Superoxide Elimination Research Articles

Biochemical and Histopathological Effects Induced by Methimazole in Rats Treated by vitamin C

In order to investigate some biochemical and histopathological changes effects of Methimazole (ME)in rats twenty-eight white female rats were equally divided into four target groups, (ME) The 1st group, was given sterile saline (0.9%) and served as a control (c). The 2nd group, was given Methimazole at dose 0.04 mg/kg B.W. orally (positive control). The 3rd group, was given Methimazole at dose 0.04 mg/kg B.W. and Vitamin C 200 mg/kg B.W. orally. The 4th group, was given only Vitamin C 200 mg/kg B.W. orally. Results aimed in the present study on the effect of Methimazole at daily intake doses to investigate possible adverse effects and modulate its possible mechanism by measuring the oxidant and antioxidant impact in brain and Ameliorative trail of vit. C. Vit C antioxidant, such as super oxide, increase super oxide elimination. examination revealed that the (ME) histopathological figures showed very small size thyroid gland that displayed atrophy, composed numerous small size follicles with normal follicular cells and C-cells. Study concludes, (vit c) can decreased thyroid damage caused by oxidative effects triggered by (ME) and linked to their antioxidant effects.

Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke.

Microglia are activated after ischemic stroke and induce neuroinflammation. The expression of the aryl hydrocarbon receptor (AhR) has recently been reported to elicit cytokine expression. We previously reported that microglial activation mediates ischemic edema progression. Thus, the purpose of this study was to examine the role of AhR in inflammation and edema after ischemia using a mouse middle cerebral artery occlusion (MCAO) model. MCAO upregulated AhR expression in microglia during ischemia. MCAO increased the expression of tumor necrosis factor α (TNFα) and then induced edema progression, and worsened the modified neurological severity scores, with these being suppressed by administration of an AhR antagonist, CH223191. In THP-1 macrophages, the NADPH oxidase (NOX) subunit p47phox was significantly increased by AhR ligands, especially under inflammatory conditions. Suppression of NOX activity by apocynin or elimination of superoxide by superoxide dismutase decreased TNFα expression, which was induced by the AhR ligand. AhR ligands also elicited p47phox expression in mouse primary microglia. Thus, p47phox may be important in oxidative stress and subsequent inflammation. In MCAO model mice, P47phox expression was upregulated in microglia by ischemia. Lipid peroxidation induced by MCAO was suppressed by CH223191. Taken together, these findings suggest that AhR in the microglia is involved in neuroinflammation and subsequent edema, after MCAO via p47phox expression upregulation and oxidative stress.

Electrochemical and Mechanistic Study of Superoxide Elimination by Mesalazine through Proton-Coupled Electron Transfer.

The elimination of superoxide radical anions (O2•−) by 5-amino-2-hydroxybenzoic acid (mesalazine, 5-ASA), 4-amino-2-hydroxybenzoic acid (4-ASA), and related compounds used for ulcerative colitis treatment was investigated using cyclic voltammetry and electron spin resonance (ESR) analyses aided by density functional theory (DFT) calculations. Quasi-reversible O2/O2•− redox was found to be modified by the compounds, suggesting that an acid–base reaction in which a hydroperoxyl radical (HO2•) is formed from O2•− occurs. However, the deprotonated 5-ASA anion can eliminate O2•− through proton-coupled electron transfer (PCET), forming a radical product. This electron transfer (ET) was confirmed by ESR analysis. The 4-aminophenol moiety in 5-ASA plays an important role in the PCET, involving two proton transfers and one ET based on π-conjugation. The electrochemical and DFT results indicated that O2•− elimination by 5-ASA proceeds efficiently through the PCET mechanism after deprotonation of the 1-carboxyl group. Thus, 5-ASA may act as an anti-inflammatory agent in the alkali intestine through PCET-based O2•− elimination.

Exogenous β-cyclocitral treatment primes tomato plants against drought by inducing tolerance traits, independent of abscisic acid.

Drought is the most devastating stress for crops. Intensity and duration of drought determine the magnitude of plant damage; similarly, plant ability to counteract drought determines its tolerance capacity. Recent studies revealed that exogenous apocarotenoid treatment confers abiotic stress tolerance to plants. However, much less is known about the role of β-cyclocitral (βCC), the major apocarotenoid, in drought tolerance. Here, we demonstrate βCC's role in improving plants' tolerance against drought stress. Tomato (Solanum lycopersicum L.) plants were independently treated with water and βCC and grown under either water-limited or irrigated conditions. The βCC-treated drought-exposed (BD) and βCC-treated irrigated (BH) plants were analysed for the major drought tolerance associated traits; water-treated drought-exposed (CD) and water-treated irrigated plants (CH) were used as controls. On exposure to drought, unlike controls, βCC-treated plants showed no wilting, higher RWC and stomatal conductance, unchanged ABA levels and stomatal closure. The BD plants had increased photosynthesis, chlorophyll content and enhanced root, but not shoot, growth. In addition, βCC treatment enhanced proline accumulation and activity of SOD in both drought-exposed and well irrigated plants. Taken together, βCC was identified as a potential candidate that improves tomato osmolyte accumulation and superoxide elimination, independent of ABA, and prepares the plant for upcoming drought stress. Our results suggest that βCC can be used to prime crops against drought stress.

Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus.

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC50 value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC50 value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity.

P-103 - The activity of proline as singlet oxygen and superoxide scavenger

Proline (Pro) is an important stress protecting molecule in plants and other cellular systems against oxidative damage. However, the actual sensitivity and selectivity of Pro against specific reactive oxygen species (ROS), especially singlet oxygen and superoxide, is debated. Here we present in vitro studies by using EPR and oxygen uptake detection of ROS showing that Pro can indeed scavenge both singlet oxygen and superoxide. The scavenging activity of Pro is compared to that of ascorbate (Asc), a known quencher of singlet oxygen and superoxide. In contrast to Asc, which a chemical quencher, Pro eliminates singlet oxygen via a physical mechanism with ca. 1% efficiency that of Asc. The superoxide scavenging activity of Pro does not proceed via H2O2 production as in case of superoxide dismutase (SOD), but similar to that of Asc that includes electron transfer, and its efficiency is ca. 6% that of Asc on equal molar basis. Since Pro can accumulate up to 10-fold higher concentrations under stress conditions than Asc, the overall singlet oxygen and superoxide elimination activity of Pro can reach 10 and 60% of the activity of Asc, respectively. In stress exposed plants this could be physiologically relevant in cell compartments with low amount of more efficient scavengers.

Cirrhotic patients with minimal hepatic encephalopathy have increased capacity to eliminate superoxide and peroxynitrite in lymphocytes, associated with cognitive impairment

Patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood. We aimed to assess whether MHE patients show alterations in different types of blood cells in (a) basal reactive oxygen and nitrogen species levels; (b) capacity to metabolise these species. To assess the mechanisms involved in the altered capacity to metabolise these species we also analysed: (c) peroxynitrite formation and d) peroxynitrite reaction with biological molecules. Levels of reactive oxygen and nitrogen species were measured by flow cytometry in blood cell populations from cirrhotic patients with and without MHE and controls, under basal conditions and after adding generators of superoxide (plumbagin) or nitric oxide (NOR-1) to assess the capacity to eliminate them. Under basal conditions, MHE patients show reduced superoxide and peroxynitrite levels and increased nitric oxide (NO) and nitrotyrosine levels. In patients without MHE plumbagin strongly increases cellular superoxide, moderately peroxynitrite and reduces NO levels. In MHE patients, plumbagin increases slightly superoxide and strongly peroxynitrite levels and affects slightly NO levels. NOR-1 increases NO levels much less in patients with than without MHE. These data show that the mechanisms and the capacity to eliminate cellular superoxide, NO and peroxynitrite are enhanced in MHE patients. Superoxide elimination is enhanced through reaction with NO to form peroxynitrite which, in turn, is eliminated by enhanced reaction with biological molecules, which could contribute to cognitive impairment in MHE. The data show that basal free radical levels do not reflect the oxidative stress status in MHE.

102 - Abnormal Methylation and Redox Homeostasis in the Onset and Progression of Combined Homocystinuria and Methylmalonic Aciduria (MMACHC)

Nutritional and genetic deficiencies of cobalamin (vitamin B12) lead to severe neurological and hematological dysfunction, and in severe cases, to death. Intracellular processing of dietary cobalamins is catalyzed by the glutathione-dependent MMACHC protein, which primes the cobalamin molecule prior to its delivery to cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MM). Dysfunction or absence of MMACHC lead to combined homocystinuria and methylmalonic aciduria (OMIM 609831) by inactivation of MS and MM, respectively. We examined the proteome of cytosolic and nuclear fractions of cultured skin fibroblasts from five healthy neonates and five patients with early onset MMACHC disease. Stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry permitted the identification of 3450 proteins. Methylated Arg, Lys and Glu residues were found in 11 histones and 126 non-histone proteins. Significant changes in protein expression levels were confirmed for enzymes of the methionine cycle, the trans-sulfuration pathway, mitochondrial respiration and the elimination of superoxide and peroxides. Targeted metabolomics confirmed functional disturbances in relevant endogenous aminothiols. A direct interaction of MMACHC with heat shock protein 90 was also demonstrated. This is the first study to investigate redox homeostasis in an inherited metabolic disorder without external induction of oxidative stress. Our data suggest that natural mutations in the MMACHC gene impair cellular methylation and redox homeostasis, which may contribute to the vascular and neurological phenotype observed in these patients.

Chronic estrogen exposure affects gene expression in the rostral ventrolateral medulla of young and aging rats: Possible role in hypertension

BackgroundChronic exposure to estradiol-17β (E2) in adult female rats increases mean arterial pressure by stimulating superoxide production in the rostral ventrolateral medulla (RVLM). However the mechanisms behind this phenomenon are unknown. We hypothesized that E2 exposure induces the gene expression of cytokines, chemokines and NADPH oxidase (Nox) in the RVLM that promotes superoxide production and aging would exacerbate this effect. MethodsYoung adult (3–4 month old) and middle-aged (6–8 month old) female Sprague Dawley rats were sham-implanted (YS and MS respectively) or implanted s.c. with slow-release E2 pellets (20ng of E2/day for 90 days; YE and ME respectively). Blood pressure (BP) was measured during the last 3 weeks of exposure in a separate set of rats. At the end of treatment, the animals were sacrificed and RVLM was isolated from the brainstem. PCR array and Quantitative RT-PCR were performed with the tissue to quantify genes associated with hypertension and superoxide production. Superoxide dismutase (SOD) activity was also measured in the RVLM from a different set of animals. ResultsE2 exposure increased mean arterial pressure in both YE and ME animals. Inflammatory genes such as interleukin-1β, interleukin-6 and monocyte chemoattractant protein-1 were significantly up-regulated in the RVLM of ME treated female rats compared to YS rats, but not in YE rats. Endothelin-1 (ET-1) gene was up-regulated in the RVLM of both YE and ME rats that were exposed to E2. Furthermore, chronic E2 treatment increased the mRNA levels of Nox1 and Nox2 genes in the RVLM of YE but not ME animals. SOD activity was reduced in MA animals, compared to young animals. E2 treatment had no significant effect on SOD activity. ConclusionChronic E2 exposure stimulates the expression of inflammatory genes in older animals and increases the expression of Nox subunits in the RVLM of younger animals. SOD activity was reduced in older animals. This suggests increased superoxide production in younger animals, but reduced superoxide elimination in older animals. On the other hand, E2 exposure stimulates ET-1 expression in both young and aging animals. These findings suggest that hypertension caused by chronic E2 exposure may involve different molecular mediators in young and aging animals, however ET-1 and superoxide could be common mediators for both age groups.

IL-6 Mediated Degeneration of Forebrain GABAergic Interneurons and Cognitive Impairment in Aged Mice through Activation of Neuronal NADPH Oxidase

BackgroundMultiple studies have shown that plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) are elevated in patients with important and prevalent adverse health conditions, including atherosclerosis, diabetes, obesity, obstructive sleep apnea, hypertension, and frailty. Higher plasma levels of IL-6, in turn, increase the risk of many conditions associated with aging including age-related cognitive decline. However, the mechanisms underlying this association between IL-6 and cognitive vulnerability remain unclear.Methods and FindingsWe investigated the role of IL-6 in brain aging in young (4 mo) and aged (24 mo) wild-type C57BL6 and genetically-matched IL-6−/− mice, and determined that IL-6 was necessary and sufficient for increased neuronal expression of the superoxide-producing immune enzyme, NADPH-oxidase, and this was mediated by non-canonical NFκB signaling. Furthermore, superoxide production by NADPH-oxidase was directly responsible for age-related loss of parvalbumin (PV)-expressing GABAergic interneurons, neurons essential for normal information processing, encoding, and retrieval in hippocampus and cortex. Targeted deletion of IL-6 or elimination of superoxide by chronic treatment with a superoxide-dismutase mimetic prevented age-related loss of PV-interneurons and reversed age-related cognitive deficits on three standard tests of spatial learning and recall.ConclusionsPresent results indicate that IL-6 mediates age-related loss of critical PV-expressing GABAergic interneurons through increased neuronal NADPH-oxidase-derived superoxide production, and that rescue of these interneurons preserves cognitive performance in aging mice, suggesting that elevated peripheral IL-6 levels may be directly and mechanistically linked to long-lasting cognitive deficits in even normal older individuals. Further, because PV-interneurons are also selectively affected by commonly used anesthetic agents and drugs, our findings imply that IL-6 levels may predict adverse CNS effects in older patients exposed to these compounds through specific derangements in inhibitory interneurons, and that therapies directed at lowering IL-6 may have cognitive benefits clinically.

Discovery of superoxide reductase: an historical perspective.

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.

Independent Generation and Study of 5,6‐Dihydro‐2′‐deoxyuridin‐6‐yl, a Member of the Major Family of Reactive Intermediates Formed in DNA from the Effects of γ‐Radiolysis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Independent generation and study of 5,6-Dihydro-2'-deoxyuridin-6-yl, a member of the major family of reactive intermediates formed in DNA from the effects of gamma-radiolysis.

Nucleobase radicals are the major family of reactive intermediates formed when nucleic acids are exposed to gamma-radiolysis. Elucidation of their reactivity is complicated by the formation of multiple species randomly throughout the biopolymers. 5,6-Dihydro-2'-deoxyuridin-6-yl (1) was generated upon photolysis (350 nm) of the respective tert-butyl ketone (2). The radical abstracts hydrogen atoms from beta-mercaptoethanol (k = 8.8 +/- 0.5 x 10(6) M(-)(1) s(-)(1)) and 2,5-dimethyltetrahydrofuran (k = 31 +/- 2.5 M(-)(1) s(-)(1)). The latter was used as a model for the 2-deoxyribose component of DNA. The major product formed in the presence of O(2) was 6-hydroxy-5,6-dihydro-2'-deoxyuridine (11), which is believed to be formed directly from the peroxy precursor and not via elimination of superoxide. Small amounts of 2-deoxyribonolactone (13) were also formed under aerobic conditions. This product is believed to result from intramolecular hydrogen atom abstraction by the C6-peroxyl radical (14) and suggests that gamma-radiolysis may indirectly result in oxidation of the C1'-position of nucleotides, despite the inaccessibility of this hydrogen in duplex DNA.

Superoxide reductase from Desulfoarculus baarsii

Superoxide radical (O2.-) is the univalent reduction product of molecular oxygen and belongs to the group of the so-called toxic oxygen derivatives. For years the only enzymatic system known to catalyze the elimination of superoxide was the superoxide dismutase (SOD), which catalyzes dismutation of superoxide radical anions to hydrogen peroxide and molecular oxygen

SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis.

We constructed a sodA-disrupted mutant of Bacillus subtilis 168, BK1, by homologous recombination. The mutant was not able to grow in minimal medium without Mn(II). The spore-forming ability of strain BK1 was significantly lower in Mn(II)-depleted medium than that of the wild-type strain. These deleterious effects caused by the sodA mutation were reversed when an excess of Mn(II) was used to supplement the medium. Moreover, the growth inhibition by superoxide generators in strain BK1 and its parent strain was also reversed by the supplementation with excess Mn(II). We therefore estimated the Mn-dependent superoxide-scavenging activity in BK1 cells. Whereas BK1 cells have no detectable superoxide dismutase (Sod) on native gel, the superoxide-scavenging activity in crude extracts of BK1 cells grown in Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) was significantly detected by the modified Sod assay method without using EDTA. The results obtained suggest that Mn, as a free ion or a complex with some cellular component, can catalyze the elimination of superoxide and that both SodA and Mn(II) are involved not only in the superoxide resistance of vegetative cells but also in sporulation.

Stimulation of soluble guanylate cyclase by superoxide dismutase is mediated by NO.

Soluble guanylate cyclase (sGC), which is found in many cells and tissues, represents the receptor for the intra- and intercellular messenger molecule NO. Superoxide dismutase (SOD), an enzyme involved in the degradation of toxic superoxide radicals, has been proposed as a non-NO activator of sGC. Here we show that SOD stimulated sGC purified from bovine lung up to 10-fold. Activation by SOD was not influenced by the hydroxyl radical scavengers mannitol and DMSO. In contrast, the presence of the NO scavengers oxyhaemoglobin and 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide, as well as the O2(-)-generating system xanthine oxidase/hypoxanthine, led to inhibition of SOD-stimulated cGMP production. NO-insensitive sGC mutants were not influenced either by SOD or by xanthine oxidase. We have previously shown that sGC was stimulated by NO present in the normal atmosphere. Here we show that the SOD effect depended on the NO concentration from the atmosphere, as the stimulation of sGC by defined NO gases (0, 120, 330 and 1000 parts per billion NO) was potentiated by SOD. NO stimulation of sGC and its potentiation by SOD were inhibited by oxyhaemoglobin to identical levels. We conclude that the SOD-mediated stimulation of sGC is due to the elimination of superoxide, thereby preventing its reaction with NO to form peroxynitrite.

Substituent effect on superoxide elimination from peroxyl radicals of adenine and methylated derivatives

The hydroxyl radical, generated by UV photolysis of 4-mercaptopyridine-N-oxide, was made to react with adenine and several methylated derivatives. The formation of the corresponding 8-hydroxy derivatives was observed in all cases and is the result of the oxidation of the respective 8-hydroxyl adducts. When this oxidation is carried out by molecular oxygen, an intermediate peroxyl radical is formed by addition of O 2 at C(4). Superoxide anion is eliminated from the peroxyl radical, followed by deprotonation, resulting in the formation of 8-hydroxyadenines (8-oxo-7,8-dihydroadenines) as the final stable products. The extent of superoxide elimination, as compared to other processes by which peroxyl radicals decay, was found to increase with the number of methyl substituents at either N 6 or N(9). This indicates that positive charge is developed in the transition state, as suggested by the negative ρ value of the Hammett relationship for this reaction.

Release of Superoxide from Nucleoside Peroxyl Radicals, a Double-Edged Sword?

5,6-Dihydrothymidin-5-yl (1) and 2‘-deoxyuridin-1‘-yl (3) were independently generated in solution under aerobic conditions. The release of superoxide (O2•-) from the respective peroxyl radicals derived from 1 and 3 was determined spectrophotometrically. Competition studies enable one to estimate that the rate constant for elimination of O2•- from the peroxyl radical (4) derived from 3 is ∼1 s-1. This process is competitive with the anticipated rate of trapping of 4 in DNA by glutathione. Relative rate studies indicate that O2•- generation resulting from the formation of 1 under aerobic conditions competes effectively with trapping of the peroxyl radical by Bu3SnH. Superoxide elimination from the peroxyl radical of 1 (2) restores the damaged nucleoside to its unaltered form, implying that this reactive intermediate has a naturally occurring detoxification pathway available to it. However, the freely diffusible superoxide can react further to generate other reactive species capable of damaging nucleic acid...