive antioxidants were added. The antioxidants used in this work were ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol. These antioxidants are capable of scavenging the superoxide radical and therefore protect the purine bases immobilized on the GCE surface. The results demonstrated that the DNA-based biosensor is suitable for the rapid assess of TAC in beverages.

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

Superoxide reductase from the syphilis spirochete Treponema pallidum

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A ° , = 106.9 ) and diffracted beyond 1.60 A ° resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A ° , = 104.9 ) and diffracted beyond 1.55 A ° . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 A ° ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

Biochem. J. (2011) 438,485–494 doi:10.1042/BJ20110836