Water restriction increases renal inner medullary manganese superoxide dismutase (MnSOD)

Oxidative stress damages cells. NaCl and urea are high in renal medullary interstitial fluid, which is necessary to concentrate urine, but which causes oxidative stress by elevating reactive oxygen species (ROS). Here, we measured the antioxidant enzyme superoxide dismutases (SODs, MnSOD, and Cu/ZnSOD) and catalase in mouse kidney that might mitigate the oxidative stress. MnSOD protein increases progressively from the cortex to the inner medulla, following the gradient of increasing NaCl and urea. MnSOD activity increases proportionately, but MnSOD mRNA does not. Water restriction, which elevates renal medullary NaCl and urea, increases MnSOD protein, accompanied by a proportionate increase in MnSOD enzymatic activity in the inner medulla, but not in the cortex or the outer medulla. In contrast, Cu/ZnSOD and TNF-α (an important regulator of MnSOD) do not vary between the regions of the kidney, and expression of catalase protein actually decreases from the cortex to the inner medulla. Water restriction increases activity of mitochondrial enzymes that catalyze production of ROS in the inner medulla, but reduces NADPH oxidase activity there. We also examined the effect of high NaCl and urea on MnSOD in Madin-Darby canine kidney (MDCK) cells. High NaCl and high urea both increase MnSOD in MDCK cells. This increase in MnSOD protein apparently depends on the elevation of ROS since it is eliminated by the antioxidant N-acetylcysteine, and it occurs without raising osmolality when ROS are elevated by antimycin A or xanthine oxidase plus xanthine. We conclude that ROS, induced by high NaCl and urea, increase MnSOD activity in the renal inner medulla, which moderates oxidative stress.     

Genetic polymorphism of manganese superoxide dismutase (MnSOD) and breast cancer susceptibility

Within mitochondria, manganese superoxide dismutase (MnSOD) provides a major defence against oxidative damage by reactive oxygen species (ROS). An alanine-9valine (Ala-9Val) polymorphism in the mitochondrial targeting sequence of MnSOD has been described and has recently been associated with risk of human breast cancer. Our present case-control study was performed to explore the association between MnSOD genetic polymorphism and individual susceptibility to breast cancer. Ala-9Val polymorphism in the signal sequence of the protein for MnSOD was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in a study population. There was no significant difference in risk for breast cancer development between patients positive and negative for the MnSOD Ala allele with adjusted odds ratio (OR): 0.86 (95% confidence interval (CI(0.43 to 1.72). When MnSOD Ala was combined with either cytochrome P450 1B1 CYP1B1*1 and catechol O-methyltransferase COMT-L (V158M) genotypes, the risk for developing breast cancer was significantly increased in patients with a body mass index (BMI) greater than 24 kg m(-2) (OR: 1.42 (95%CI=1.04-1.93)).     

Association between manganese superoxide dismutase (MnSOD) gene polymorphism and elderly obesity

Evidence suggests an association between obesity and oxidative stress caused by superoxide production. Since the dismutation of superoxide is catalyzed by superoxide dismutase enzymes, we tested the association between obesity and Ala16Val manganese-dependent superoxide dismutase gene (MnSOD) polymorphism. We analyzed 815 free-living community subjects (≥60 years old) grouped into subjects who were either obese (BMI ≥ 30 kg/m²) or non-obese (BMI < 25 kg/m²). Additionally, we investigated the possible interaction between the Ala16Val MnSOD gene polymorphism and obesity in the modulation of biochemical and nutritional variables. We found a positive association between MnSOD polymorphism and obesity, since higher VV frequency (28.2%) was observed in the obese group (P = 0.002, odds ratio 1.949, 95% CI: 1.223–3.008). This result was independent of sex, age, diabetes, dyslipidemia, hypertension, and metabolic syndrome. A possible biological explanation of the association described here could be a chronic state of superoxide enzyme imbalance present in VV carriers, which could affect differential metabolic pathways contributing to the obese state.     

Model mice for tissue-specific deletion of the manganese superoxide dismutase (MnSOD) gene

Manganese superoxide dismutase (MnSOD) is the enzyme that converts toxic O(2)(-) to H(2)O(2) in mitochondria. Previous reports showed that a deficiency of MnSOD in mice was neonatal lethal. Therefore, a model mouse was not available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type model mouse, we designed tissue-specific MnSOD conditional knockout mice using a Cre-loxp system. First, we crossbred MnSOD flox mice with transgenic mice expressing Cre recombinase under the control of the chicken actin promoter (CAG). We confirmed that CAG MnSOD knockout mice were completely deficient in MnSOD and died as neonates, validating the use of the Cre-loxp system. Next, we generated liver-specific MnSOD-deficient mice by crossbreeding with Alb-Cre transgenic mice. MnSOD activity and protein were both significantly downregulated in the liver of liver-specific MnSOD knockout mice. However, no obvious morphological abnormality was observed in the liver when biochemical alterations such as lipid peroxidation were not detectable, suggesting a redundant or less important physiological role for MnSOD in the liver than previously thought. In the present study, we successfully generated tissue-specific MnSOD conditional knockout mice that would provide a useful tool for the analysis of various age-associated diseases such as diabetes mellitus, Parkinson's disease, stroke, and heart disease, when crossbred with tissue-specific transgenic Cre mice.     

Heat-shock protein 25 (Hspb1) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB)

We previously demonstrated that overexpression of HSP25 (now known as Hspb1) conferred increased resistance to ionizing radiation (Radiat Res. 154, 421-428, 2000). In the present study, L929 cells overexpressing Hspb1 were shown to have increased expression of the manganese superoxide dismutase gene (now known as SOD2) and its enzyme activity. To elucidate Hspb1-induced pathways leading to activation of these antioxidant enzymes, the production of the tumor necrosis factor alpha (Tnf) and interleukin 1 beta (Il1b) genes was examined. Increased expression of Tnf and Il1b resulting from Hspb1 overexpression was detected by RT-PCR. Increased activation of Nfkb (degradation of Ikb, a member of the Nfkb family) was also found in Hspb1-overexpressing cells. When treated with Tnf, Nfkb activation and SOD2 gene expression were increased more by Hspb1 overexpression. Moreover, transfection with the Hspb1 antisense gene abrogated all of the Hspb1-mediated phenomena. To further elucidate the exact relationship between induction of SOD2 and Nfkb activation, a dominant negative I-kBalpha (now known as Nfkb1a) construct was transfected into Hspb1-overexpressing cells. The dominant negative Nfkb1a inhibited Hspb1-mediated SOD2 gene expression. In addition, Hspb1-mediated radioresistance was blocked by dominant negative Nfkb1a transfection. When the SOD2 gene was transfected into L929 cells, a somewhat increased radioresistance was detected by a clonogenic survival assay compared to control cells. Hspb1 produced Tnf and Il1b and facilitated SOD2 gene expression through Nfkb activation, possibly resulting in Hspb1-mediated radioresistance.     

Immunohistochemical expression of superoxide dismutase (MnSOD) anti-oxidant enzyme in invasive breast carcinoma

The most important cellular protective mechanisms against oxidative stress are antioxidant enzymes. Their action is based on decomposal of reactive oxygen species (ROS) and their transformation to H2O2. Within the mitochondria manganese superoxide dismutase (MnSOD) affords the major defense against ROS. In this study we investigated tissue sections from 101 breast carcinomas for the immunohistochemical expression of MnSOD protein and these results were assessed in relation to various clinicopathological parameters, in order to clarify the prognostic value of this enzyme. The possible relationship to hormone receptor content, anti-apoptotic protein bcl-2, p53 and cell proliferation was also estimated. High expression levels were observed, as 79/101 (78,2%) cases expressed strong immunoreactivity. In this study MnSOD increased in a direct relationship with tumor grade and is therefore inversely correlated with differentiation (p=0.0004). Furthermore, there was a strong positive correlation between MnSOD expression and p53 protein immunoreactivity (p=0.0029). The prognostic impact of MnSOD expression in determining the risk of recurrence and overall survival with both univariate (long-rang test) and multivariate (Cox regression) methods of analysis was statistically not significant. These results indicate that neoplastic cells in breast carcinomas retain their capability to produce MnSOD and thus protected from the possible cellular damage provoked by reactive oxygen species. In addition, MnSOD content varies according to the degree of differentiation of breast carcinoma.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.     

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.     

Manganese superoxide dismutase, MnSOD and its mimics

Increased understanding of the role of mitochondria under physiological and pathological conditions parallels increased exploration of synthetic and natural compounds able to mimic MnSOD - endogenous mitochondrial antioxidant defense essential for the existence of virtually all aerobic organisms from bacteria to humans. This review describes most successful mitochondrially-targeted redox-active compounds, Mn porphyrins and MitoQ(10) in detail, and briefly addresses several other compounds that are either catalysts of O(2)(-) dismutation, or its non-catalytic scavengers, and that reportedly attenuate mitochondrial dysfunction. While not a true catalyst (SOD mimic) of O(2)(-) dismutation, MitoQ(10) oxidizes O(2)(-) to O(2) with a high rate constant. In vivo it is readily reduced to quinol, MitoQH(2), which in turn reduces ONOO(-) to NO(2), producing semiquinone radical that subsequently dismutes to MitoQ(10) and MitoQH(2), completing the "catalytic" cycle. In MitoQ(10), the redox-active unit was coupled via 10-carbon atom alkyl chain to monocationic triphenylphosphonium ion in order to reach the mitochondria. Mn porphyrin-based SOD mimics, however, were designed so that their multiple cationic charge and alkyl chains determine both their remarkable SOD potency and carry them into the mitochondria. Several animal efficacy studies such as skin carcinogenesis and UVB-mediated mtDNA damage, and subcellular distribution studies of Saccharomyces cerevisiae and mouse heart provided unambiguous evidence that Mn porphyrins mimic the site and action of MnSOD, which in turn contributes to their efficacy in numerous in vitro and in vivo models of oxidative stress. Within a class of Mn porphyrins, lipophilic analogs are particularly effective for treating central nervous system injuries where mitochondria play key role. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(?-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20?°C, under alkaline condition (pH 9) with a specific activity of 3299?U?mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.     

A mimic of superoxide dismutase activity based upon desferrioxamine B and manganese(IV)

MnO2 reacted with desferrioxamine B yielding a green, water-soluble complex, with absorption maxima at 315 and 635 nm whose extinction coefficients were 925 and 60 M-1 cm-1, respectively. Increasing the proportion of ligand to metal increased both color yield and ability to scavenge O2-, with maximal color yield and activity being achieved at a 1:1 ratio. The complex catalyzed the dismutation of O2- and 1 microM was equivalent to 1 unit of superoxide dismutase activity in the xanthine oxidase-cytochrome c assay. The complex thus exhibited approximately 0.1% as much activity as did the manganese-containing superoxide dismutase, on the basis of manganese content. The activity of the complex was not suppressed by bovine serum albumin or by the soluble proteins extracted from Lactobacillus plantarum. In contrast, the activities of Cu(II) complexes of salicylate or Gly-His-Lys were suppressed by these proteins.     

Tissue-specific activity of two manganese superoxide dismutase promoters in transgenic tobacco.

In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.     

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.     

Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis

The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.