Circadian Protection against Reactive Oxygen Species Involves Changes in Daily Levels of the Manganese- and Iron-Containing Superoxide Dismutase Isoforms in Lingulodinium polyedrum

A circadian rhythm in the total activity of superoxide dismutase (SOD; EC 1.15.1.1) from the unicellular alga Lingulodinium polyedrum is shown to be attributable to the mitochondrial MnSOD and chloroplastic FeSOD isoforms. Activity gels and labelling with polyclonal antibodies against pure CuZnSOD, MnSOD and FeSOD revealed a distinct circadian pattern in the abundance of the latter two isoforms, with peak values in early photophase 5 times greater than at the dark phase. However, no such changes were detected for the CuZnSOD isoform, which remained at constant levels over the 24-h light/dark cycle. These SOD isoforms might provide protection against damage from photochemically generated oxygen radicals, thus preventing subcellular oxidative stress.     

Characterization of genomic clones and expression analysis of the three types of superoxide dismutases during nodule development in Lotus japonicus

Superoxide dismutases (SODs) are metalloenzymes that play a primary role in the protection against oxidative stress in plants and other organisms. We have characterized four SOD genes in Lotus japonicus and have analyzed their expression in roots and four developmental stages of nodules. The expression of cytosolic CuZnSOD, at the mRNA, protein, and enzyme activity levels, decreases with nodule age, and the protein is localized in the dividing cells and infection threads of emergent nodules and in the infected cells of young nodules. The mitochondrial MnSOD was downregulated, whereas the bacteroidal MnSOD displayed maximal protein and enzyme activity levels in older nodules. Two additional genes, encoding plastidic (FeSOD1) and cytosolic (FeSOD2) FeSOD isoforms, were identified and mapped. The genes are located in different chromosomes and show differential expression. The FeSOD1 mRNA level did not change during nodule development, whereas FeSOD2 was upregulated. The distinct expression patterns of the SOD genes may reflect different regulatory mechanisms of the enzyme activities during nodule ontogeny. In particular, at the mRNA and activity levels, the virtual loss of cytosolic CuZnSOD in mature and old nodules, concomitant with the induction of FeSOD2, suggests that the two enzymes may functionally compensate each other in the cytosol at the late stages of nodule development.     

Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicide mancozeb: an Italian study

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical–pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.     

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.     

Effect of hypoxia on photosynthetic activity and antioxidative response in gametophores of <Emphasis Type="Italic">Mnium undulatum</Emphasis>

The effects of hypoxia caused by complete submerging of Mnium undulatum gametophores in water, on their photosynthetic activity and the activity of two antioxidative enzymes: superoxide dismutase (SOD) and catalase (CAT) were investigated. The net photosynthesis was strongly inhibited throughout the experiment, and the strong drop in the maximum quantum yield of the PSII (F_v/F_m) was also observed. Three classes of SOD: MnSOD, FeSOD, Cu/ZnSOD and three isoforms of Cu/ZnSOD were identified. A significant decrease in activity of MnSOD, FeSOD and one Cu/ZnSOD isoform was observed after 24 and 48 h of hypoxia. FeSOD activity decreased already after 1 h of submerging in water and its activity remained at the low level during whole period of the experiment. CAT activity was also strongly inhibited in response to hypoxia stress. The obtained results suggest relationships between photosynthetic activity and antioxidative system in M. undulatum gametophores under oxygen deficiency stress.     

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical-pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.     

Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase

There are two types of intracellular superoxide dismutases: the mitochondrial manganese SOD (MnSOD) and the cytoplasmic copper/zinc SOD (CuZnSOD). Mutant mice that lack MnSOD die shortly after birth because of cardiomyopathy and mitochondrial injury. In order to verify if CuZnSOD could compensate for MnSOD deficiency, a new mutant mouse that overexpresses CuZnSOD but is deficient in MnSOD was generated by crossing MnSOD knockout mice with CuZnSOD transgenic mice. CuZnSOD activity was significantly increased in the blood, brain, liver, and heart of MnSOD knockout, CuZnSOD transgenic mice when compared with nontransgenic mice. However, overexpression of CuZnSOD did not prevent neonatal lethality in mice that lack MnSOD, nor did it prevent oxidative aconitase inactivation, nor did it rescue MnSOD-deficient astrocytes in culture. Based on our findings, which emphasize the strong enzymatic compartmentalization of CuZnSOD and MnSOD, therapeutic antioxidant strategies should consider the final intracellular localization of the antioxidant used, especially when those strategies are directed against mitochondrial diseases.     

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O₂ tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Irontype SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O₂ tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.     

Senescence-related changes in the antioxidant status of ginkgo and birch leaves during autumn yellowing

The antioxidant status of birch and ginkgo leaves during autumnal senescence was characterized by the activities of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD). The contents of leaf H₂O₂ and ascorbate were used as indicators of oxidative stress. Degradation of chlorophyll (chl) during natural senescence was not accompanied either by an increase of H₂O₂ or by a decrease of reduced ascorbate. A transient decrease of reduced ascorbate in ginkgo and birch leaves in early senescence was accompanied by CAT inactivation. The activity of ionically-bound PODs was stimulated in late senescence in both species, when more than 30% of chl was degraded. Induction of MnSOD in both species and new isoforms of CuZnSOD in birch in late senescence was accompanied by the disappearance of other CuZnSOD isoforms in birch and FeSOD in ginkgo. The role of antioxidative enzymes in keeping ascorbate reduced and endogenous H₂O₂ at low levels in senescent leaves of deciduous trees was discussed.     

The virulence of Vibrio vulnificus is affected by the cellular level of superoxide dismutase activity

The virulence of superoxide dismutase (SOD) mutants of Vibrio vulnificus, as tested by intraperitoneal injection into mice, decreases in the order of sodC mutant, sodA mutant, and sodB mutant lacking CuZnSOD, MnSOD, and FeSOD, respectively. The survival of SOD mutants under superoxide stress also decreases in the same order. The virulence of soxR mutant, which is unable to induce MnSOD in response to superoxide, is similar to that of the sodA mutant, as the survival of the soxR mutant under superoxide stress is similar to that of the sodA mutant. Consistently, the lowered survival of the soxR mutant is complemented not only with soxR but also with sodA. Thus, the virulence of V. vulnificus is significantly affected by the cellular level of SOD activity, and an increase in SOD level through MnSOD induction by SoxR under superoxide stress is essential for virulence.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Purification and identification of the type of superoxide dismutase fromGlœocapsa sp.

Superoxide dismutase purified from the photoautotrophic cyanobacteriumGlœocapsa sp. has a molar mass of 38 kDa, as estimated by gel filtration. Inhibition pattern indicated limited resistance to H₂O₂ under conditions which entirely inhibited FeSOD. The enzyme is insensitive to cyanide and is inhibited by azide. This suggested the presence of a hybrid of two isozymes, FeSOD and a minor constituent of MnSOD. Amino acid composition of the purified SOD showed structural homology to FeSOD and MnSOD apoenzymes.     

Induction of new isoforms of superoxide dismutase and catalase enzymes in the flag leaf of wheat during monocarpic senescence.

Leaf senescence is a programmed cell death phenomenon and involves oxidative stress. Superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT EC 1.11.1.6) activities were studied in the flag leaf of Triticum aestivum cv. Kundan at different stages of grain development. Both SOD and CAT activities showed a decline during monocarpic senescence. Three SOD isozymes were observed in the cytosol, of which one isozyme was observed in the chloroplasts as well. Mitochondria showed the presence of three low abundant SOD isoforms. Inhibitor studies revealed the cytosolic and chloroplastic isoforms to be Cu/Zn SODs. In mitochondria however, two isozymes were MnSOD and one of them appeared to be FeSOD. These isoforms present in the mitochondria increased in activity as senescence progressed. Three isoforms of CAT were observed in peroxisomes which responded differentially during monocarpic senescence. The changes in the kind and pattern of the antioxidant enzymes supported the ordered sequence of events during leaf senescence. This is the first report showing an increase in mitochondrial FeSOD activity during leaf senescence.     

Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity

Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage. The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC). The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO. We found that the Cys-MCO system caused the release of free copper from CuZnSOD. CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system.     

Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.     

Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12.

Superoxide dismutases are enzymes that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous superoxide dismutases, one containing manganese (MnSOD) and the other iron (FeSOD). Although E. coli Mn and FeSOD catalyze the dismutation of superoxide with comparable rate constants, it is not known if they are physiologically equivalent in their protection of cellular targets from oxyradical damage. To address this issue, isogenic strains of E. coli containing either Mn or FeSOD encoded on a plasmid and under the control of tac promoter were constructed. SOD specific activity in the Mn and FeSOD strains could be controlled by the concentration of isopropyl beta-thiogalactoside in the medium. The tolerance of these strains to oxidative stress was compared at equal Mn and FeSOD specific activities. Our results indicate that E. coli Mn and FeSOD are not functionally equivalent. The MnSOD is more effective than FeSOD in preventing damage to DNA, while the FeSOD appears to be more effective in protecting a cytoplasmic superoxide-sensitive enzyme. These data are the first demonstration that Mn and FeSOD are adapted to different antioxidant roles in E. coli.     

Acute and/or chronic stress models modulate CuZnSOD and MnSOD protein expression in rat liver

Cellular protection against oxidative stress is afforded by the enzyme superoxide dismutase (SOD). In this study, the protein levels of copper–zinc SOD (CuZnSOD) in the cytosolic and nuclear fraction, manganese SOD (MnSOD) in the mitochondrial, and cytosolic fraction and cytochrome c (cyt c) in the liver of male rats exposed to 2 h of acute immobilization (IM) or Cold stress, 21 days chronic isolation or their combinations (chronic/acute stress) were examined. The serum corticosterone (CORT) level was measured, as an indicator of stress stimuli. Both acute stressors with elevated CORT levels caused a decrease of mitochondrial MnSOD, while acute IM resulted in redistribution of the CuZnSOD protein level between the cytosolic and nuclear fraction. Chronic isolation, during which the CORT level was close to control value, resulted in an increase of cytosolic CuZnSOD, whereas a decrease of MnSOD in mitochondrial and its corresponding increase in cytosol fraction was found. In both combined stress regimes, an increase of the CuZnSOD and MnSOD levels in the cytosolic fraction was recorded whereby increase of the CORT level was observed only in the chronic isolation followed by acute IM. The data indicate that acute and/or chronic stress models have different degrees of influence on serum CORT and SOD subcellular protein levels. Increased cytosolic CuZnSOD protein level under chronic isolation suggests that state of oxidative stress may also exist under CORT level similar to the basal value. The presence of MnSOD and cyt c in the cytosolic fraction could serve as useful parameters for mitochondrial dysfunction.