Heterozygous Superoxide Dismutase Research Articles

Astragalus polysaccharides improve cardiomyopathy in STZ-induced diabetic mice and heterozygous (SOD2+/-) knockout mice

Oxidative stress plays an important role in the development of diabetic cardiomyopathy. In the present study, we determined whether the effect of astragalus polysaccharides (APS) on diabetic cardiomyopathy was associated with its impact on oxidative stress. Streptozotocin (STZ)-induced diabetic mice and heterozygous superoxide dismutase (SOD2+/-) knockout mice were administered APS. The hemodynamics, cardiac ultrastructure, and the apoptosis, necrosis and proliferation of cardiomyocytes were assessed to evaluate the effect of APS on diabetic and oxidative cardiomyopathy. Furthermore, H2O2 formation, oxidative stress/damage, and SOD activity in cardiomyocytes were evaluated to determine the effects of APS on cardiac oxidative stress. APS therapy improved hemodynamics and myocardial ultrastructure with reduced apoptosis/necrosis, and enhanced proliferation in cardiomyocytes from both STZ-induced diabetic mice and heterozygous SOD2+/- knockout mice. In addition, APS therapy reduced H2O2 formation and oxidative stress/damage, and enhanced SOD activity in both groups of mice. Our findings suggest that APS had benefits in diabetic cardiomyopathy, which may be partly associated with its impact on cardiac oxidative stress.

AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function

Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2(+/-)) mice had no evidence of increased renal disease, and Ampka2(-/-) mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.

Association of Superoxide Anions with Retinal Pigment Epithelial Cell Apoptosis Induced by Mononuclear Phagocytes

Oxidative stress of the retinal pigment epithelium by reactive oxygen species and monocytic infiltration have been implicated in age-related macular degeneration. The purpose of this study was to determine the role of superoxide anions (O(2)(-)) in mononuclear phagocyte-induced RPE apoptosis. Mouse RPE cell cultures were established from wild-type and heterozygous superoxide dismutase 2-knockout (Sod2(+/-)) mice. The intracellular reactive oxygen species, O(2)(-) and hydrogen peroxide, were measured by using dihydroethidium assay and 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate, acetyl ester assay, respectively. RPE apoptosis was evaluated by Hoechst staining and terminal deoxynucleotidyltransferase dUTP nick-end labeling assay. Changes in mitochondrial membrane potential were detected by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide dye. Activated caspases and caspase-3 were detected in situ by FITC-VAD-fmk staining and caspase-3 substrate, respectively. Mononuclear phagocytes and interferon-gamma-activated mononuclear phagocytes induced the production of intracellular RPE O(2)(-), a decrease in RPE mitochondrial membrane potential, caspase activation, and apoptosis of mouse RPE cells. All theses changes were significantly enhanced in the Sod2(+/-) RPE cells. Activated mononuclear phagocytes induced more of these oxidative and apoptotic changes in RPE cells than did unstimulated mononuclear phagocytes. The authors have shown that the decreased expression of SOD2 and increased superoxide production correlate with RPE apoptosis induced by unstimulated and activated mononuclear phagocytes. The authors suggest that elevated O(2)(-) levels due to genetic abnormalities of SOD2 or immunologic activation of mononuclear phagocytes lead to greater levels of RPE apoptosis. The present study could serve as a useful model to characterize RPE/phagocyte interaction in AMD and other retinal diseases.

Sensitivity of Liver Injury in Heterozygous Sod2 Knockout Mice Treated with Troglitazone or Acetaminophen

Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/- mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/- mice. Although we conducted a repeated dose toxicity study in Sod2+/- mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/- mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/- mice. These results suggest that Sod2+/- mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

Proteomics profiling of hepatic mitochondria in heterozygous Sod2+/− mice, an animal model of discreet mitochondrial oxidative stress

The heterozygous superoxide dismutase 2 (SOD2) gene knockout (Sod2+/-) mouse model has been increasingly used in cardiovascular and age research, neurobiology, and pharmacology/toxicology. These mutant mice exhibit mild oxidant stress in mitochondria but remain clinically inconspicuous. Although the Sod2+/- mouse has been characterized with respect to mitochondrial function and transcript expression of certain individual genes, the effects of the singular loss of the Sod2 allele on the global expression of hepatic mitochondrial proteins remains unknown. We therefore performed a differential analysis of the hepatic mitochondrial proteome from Sod2+/- mice and wild-type mice in order to identify the consequences of partial Sod2 deletion. Using 2-D difference gel electrophoresis (DIGE) coupled with MALDI-MS/MS, we found approximately 1500 protein spots, of which 57 were differentially expressed (> or =1.5-fold change). Both SOD 1 and 2 were downregulated, but other antioxidant enzymes and related proteins were upregulated (<two-fold). The data indicate that heterozygous Sod2+/- mice exhibit a mild mitochondrial oxidative stress which is partly compensated by the antioxidant defense system linked to the tricarboxylic acid (TCA) cycle, urea cycle, beta-oxidation, and oxidative phosphorylation (OXPHOS). The results of this study are compatible with our hypothesis that the Sod2+/- mouse is a suitable animal model for studying clinically silent mitochondrial abnormalities.

Troglitazone-Induced Hepatic Necrosis in an Animal Model of Silent Genetic Mitochondrial Abnormalities

Troglitazone, a first-generation thiazolidinedione antidiabetic drug, was withdrawn from the market due to an unacceptable risk of idiosyncratic hepatotoxicity. Troglitazone does not cause hepatotoxicity in normal healthy rodents, but it produces mitochondrial injury in vitro at high concentrations. The aim of this study was to explore whether genetic mitochondrial abnormalities might sensitize mice to hepatic adverse effects of troglitazone. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model of clinically silent mitochondrial stress. Troglitazone was daily administered for 4 weeks (0, 10 or 30 mg/kg/day, ip). We found that troglitazone caused overt liver injury in the high-dose group, manifested by increased serum alanine aminotransferase activity (> twofold) and midzonal areas of hepatic necrosis, in Sod2(+/-) but not in wild-type mice. No signs of hepatotoxicity were apparent at 2 weeks of treatment. Hepatic mitochondria isolated from troglitazone-treated mice exhibited decreased activities of aconitase (by 45%) and complex I (by 46%) and increased (by 58%) protein carbonyls, indicative of enhanced mitochondrial oxidant stress. This was paralleled by compensatory increases in mitochondrial glutathione levels. Finally, in hepatocytes isolated from untreated Sod2(+/-), but not wild-type mice, troglitazone caused a concentration-dependent increase in superoxide anion levels as demonstrated with a selective mitochondria-targeting fluorescent probe. In conclusion, prolonged administration of troglitazone can superimpose oxidant stress, potentiate mitochondrial damage, and induce delayed hepatic necrosis in mice with genetically compromised mitochondrial function. These data are consistent with our hypothesis that inherited or acquired mitochondrial abnormalities may be one of the contributing determinants of susceptibility to troglitazone-induced idiosyncratic liver injury.

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2+/− mice

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2 +/− ) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2 +/− , but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2 +/− , but not Sod2 +/+ mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.