Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase

Enrichment of the growth medium with iron partially relieves the phenotypic deficits imposed on Escherichia coli by lack of both manganese and iron superoxide dismutases. Thus iron supplementation increased the aerobic growth rate, decreased the leakage of sulfite, and diminished sensitivity toward paraquat. Iron supplementation increased the activities of several [4Fe-4S]-containing dehydratases, and this was seen even in the presence of 50 microg/ml of rifampicin, an amount which completely inhibited growth. Assessing the O-2 scavenging activity by means of lucigenin luminescence indicated that the iron-enriched sodAsodB cells had gained some means of eliminating O-2, which was not detectable as superoxide dismutase activity in cell extracts. It is noteworthy that iron-enriched cells were not more sensitive toward the lethality of H2O2 despite having the usual amount of catalase activity. This indicates that iron taken into the cells from the medium is not available for Fenton chemistry, but is available for reconstitution of iron-sulfur clusters. We suppose that oxidation of the [4Fe-4S] clusters of dehydratases by O-2 and their subsequent reductive reconstitution provides a mechanism for scavenging O-2 and that speeding this reductive reconstitution by iron enrichment both spared other targets from O-2 attack and maintained adequate levels of these enzymes to meet the metabolic needs of the cells.     

In vivo architecture of the manganese superoxide dismutase promoter

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellular defense against damaging superoxide radicals generated by aerobic metabolism and as a consequence of inflammatory disease. Elevated expression of Mn-SOD therefore provides a potent cytoprotective advantage during acute inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less promoter characteristic of a housekeeping gene. In contrast, however, Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1. To understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS) site analysis, which revealed seven hypersensitive sites throughout the gene. Following high resolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treatment. Dimethyl sulfate in vivo footprinting identified 10 putative constitutive protein-DNA binding sites in the proximal Mn-SOD promoter as well as two stimulus-specific enhanced guanine residues possibly due to alterations in chromatin structure. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kr uppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.     

Expression of manganese superoxide dismutase in esophageal and gastric cancers

The tumor-killing activity of radiotherapy and chemotherapy for cancer is closely associated with the production of active oxygen, and the relation between therapeutic resistance and active oxygen scavengers produced by the tumor itself is gaining more attention. It is considered that manganese superoxide dismutase (MnSOD) protects host cells from oxidative stress, in synergy with other antioxidant enzymes. In this study, we used a quantitative polymerase chain reaction assay to measure MnSOD mRNA in resected specimens from patients with esophageal and gastric cancers. In both esophageal and gastric cancers, the level of MnSOD mRNA was significantly elevated in cancer tissue compared to non-cancer tissue (P < 0.01). In gastric cancer tissue, the MnSOD mRNA level was significantly higher than in esophageal cancer tissue (P < 0.01). The significance of MnSOD in cancer tissue was investigated further by measuring MnSOD content in resected specimens using an enzyme-linked immunosorbent assay, and by examining its location by an immunohistochemical method. Upregulation of MnSOD in cancer tissue most likely serves as a protective mechanism against anti-cancer therapies known to produce superoxide radicals as a key component of their tumor-killing activity.     

Induction of manganese superoxide dismutase in BV-2 microglial cells

The regulation of the manganese-dependent superoxide dismutase (Mn-SOD) was studied in immortalized microglial cells (line BV-2). BV-2 cells, activated with lipopolysaccharide (LPS), exhibited an increase in Mn-SOD-like immunoreactivity, that was associated with an accumulation of nitrite in the culture medium and an increase in immunoreactivity for the inducible type of nitric oxide synthase (i-NOS). The i-NOS inhibitor L-N6-(1-iminoethyl)-lysine (NIL, 600 microM) suppressed the nitrite accumulation and the increase in Mn-SOD-like immunoreactivity in activated cells without significant effect on the level of i-NOS-like immunoreactivity. The NO donor sodium nitroprusside dose-dependently increased Mn-SOD-like immunoreactivity in NIL-pretreated BV-2 cells. These results indicate that the induction of Mn-SOD in activated BV-2 cells is mediated in part by NO, or its metabolites.     

Potato mitochondrial manganese superoxide dismutase is an RNA-binding protein

We have investigated the toxicity of the cholesterol oxidation products (oxysterols), 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol and 26-hydroxycholesterol to human monocyte-macrophages in vitro. The 7-position derivatives are present in low density lipoprotein (LDL) oxidised with copper (II) sulphate and macrophages, and in extracts of human atherosclerotic lesions, which also contain 26-hydroxycholesterol. We have also assessed 25-hydroxycholesterol for toxicity because it has often been used in studies of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition and LDL receptor down-regulation. Measurement of radioactivity release from monocyte-macrophages preloaded with tritiated adenine, as a means of assessing cytotoxicity that all the oxysterols showed time- and concentration-dependent toxicity. The cytotoxic potency of 26-hydroxycholesterol was the greatest. The 7-position derivatives also produced marked cell damage, though at higher concentrations than for 26-hydroxycholesterol. Of the oxysterols assessed, the toxicity of 25-hydroxycholesterol was the least. The cytotoxicity of 7 beta-hydroxycholesterol and 26-hydroxycholesterol was also shown using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay which confirmed that 26-hydroxycholesterol was more toxic than 7 beta-hydroxycholesterol. Incubation of monocyte-macrophages with cholesterol added to the different oxysterols gave varying results. Cholesterol, which was not itself toxic, inhibited the toxicity of 25-hydroxycholesterol and 26-hydroxycholesterol, but the toxicity of the 7-position derivatives was not affected. The possible relevance of these molecules to the death of macrophages seen in atherosclerosis is discussed.     

The manganese superoxide dismutase from the penicillin producer Penicillium chrysogenum

A survey was conducted to clarify the tuberculosis (TB) situation among the homeless during the period from 1991 to 1995 in Nagoya city, using 5,222 registration cards of TB cases registered at one of Nagoya City's 16 Health Centers. Out of 5,222 TB cases, there were 269 homeless cases (267 male and 2 female). Ninety-seven percent of them were pulmonary TB cases. The incidence and prevalence rates of TB per 100,000 among the homeless were estimated at around 1,500 and around 2,400, respectively, around 20 times higher than those of the non-homeless male over the 19 years of age. A decrease in the incidence rate of TB cases among the homeless was not seen, although the rate among the non-homeless decreased gradually. The percentage of infectious (bacillary and/or cavitary) tuberculous cases among the homeless was higher than in the non-homeless. In the infectious cases, the percentage of smear-positive bacillary cases or far advanced cavitary cases was 52.1% or 9.4% among the homeless compared to 48.1% or 2.6% among the non-homeless, respectively. The detection rate by chest X-ray examination of the homeless was 3.9%.     

Low-temperature thermochromism marks a change in coordination for the metal ion in manganese superoxide dismutase

We have observed thermochromism (temperature-dependent absorption) for anion complexes of manganese superoxide dismutase indicating a change in coordination number for the metal complex at low temperatures. The ligand field spectra for the Mn(III) ion, characteristic of five-coordination for the azide complex at 295 K, cleanly convert to spectra reflecting six-coordination at low temperature, with a midpoint for the transition near 200 K. The active site structure is temperature-dependent, a relatively rigid, distorted octahedral low-temperature Mn complex melting with dehydration (or displacement of one of the protein ligands) to form a five-coordinated complex under physiological conditions. Thermodynamic parameters for the transition estimated from van't Hoff analysis (delta HvH = 5 kcal/mol; delta SvH = 22 cal/mol K) are consistent with reduced chemical binding and increased fluxionality at room temperature. This thermochromism of MnSD demonstrates the existence of distinct isomeric forms of the active site metal complex, whose relative stability depends on the degree of vibrational excitation. The marginal destabilization of the six-coordinate anion complex under physiological conditions suggests that the enzyme may thermally control the stability of intermediates in a dissociative displacement mechanism for substrate binding and redox.     

Induction of manganese superoxide dismutase gene expression in bronchoepithelial cells after rockwool exposure

Superoxide dismutases play an important protective role in the lung defense against the pro-oxidative effect of fibrous dusts (e.g. crocidolite fibers). Particularly crocidolite, but also other asbestos fibers, are known to induce cellular antioxidant defense. Although rockwool, a man-made fiber made from rock, is used widely for insulation purposes, its effects on the superoxide dismutases in bronchoepithelial cells have not been investigated. Thus, the purpose of this study was to determine whether human bronchoepithelial cells (BEAS 2B) respond to rockwool fibers (115-4 experimental rockwool fiber) by induction of MnSOD mRNA and an increase of MnSOD activity levels. The results were compared with BEAS 2B cells exposed to silica (alpha-quartz: DQ12; SiO2) and UICC (Union Internationale Contre le Cancer) crocidolite (concentrations of all dusts: 0, 2, 5, 10, 25, 50 micrograms/cm2 = 0, 2.4, 6, 12, 30, 60 micrograms/ml; 24-h exposure) as control fibers. Scanning electron microscopy confirmed close dust cell contact under all experimental settings. Very low MnSOD mRNA baseline levels rose significantly (p < 0.001) in BEAS 2B cells exposed to all three dusts at 2 micrograms/cm2. However, at > 25 micrograms/cm2 MnSOD mRNA levels in silica- and crocidolite- but not in rockwool-exposed cells decreased. Slight (no significance) increases of MnSOD activity were observed which decreased at higher dust (> 5 micrograms/cm2) concentrations. These results suggest that: (1) like crocidolite and silica, rockwool accelerates MnSOD gene expression in bronchoepithelial cells; (2) an increase of MnSOD mRNA levels is not accompanied by MnSOD activity elevation; (3) in contrast to rockwool, high concentrations (> or = 25 micrograms/cm2) of crocidolite and silica reduced MnSOD activity and MnSOD mRNA levels. Because oxidants (H2O2) and crocidolite fibers were shown to reduce SOD activity, lack of active MnSOD protein may be caused by inactivation on a post-translational level. Furthermore, the decline of MnSOD mRNA and MnSOD activity levels coincides with increasing cytotoxicity. In conclusion, rockwool was demonstrated to induce MnSOD gene expression, perhaps because of its pro-oxidative effect in bronchoepithelial cells. In contrast to crocidolite and silica, rockwool fibers are not cytotoxic in this experimental setting.     

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.     

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.     

The 3' untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element

A redox-sensitive protein that binds to the 3' untranslated region (UTR) of manganese superoxide dismutase (MnSOD) RNA has been described previously [Fazzone, H., Wangner, A., and Clerch, L. B. (1993) J. Clin. Invest. 92, 1278-1281; Chung, D. J., and Clerch, L. B. (1997) Am. J. Physiol. 16, L714-L719]. In the present study, cross-competition gel retardation and RNase H assays were used to identify a 41-base region located 111 bases downstream of the stop codon as the 3' UTR cis element involved in protein binding. The base sequence of this region is approximately 75% conserved among the 3' UTRs of rat, mouse, cow, and human MnSOD mRNAs at approximately the same distance downstream of the stop codon. The role of this protein-binding region in RNA translation was assessed in an in vitro rabbit reticulocyte lysate system. Translation of MnSOD RNA from which the 3' UTR element was deleted decreased 60% compared with translation of MnSOD RNA containing the 3' UTR cis element. In the presence of a specific competitor oligoribonucleotide that inhibits MnSOD RNA protein-binding activity, translation of MnSOD RNA containing the 3' UTR was decreased by 65%. Thus, both the cis element and RNA protein-binding activity were required for more efficient translation of the MnSOD. An analysis of ribosomal profiles suggests the MnSOD RNA-binding protein participates in the formation of the translation initiation complex. When MnSOD RNA-binding activity was inhibited, initiation complex formation was decreased by 50%. From the data obtained in this study, we propose that the 3' UTR cis element of MnSOD through its interaction with MnSOD RNA-binding protein may function as a translational enhancer.     

Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients. In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen. Two isoforms of superoxide dismutase are synthesized by P. aeruginosa; they differ by the metal present at their active site, which is either iron or manganese. To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P. aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient. The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain. It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat. These results suggest that MnSOD may participate in the adaptation of mucoid strains of P. aeruginosa to the stationary phase of growth in the lungs of CF patients.     

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues

Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.     

Lipopolysaccharide induces manganese superoxide dismutase in the rat pancreas: its role in caerulein pancreatitis

Intraperitoneal injection of lipopolysaccharide (LPS) at a dose of 50 micrograms/kg increased the activity and the mRNA level of manganese superoxide dismutase (Mn-SOD) but did not change those of copper/zinc-SOD (Cu/Zn-SOD) in the rat pancreas. Both the formation of pancreatic edema and the elevation of serum amylase during caerulein pancreatitis were significantly relieved in the rats pretreated with LPS (50 micrograms/kg) compared with the rats without the pretreatment. These results support the view that superoxides play a key role in the pathogenesis of caerulein pancreatitis, and that Mn-SOD in the pancreas may work as a defense against the development of this disease.     

Protein kinase Cdelta-dependent induction of manganese superoxide dismutase gene expression by microtubule-active anticancer drugs

Bacterial lipopolysaccharide can induce manganese superoxide dismutase (MnSOD) gene expression in a variety of cells. Paclitaxel (taxol) shares many properties of lipopolysaccharide. Here we report that paclitaxel can induce MnSOD gene expression in human lung adenocarcinoma cell line A549 in a time- and dose-dependent manner. Additional anticancer drugs, vinblastine and vincristine, also induced MnSOD gene expression. We have shown previously (Das, K. C., and White, C. W. (1997) J. Biol. Chem. 272, 14914-14920) that these drugs can activate protein kinase C (PKC). The PKC agonists thymeleatoxin (0.5 microM) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA; 10 nM) potently induced MnSOD gene expression. Calphostin C and GF109203X, both specific inhibitors of PKC, each inhibited MnSOD gene expression by anticancer agents. Down-regulation of PKC by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) also inhibited induction of MnSOD by anticancer drugs, indicating an important role of PKC in MnSOD signaling by these agents. Of 11 PKC isoenzymes, only PKCdelta translocated to the cell membrane after stimulation with anticancer drugs. By contrast, dPPA, PMA, and thymeleatoxin caused translocation of PKCalpha, betaI, delta, and mu isotypes. Anticancer drug-stimulated cells also had increased total PKC activity in membrane and cytosolic fractions. Thus, paclitaxel, vinblastine, and vincristine each specifically activate PKCdelta, whereas PMA, thymeleatoxin, and dPPA activate multiple isoenzymes. PKCdelta was the only isoform activated by each agent in both groups of compounds effective in MnSOD induction.