Loss Of Superoxide Dismutase Activity Research Articles

Metabolomic analysis of oxidative stress: Superoxide dismutase mutation and paraquat induced stress in Drosophila melanogaster

Oxidative stress results in substantial biochemical and physiological perturbations in essentially all organisms. To determine the broad metabolic effects of oxidative stress, we have quantified the response in Drosophila melanogaster to both genetically and environmentally derived oxidative stress. Flies were challenged with loss of Superoxide dismutase activity or chronic or acute exposure to the oxidizing chemical paraquat. Metabolic changes were then quantified using a recently developed chemical isotope labeling (CIL) liquid chromatography - mass spectrometry (LC-MS) platform that targets the carboxylic acid and amine/phenol submetabolomes with high metabolic coverage. We discovered wide spread changes in both submetabolomes in response to all three types of stresses including: changes to the urea cycle, tryptophan metabolism, porphyrin metabolism, as well as a series of metabolic pathways involved in glutathione synthesis. Strikingly, while there are commonalities across the conditions, all three resulted in different metabolomic responses, with the greatest difference between the genetic and environmental responses. Genetic oxidative stress resulted in substantially more widespread effects, both in terms of the percent of the metabolome altered, and the magnitude of changes in individual metabolites. Chronic and acute environmental stress resulted in more similar responses although both were distinct from genetic stress. Overall, these results indicate that the metabolomic response to oxidative stress is complex, reaching across multiple metabolic pathways, with some shared features but with more features unique to different, specific stressors.

Hydrogen peroxide produced by superoxide dismutase SOD-2 activates sperm in Caenorhabditis elegans

Superoxide dismutase (SOD) is a ubiquitous antioxidant enzyme that catalytically converts the superoxide radical to hydrogen peroxide (H2O2). In mammals, high SOD activity is detectable in sperm and seminal plasma, and loss of SOD activity has been correlated with male infertility; however, the underlying mechanisms of sperm infertility remain to be clarified. Here we report that the deletion of two major SOD genes in Caenorhabditis elegans, sod-1 and sod-2, causes sperm activation defects, leading to a significant reduction in brood size. By examining the reactivity to the sperm activation signals Pronase and triethanolamine, we found that sod-1;sod-2 double mutant sperm cells display defects in pseudopod extension. Neither the content nor oxidative modification of major sperm protein, an essential cytoskeletal component for crawling movement, were significantly affected in sod-1;sod-2 mutant sperm. Surprisingly, H2O2, the dismutation product of SOD, could activate sod-1;sod-2 mutant sperm treated with Pronase. Moreover, the H2O2 scavenger ebselen completely inhibited pseudopod extension in wild-type sperm treated with Pronase, and H2O2 could directly induce pseudopod extension in wild-type sperm. Analysis of Pronase-triggered sperm activation in sod-1 and sod-2 single mutants revealed that sod-2 is required for pseudopod extension. These results suggest that SOD-2 plays an important role in the sperm activation of C. elegans by producing H2O2 as an activator of pseudopod extension.

Oxidant-antioxidant balance in asthma and the potential of antioxidant therapies

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Superoxide dismutase is dispensable for normal animal lifespan

Reactive oxygen species (ROS) are toxic oxygen-containing molecules that can damage multiple components of the cell and have been proposed to be the primary cause of aging. The antioxidant enzyme superoxide dismutase (SOD) is the only eukaryotic enzyme capable of detoxifying superoxide, one type of ROS. The fact that SOD is present in all aerobic organisms raises the question as to whether SOD is absolutely required for animal life and whether the loss of SOD activity will result in decreased lifespan. Here we use the genetic model organism Caenorhabditis elegans to generate an animal that completely lacks SOD activity (sod-12345 worms). We show that sod-12345 worms are viable and exhibit a normal lifespan, despite markedly increased sensitivity to multiple stresses. This is in stark contrast to what is observed in other genetic model organisms where the loss of a single sod gene can result in severely decreased survival. Investigating the mechanism underlying the normal lifespan of sod-12345 worms reveals that their longevity results from a balance between the prosurvival signaling and the toxicity of superoxide. Overall, our results demonstrate that SOD activity is dispensable for normal animal lifespan but is required to survive acute stresses. Moreover, our findings indicate that maintaining normal stress resistance is not crucial to the rate of aging.

Antioxidant properties of pioglitazone limit nicotinamide adenine dinucleotide phosphate hydrogen oxidase and augment superoxide dismutase activity in cardiac allotransplantation

Many non-immunologic factors contribute to the development of cardiac allograft vasculopathy (CAV), chief among them being ischemia-reperfusion injury associated with oxidative stress. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ agonist, could attenuate graft oxidant stress in cardiac transplantation. Experiments were performed using a murine heterotopic cardiac allotransplantation model. Pioglitazone was administered to recipients once daily, beginning 1 day before transplantation. At 4 hours after transplantation, pioglitazone significantly reduced the expression of endothelial cell adhesion receptors and infiltration of polymorphonuclear leukocytes (PMNs). The anti-oxidant balance in pioglitazone-treated cardiac allografts was significantly bolstered by reduced nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (Nox1 and p22(phox) sub-units) activity and preservation of manganese superoxide dismutase (SOD) activity, resulting in the mitigation of oxidative damage at the level of lipids, proteins, and DNA. At 7 days after transplantation, PPAR-γ was significantly up-regulated by pioglitazone, but nuclear factor-κB and inducible nitric oxide synthase were significantly down-regulated. A concomitant reduction of inflammatory cytokines and chemokines and graft leukosequestration was noted. Pioglitazone consequently prolonged cardiac allograft survival and attenuated CAV development. In vitro experiments demonstrated that pioglitazone decreased transendothelial PMN migration, NADPH oxidase activity, and loss of SOD activity in PMNs and endothelial cells. Pioglitazone can suppress the oxidative stress and damage and can stimulate antioxidant capacity in cardiac allografts after transplantation. Mitigation of graft oxidant stress could be an important mechanism through which pioglitazone confers benefit after cardiac transplantation.

Processing of semen can result in increased sperm DNA fragmentation

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

Rapid loss of superoxide dismutase activity during antigen-induced asthmatic response

Loss of superoxide dismutase activity occurs within minutes of an acute asthmatic response to segmental antigen instillation into the lung of individuals with atopic asthma. Decreased activity undoubtedly contributes to airway inflammation and injury through increased formation of reactive oxygen and nitrogen species, and suggests that enrichment of lung antioxidants is therapeutic for asthma.

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages.

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2.-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2.- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants.

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.

Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation.

Cryopreservation of human sperm, now generally required in donor insemination programs, adversely affects the sperm in terms of standard sperm evaluation parameters and fertilizing ability. The freeze-thaw process appears to produce sublethal damage that appears only after a delay. The authors hypothesized that cryopreservation enhanced peroxidation of sperm membrane lipids, based on previous studies of sperm lipid peroxidation, which showed that the effects of peroxidative damage became evident only after a delay, depending on the peroxidation rate. The effect of cryopreservation on the phospholipid content, the composition of the acyl moieties of the phospholipids, and the activities of the peroxidation protective enzymes, superoxide dismutase (SOD) and glutathione peroxidase plus reductase, in human sperm were examined to test the hypothesis. Parallel determinations were made of the percent motility, the average path velocity of the motile cells, and the time to loss of motility under specified aerobic incubation conditions, which gives a good estimate of the lipid peroxidation rate. The phospholipid content decreases after cryopreservation, with loss of phosphatidylcholine and phosphatidylethanolamine being the more pronounced. Polyunsaturated acyl moieties were also preferentially lost. This loss pattern is observed also from lipid peroxidation. The activities of glutathione peroxidase plus reductase remained unchanged. The sperm SOD activities varied widely between samples before cryopreservation. In all samples there was a decline in SOD activity after freeze-thaw, but the extent of the decline was also widely variable. The time to loss of motility declined in parallel with SOD activity, and a strong correlation (R2 greater than 0.9) between SOD activity and time to loss of motility was found for all samples, before and after freeze-thaw. The authors conclude that cryopreservation does enhance lipid peroxidation in human sperm, as hypothesized, and that this enhancement is mediated at least in part by the loss of SOD activity occurring during the process.

In vitro effects of Thiram on liver antioxidant enzyme activities of rainbow trout ( Oncorhynchus mykiss)

The effects of the dithiocarbamate Thiram on the activities of Superoxide dismutase, catalase and glutathione peroxidase in young rainbow trout livers were studied in vitro. Catalase and glutathione peroxidase activities were evaluated with standardized methods. Superoxide dismulase was assayed by a method based on the inhibiting action of the enzyme on the rate of oxidation of NADH. The activities of superoxide dismutase, catalase and glutathione peroxidase were found to be 11.3 μg/min/mg, 33.1 μmol/min/mg and 12.2 nmol/min/mg protein respectively, in control animals. Incubation of liver supernatants with 10−6 M, 10−5 M and 10−4 M Thiram for 1.5 h resulted in a total loss of Superoxide dismutase activity at 10−4 M, whereas catalase activity showed no significant reaction to the same range of toxic concentrations. The activity of glulathione peroxidase decreased at 10−6 M of Thiram but was not further reduced at higher concentrations of dithiocarbamate.

Effect of Streptococcus lactis preparation in diet of mice on inhibiting age-dependent loss of superoxide dismutase activity in tissues

SL-diet was prepared using commercial pellets supplemented with 1% pulverized Streptococcus lactis (SL) fed group of ICR strain mice were bred from 8 weeks up to 75 weeks. End-point age for survival in these mice was approximately 75 weeks. No significant differences in survival rate were observed at 75 weeks between the commercial diet and SL-diet group. The superoxide dismutase (SOD) activities in the liver, brain and erythrocytes of 75-week-old mice significantly decreased compared with those aged 10 weeks. Cu·.Zn-SOD and Mn-SOD activities in the liver and brain of SL-diet mice were maintained in similar degree in the juvenile group, however, decreased significantly in the commercial diet group. SOD in erythrocytes was slightly decreased and no significant difference was noted in either group.

Enzymatic changes in rat erythrocytes with increasing cell and donor age: Loss of superoxide dismutase activity associated with increases in catalytically defective forms

Erythrocytes from young (6–8 month old) and old (28–31 month old) WF rats were separated into four age fractions by centrifugation on a discontinuous, isotonic, arabinogalactan density gradient. Specific activities of super-oxide dismutase (SOD) with respect to activity per unit hemoglobin (Hb) and activity per cell were determined for each cell age group. SOD activity was found to diminish with respect to erythrocyte age. More significantly, it was found that young erythrocytes of old animals already contain considerably reduced SOD activity as compared to cells of young animals. The level of SOD catalytic activity per unit enzyme antigen was also found to decrease with both increasing cell and animal age. Young cells of old animals contain significant amounts of catalytically altered molecules.

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.