The Use of Transgenic and Mutant Mice to Study Oxygen Free Radical Metabolism
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.
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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
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ABSTRACT: Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2(-/-)) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2(-/-) mice, we examined the growth of isolated mouse embryonic fibroblasts (MEFs) from Sod2(-/-) mice. We found that the proliferation of Sod2(-/-) MEFs was significantly decreased compared with wild-type MEFs despite the absence of morphological differences. The Sod2(-/-) MEFs produced less cellular ATP, had lower O(2) consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G-protein-coupled receptor-mediated intracellular Ca(2+) signal transduction was also severely suppressed in Sod2(-/-) MEFs. Finally, the ratio of microtubule-associated protein light chain 3 (LC3)-II/LC3-I, an index of autophagic activity, was increased in Sod2(-/-) MEFs, consistent with a reduction in mTOR signal transduction. These data demonstrate that MnSOD deficiency results in alterations in several key signaling pathways, which may contribute to the lethal phenotype of Sod2(-/-) mice.Free Radical Biology and Medicine 11/2010; 49(8):1255-62. DOI:10.1016/j.freeradbiomed.2010.07.006 · 5.71 Impact Factor
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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