Overexpression Of Mn-superoxide Dismutase Research Articles

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Overexpression of Mn-superoxide dismutase in Oxya chinensis mediates increased malathion tolerance

Superoxide dismutase (SOD) is the first line of defense against oxidative damage. Malathion is an organophosphate insecticide and can induce the production of reactive oxygen species (ROS) and cause the intracellular oxidative stress. The present study was undertaken to examine the effects of malathion on SODs activity and their transcriptional levels in Oxya chinensis (Thunberg) (Orthoptera: Acrididae). The results showed that total SOD and MnSOD activities increased as a dose-dependent manner while CuZnSOD activity has no significant changes after malathion treatments. Total SOD and MnSOD activities were the highest at the concentration of 0.8 μg μL−1 malathion treatment and increased significantly about 1.81- and 2.48-fold compared with the control, respectively. Increased mRNA expression of MnSOD, ecCuZnSOD1, and ecCuZnSOD2 were observed after malathion treatments. Moreover, the alteration of MnSOD transcript was similar to the profiles of MnSOD activity. These results suggested that the up-regulation expression of MnSOD transcript led to the increase of MnSOD activity in order to eliminate the excessive ROS caused by malathion. In addition, we evaluated the role of individual SOD gene in malathion stress by using RNAi and recombinant SOD proteins. The results showed that ROS contents increased significantly after the silencing of MnSOD and ecCuZnSOD1 genes. The OD values of the E. coli cells transformed with pET-28a-OcMnSOD plasmid were 1.13–1.31-fold and 1.08–1.33-fold higher than those of cells with pET-28a plasmids under 0.4 and 0.8 μg μL−1 malathion treatments, respectively. These findings indicated that MnSOD exerted an important role in defense oxidative stress caused by malathion.

Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.

Overexpression of Mn superoxide dismutase attenuates age related up‐regulation of TGF‐ß and remodeling in the aging heart

Aging results in accumulation of extracellular matrix proteins and impairment of cardiac function. Oxidative stress is a potential mechanism in age‐associated cardiac fibrosis. We have previously show exercise training reduces fibrosis and expression of transforming growth factor‐beta (TGF‐ß1) in the aging heart, concomitant with a reduction in oxidative stress and upregulation of the manganese‐isoform of superoxide dismutase (MnSOD). However, the causal relationship between MnSOD and TGF‐ß1 in the aging heart is unknown. Thus, we tested the hypothesis that overexpression of Mn‐SOD would reduce TGF‐ß1 and markers of fibrosis in the aging heart. Old (27 mo) wild type (OWT) animals had larger cardiac myocyte cross‐sectional areas (CSA) that young (4 mo) wild type mice (YWT), an effect reduced in Old Mn‐SOD transgenic mice animals (Otg). TGF‐β1 and collagen I abundance was higher in OWT vs. YWT, while overexpression of MnSOD attenuated TGF‐β1 and collagen I in hearts of old mice. Matrix metalloproteinase‐9 (MMP‐9) was lower in OWT, with age dependent suppression of MMP‐9 prevented by Mn‐SOD. Results indicate that overexpression of Mn‐SOD overexpression protects against age‐related upregulation of TGF‐ß1 and fibrosis, suggestive that elevation of MnSOD with exercise training may indeed be cardioprotective against fibrosis and remodeling. Supported by AHA 0855158F; NIH AR054084

Iron induces bimodal population development by Escherichia coli

Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air-biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H(2)O(2) toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

Mitochondrial oxidant stress triggers cell death in simulated ischemia–reperfusion

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia–reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O 2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X L in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X L, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Overexpression of Mn Superoxide Dismutase Does Not Increase Life Span in Mice

Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4-6 months) and old (26-28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.

Overexpression of manganese superoxide dismutase by N-acetylcysteine in hyperoxic lung injury

Exposure of animals to hyperoxia causes lung injury, characterized by diffuse alveolar damage and exudation of plasma into the alveolar space. Reactive oxygen species (ROS) play an important role in the development of hyperoxic lung injury. Mitochondrial oxidative phosphorylation is one of the major sources of ROS. N-acetylcysteine (NAC) is a precursor of glutathione (GSH), which functions as an antioxidant by reducing hydrogen peroxide to water and alcohols. NAC has been shown to diminish lung injury in a large variety of animal models. We elucidated the mechanism underlying the protective effects of NAC in hyperoxia-induced lung injury. Male BALB/c mice were exposed to 98% oxygen for 72 h. The mice inhaled NAC or saline twice a day from 72 h before oxygen exposure to the end of experiment. Inhaled NAC increased the GSH level in lung homogenate. NAC also attenuated cellular infiltrations in both bronchoalveolar lavage fluid (BALF) and lung tissue. The total protein level in BALF and the level of 8-isoprostane, a marker of lipid peroxidation, in lung homogenate were decreased by inhalation of NAC. Inhaled NAC induced the overexpression of Mn superoxide dismutase (MnSOD) mRNA and protein, but did not alter the expressions of other antioxidant enzymes, including CuZnSOD, extracellular SOD, and glutathione peroxydase 1. These findings suggest that the antioxidant properties of NAC in hyperoxic lung injury involve a decrease in mitochondrial ROS in association with the induction of MnSOD, in addition to its role as a precursor of GSH.

Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.

Over-expression of Mn-superoxide dismutase as a marker of oxidative stress in lesional skin of chronic idiopathic urticaria.

We studied the involvement of oxidative stress in chronic idiopathic urticaria (CIU), assessing the activities of superoxide dismutase (SOD) and glutathione and the levels of malondialdeyde (MDA), a marker of lipid peroxidation, in samples taken from lesional skin (n = 16) and nonlesional skin (n = 11) of CIU patients. The activity of SOD and glutathione and the levels of MDA were markedly increased in lesional skin as compared with skin of healthy subjects, whereas no differences were detected between nonlesional skin of CIU patients and control samples. Immuno-dot blot assay revealed an up-regulation of Mn-SOD expression in lesional skin. These findings show that oxidative stress is crucially involved in CIU. The evidence of lipid peroxidation and compensatory increase of Mn-SOD and glutathione activities in lesional skin, in the absence of any alteration in uninvolved skin, suggests that oxidative stress is secondary to the development of inflammation.

Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities

The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1+ and TG2+. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 degrees C) and sub-optimal (18, 14 and 10 degrees C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 degrees C and 18 degrees C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants.

Overexpression of Mn-Containing Superoxide Dismutase in Transgenic Drosophila melanogaster

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5–116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4–5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu–Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.

Overexpression of manganese-superoxide dismutase prevents methylmercury tox1city in hela cells

HeLa cells were stably transformed with plasmid constructs that allowed constitutive expression of antioxidant enzymes such as catalase, glutathione peroxidase (GSH-Px), Cu.Zn-superoxide dismutase (Cu,Zn-SOD) or Mn-superoxide dismutase (Mn-SOD) to examine the involvement of reactive oxygen generation in methylmercury toxicity. Overexpression of catalase, GSH-Px or Cu,Zn-SOD did not affect the sensitivity of HeLa cells against methylmercury. However, the sensitivity of HeLa cells against methylmercury was decreased by overexpression of Mn-SOD, an enzyme localized in matrix of mitochondria and which decomposes superoxide anions. These results suggest that formation of superoxide anions in the mitochondria might be involved in the mechanism of the cytotoxicity of methylmercury.

Manganese-superoxide dismutase in endothelial cells: localization and mechanism of induction

Mechanisms of Mn-superoxide dismutase (Mn-SOD) expression in human umbilical endothelial cells were investigated by Northern blot analysis, enzyme-linked immunosorbent assay, and immunoelectron microscopy. The Mn-SOD in human endothelial cells was markedly induced by the cytokines tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide as well as by phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. The induction was partially blocked by dexamethasone and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a potent inhibitor of protein kinase C (PKC). In endothelial cells in which PKC had been desensitized to TPA by pretreatment for 24 h, addition of TNF caused overexpression of Mn-SOD. These facts suggested that at least two separate signal-transducing pathways are involved in expression of the Mn-SOD gene. Immunoelectron-microscopic studies showed that Mn-SOD was localized to the mitochondrial matrix of the capillary vascular endothelial cells of cardiac tissues and cultured endothelial cells. Mn-SOD, which is normally abundant in endothelial cells relative to other cell types, may play an important protective role against stresses such as ischemia and inflammation.