The Use of Transgenic and Mutant Mice to Study Oxygen Free Radical Metabolism

Department of Pediatrics, University of California, San Francisco, California 94143-0748, USA
Annals of the New York Academy of Sciences (Impact Factor: 4.31). 02/2006; 893(1):95 - 112. DOI: 10.1111/j.1749-6632.1999.tb07820.x

ABSTRACT To distinguish the role of Mn superoxide dismutase (MnSOD) from that of cytoplasmic CuZn superoxide dismutase (CuZnSOD), the mouse MnSOD gene (Sod2) was inactivated by homologous recombination. Sod2−/− mice on a CD1 (outbred) genetic background die within the first 10 days of life (mean, 5.4 days) with a complex phenotype that includes dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, metabolic acidosis and ketosis, and a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. On the other hand, Lebovitz et al. reported an independently derived MnSod null mouse (Sod2tmlLeb) on a mixed C57BL/6 and 129Sv background with a different phenotype. Because a difference in genetic background is the most likely explanation for the phenotypic differences, the two mutant lines were crossed into different genetic backgrounds for further analyses. To study the phenotype of Sod2tmlLeb mice CD1 background, the Sod2tmlLeb mice were crossed to CD1 for two generations before the −/+ mice were intercrossed to generate −/− mice. The life span distribution of CD1〈Sod2−/−〉Leb was shifted to the left, indicating a shortened life span on the CD1 background. Furthermore, the CD1〈Sod2−/−〉Leb mice develop metabolic acidosis at an early stage as was observed with CD1〈Sod2−/−〉Cje. When Sod2tmlCje was placed on C57BL/6J (B6) background, the −/− mice were found to die either during midgestation or within the first 4 days after birth. However, when the B6〈Sod2−/+〉Cje were crossed with DBA/2J (D2) for the generation of B6D2F2〈Sod2−/−〉Cje mice, an entirely different phenotype, similar to that described by Lebovitz et al., was observed. The F2 Sod−/− mice were able to survive up to 18 days, and the animals that lived for more than 15 days displayed neurological abnormalities including ataxia and seizures. Their hearts were not as severely affected as were those of the CD1 mice, and neurological degeneration rather than heart defect appears to be the cause of death.

  • Source
    • "O 2 @BULLET− is involved in both physiological and pathological processes [23], with O 2 @BULLET− overproduction implicated in a range of inflammatory states such as rheumatoid arthritis, osteoarthritis, arteriosclerosis, and ischemia– reperfusion [24]. The toxicity of increased O 2 @BULLET− levels is evident in homozygous SOD2 knockout mice, which die within the first 3 weeks of age [25]. O 2 @BULLET− can inactivate a range of enzymes in addition to causing direct molecular damage by initiating lipoperoxidation, leading to the destruction of neurotransmitters and hormones, and DNA single-strand damage [20]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Superoxide (O(2)(•-)) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O(2)(•-) at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O(2)(•-) levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O(2)(•-) sources, including increased Hcy levels. Hcy increased O(2)(•-) levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10-50nM) prevented Hcy-induced increases in O(2)(•-) and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O(2)(•-) production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O(2)(•-) levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O(2)(•-) levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O(2)(•-) scavenger.
    Free Radical Biology and Medicine 06/2011; 51(4):876-83. DOI:10.1016/j.freeradbiomed.2011.05.034 · 5.71 Impact Factor
  • Source
    • "SOD1 is an antioxidant isoenzyme mainly localized in the cytosol that dismutates superoxide anions to hydrogen peroxide (Fridovich 1975). SOD1 is constitutively present in all cells (Huang et al. 1999). In animals that overexpress SOD1, cytochrome c release and neuronal death were highly inhibited after FCI (Kinouchi et al. 1991; Chan 1996; Fujimura et al. 2000), tGCI (Murakami et al. 1997; Chan et al. 1998; Endo et al. 2006b), and subarachnoid hemorrhage (Endo et al. 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Reactive oxygen species generated from mitochondria have been implicated in acute brain injuries such as stroke and neurodegeneration. Recent studies have shown that mitochondrially-formed oxidants are mediators of molecular signaling, which is implicated in the mitochondria-dependent apoptotic pathway that involves pro- and antiapoptotic protein binding, the release of cytochrome c, and transcription-independent p53 signaling, leading to neuronal death. Oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves phosphatidylinositol 3-kinase/Akt and downstream signaling, which lead to neuronal survival. Genetically modified mice or rats that over-express or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.
    Journal of Neurochemistry 05/2009; 109 Suppl 1(1):133-8. DOI:10.1111/j.1471-4159.2009.05897.x · 4.24 Impact Factor
  • Source
    • "Several animal models have been developed to investigate the role of MnSOD in oxidative stress, neurodegenerative diseases, and aging. MnSOD2-deficient mice on a CD1 background have a foreshortened life span and display a multifaceted phenotype including lipid accumulation in liver and skeletal muscle, a marked decrease in both succinate dehydrogenase and aconitase in the heart, as well as metabolic acidosis and ketosis [29]. In addition, wild-type mice and murine cells treated with MnSOD gene therapy showed increased survival after radiation exposure compared to untreated mice and cells and reduced radiation-induced skin injury in mice [30] [31]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We tested several classes of antioxidant manganese compounds for radioprotective effects using human lymphoblastoid cells: six porphyrins, three salens, and two cyclic polyamines. Radioprotection was evaluated by seven assays: XTT, annexin V and propidium iodide flow cytometry analysis, gamma-H2AX immunofluorescence, the neutral comet assay, dichlorofluorescein and dihydroethidium staining, resazurin, and colony survival assay. Two compounds were most effective in protecting wild-type and A-T cells against radiation-induced damage: MnMx-2-PyP-Calbio (a mixture of differently N-methylated MnT-2-PyP+ from Calbiochem) and MnTnHex-2-PyP. MnTnHex-2-PyP protected WT cells against radiation-induced apoptosis by 58% (p = 0.04), using XTT, and A-T cells by 39% (p = 0.01), using annexin V and propidium iodide staining. MnTnHex-2-PyP protected WT cells against DNA damage by 57% (p = 0.005), using gamma-H2AX immunofluorescence, and by 30% (p < 0.01), using neutral comet assay. MnTnHex-2-PyP is more lipophilic than MnMx-2-PyP-Calbio and is also >10-fold more SOD-active; consequently it is >50-fold more potent as a radioprotectant, as supported by six of the tests employed in this study. Thus, lipophilicity and antioxidant potency correlated with the magnitude of the beneficial radioprotectant effects observed. Our results identify a new class of porphyrinic radioprotectants for the general and radiosensitive populations and may also provide a new option for treating A-T patients.
    Free Radical Biology and Medicine 05/2009; 47(3):250-60. DOI:10.1016/j.freeradbiomed.2009.04.018 · 5.71 Impact Factor
Show more