Manganese Superoxide Dismutase Knockout Mice Research Articles

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Abstract 416: Mnsod Cardiomyocyte-specific Knockout Live Up to Four Months: Role in Heart Failure Development and Progression

In our earlier investigation, we have shown that the manganese superoxide dismutase (MnSOD) mimetic, MnTnBuOE-2-PyP 5+ distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP5+ to quench O2 •- parallels its ability to reduce ONOO – and CO3 – . The absence of SOD2 (a gene that encodes MnSOD) knocked out globally is found to be embryonic lethal. We have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2 Δ ). Amazingly, we could prolong the lethality for up to four months in these conditional specific knockout mice (Mhy6-SOD2 Δ ) as compared to global knockout mice. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2 Δ to the wild type. We then examined Oxidative phosphorylation (OXPHOS) levels in Mhy6-SOD2 Δ v.s. Wild-Type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was significantly lower in Mhy6-SOD2 Δ cardiomyocytes than that in wild-type. The most noticeable difference in the O 2 consumption was found in the presence of FCCP (H + ionophore/uncoupler). Remarkably, while the FCCP treatment increased O 2 consumption in wild-type, the treatment showed no effect on the O 2 consumption in the Mhy6-SOD2 Δ cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2 Δ was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidification (ECAR), and the pattern precisely opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Mhy6-SOD2 Δ and wild-type. Herein we present the first study ever done to show the existence of cardiac-specific MnSOD knockout mice and the importance of antioxidant enzyme MnSOD that is guarding/protecting the myocardium from cardiac failures.

Age-related NMDA signaling alterations in SOD2 deficient mice

Oxidative stress affects the survival and function of neurons. Hence, they have a complex and highly regulated machinery to handle oxidative changes. The dysregulation of this antioxidant machinery is associated with a wide range of neurodegenerative conditions. Therefore, we evaluated signaling alterations, synaptic properties and behavioral performance in 2 and 6-month-old heterozygous manganese superoxide dismutase knockout mice (SOD2+/− mice). We found that their low antioxidant capacity generated direct oxidative damage in proteins, lipids, and DNA. However, only 6-month-old heterozygous knockout mice presented behavioral impairments. On the other hand, synaptic plasticity, synaptic strength and NMDA receptor (NMDAR) dependent postsynaptic potentials were decreased in an age-dependent manner. We also analyzed the phosphorylation state of the NMDAR subunit GluN2B. We found that while the levels of GluN2B phosphorylated on tyrosine 1472 (synaptic form) remain unchanged, we detected increased levels of GluN2B phosphorylated on tyrosine 1336 (extrasynaptic form), establishing alterations in the synaptic/extrasynaptic ratio of GluN2B. Additionally, we found increased levels of two phosphatases associated with dephosphorylation of p-1472: striatal-enriched protein tyrosine phosphatase (STEP) and phosphatase and tensin homolog deleted on chromosome Ten (PTEN). Moreover, we found decreased levels of p-CREB, a master transcription factor activated by synaptic stimulation. In summary, we describe mechanisms by which glutamatergic synapses are altered under oxidative stress conditions. Our results uncovered new putative therapeutic targets for conditions where NMDAR downstream signaling is altered. This work also contributes to our understanding of processes such as synapse formation, learning, and memory in neuropathological conditions.

426 - Lack of Manganese Superoxide Dismutase Impairs Brown Adipose Tissue Function in Mice

Manganese superoxide dismutase (MnSOD) is an essential member of the cell antioxidant defense system, since it efficiently catalyzes the dismutation of superoxide radical (O2–) to H2O2 and O2 in the mitochondrial matrix. Previous studies from our group have revealed that MnSOD deletion in adipocytes confers resistance to HFD-induced obesity and insulin resistance, due in part to enhanced fatty acid oxidation, mitochondrial respiration and mitochondrial biogenesis in WAT, but resulted in brown adipose tissue (BAT) atrophy and cold intolerance. In order to gain insights into the mechanisms behind BAT atrophy in adipocytes-specific MnSOD knockout mice, we generated mice with BAT-specific deletion of MnSOD using Ucp1-cre line (USod2 KO mice). As expected MnSOD protein was absent from BAT but not from WAT of USod2 KO mice. In addition, a significant decrease in adipogenic markers, such as PPARγ and FABP-4 was observed in BAT of KO mice compared to BAT of control mice. Although no changes were observed in body weight and body composition, USod2 KO mice were cold intolerant, had lower metabolic rate and reduced heat production. Despite lower energy expenditure, USod2 KO mice have improved insulin and glucose tolerance. Taken together, these results indicate that lack of MnSOD in BAT impairs its function.

Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells

Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation.

Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression

To understand the role of mitochondrial-produced superoxide (O2 (•-)) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2 (+/-)) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w/v) ethanol in the drinking water or plain water (control) for 7 d. Total cellular O2 (•-) levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2 (•-)-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2 (•-) in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. Sod2 (+/-) mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 (+/-) and LMC mice. O2 (•-) levels in hepatocytes of untreated Sod2 (+/-) mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2 (•-) levels in mitochondria of Sod2 (+/) mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O2 (•-) levels in hepatocytes of LMC mice than in Sod2 (+/-) mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O2 (•-) levels in both Sod2 (+/-) and control mice. Mitochondrial O2 (•-) is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O2 (•-) levels in hepatocytes, which appears not to originate from mitochondria.

Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention

Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention

Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse

Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.

Model mice for tissue‐specific deletion of the manganese superoxide dismutase gene

Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that converts toxic O(2)(-) to H(2)O(2). Previous studies have reported that a systemic deficiency in Mn-SOD causes neonatal lethality in mice. Therefore, no mouse model is available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type mouse model, we generated tissue-specific Mn-SOD conditional knockout mice using a Cre-loxp system. First, we generated liver-specific Mn-SOD-deficient mice by crossbreeding with albumin-Cre transgenic mice. Mn-SOD proteins were significantly downregulated in the liver of liver-specific Mn-SOD knockout mice. Interestingly, the mutant mice showed no obvious morphological abnormalities or biochemical alterations in the liver, suggesting a redundant or less important physiological role for Mn-SOD in the liver than previously thought. Next, we generated heart/muscle-specific Mn-SOD-deficient mice by crossbreeding muscle creatine kinase-Cre transgenic mice. The mutant mice developed progressive dilated cardiomyopathy with specific molecular defects in mitochondrial respiration. Furthermore, brain-specific Mn-SOD-deficient mice that had been developed by crossbreeding with nestin-Cre transgenic mice developed a spongiform encephalopathy-like pathology associated with gliosis and died within 3 weeks of birth. These results imply that the superoxide generated in mitochondria plays a pivotal role in the development and progression of pathologies in the heart and brain, but not in the liver. In conclusion, we successfully generated various tissue-specific Mn-SOD conditional knockout mice that provide useful tools for the analysis of various oxidative stress-associated diseases.

Characterization of novel kidney specific manganese superoxide dismutase knockout mice

Inactivation of manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant, is a key issue in renal failure following transplantation or ischemia/reperfusion (IR) injury. Homozygous MnSOD knock out (KO) mice die shortly after birth, and heterozygous MnSOD KO mice have decreased MnSOD activity in all tissues/organs. To address the role MnSOD inactivation has on renal injury, we generated bi‐transgenic kidney specific MnSOD KO (50% and 100%) mice by breeding KidneyCre mice1 and floxed MnSOD mice2. Characterization of these KOs revealed that phenotypically the mice are smaller in size compared to littermate controls, but show no survival differences. Immunohistochemistry data showed that the Cre‐recombinase mediated MnSOD deletion occurred specifically in the kidney and was located predominantly in the medullary region. The kidney specific KOs exhibited histologic evidence of mild renal damage, increased tyrosine nitration, and renal cell death. KO mice renal function was not altered; however, more renal damage was observed following IR injury compared to littermate controls. In summary, we have developed a novel kidney specific MnSOD KO mouse model that will help define the molecular pathways that occur downstream of MnSOD inactivation during renal injury.Supported by NIH grant 1RO1DK0789361.

Anti-aging Research Using Mn-SOD Conditional Knockout Mice

Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that converts toxic O(2)(-) to H(2)O(2). Previous studies have reported that a systemic deficiency in Mn-SOD causes neonatal lethality in mice. Therefore, no mouse model is available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type mouse model, we generated tissue-specific Mn-SOD conditional knockout mice using a Cre-loxp system. First, we generated liver-specific Mn-SOD-deficient mice by crossbreeding with albumin-Cre transgenic mice. Mn-SOD proteins were significantly downregulated in the liver of liver-specific Mn-SOD knockout mice. Interestingly, the mutant mice showed no obvious morphological abnormalities or biochemical alterations in the liver, suggesting a redundant or less important physiological role for Mn-SOD in the liver than previously thought. Next, we generated heart/muscle-specific Mn-SOD-deficient mice by crossbreeding with muscle creatine kinase-Cre transgenic mice. The mutant mice developed progressive dilated cardiomyopathy with specific molecular defects in mitochondrial respiration. Furthermore, skeletal muscle-specific Mn-SOD-deficient mice that had been generated by crossbreeding with human skeletal actin-Cre transgenic mice developed a severe physical disturbance associated with impaired cellular ATP metabolism. These results imply that the superoxide generated in mitochondria plays a pivotal role in the development and progression of pathologies in the heart and skeletal muscle, but not in the liver. In conclusion, we successfully generated various tissue-specific Mn-SOD conditional knockout mice that provide useful tools for the analysis of various oxidative stress-associated diseases.

Deficiency of MnSOD in Hematopoietic Stem Cells Causes a Sideroblastic Anemia-Like Phenotype.

Abstract 1991Poster Board I-1013 Introduction:Sideroblastic anemia is caused by an inappropriate incorporation of iron into hemoglobin, and as such a phenotype demonstrating ringed sideroblasts, improper iron deposition, and malignancy is observed. The only known hereditary cause of this disease is a mutation in delta-aminolevulinate synthase 2 (ALAS2), which is a mitochondrially located enzyme that catalyzes the first step in heme synthesis, while the causes of the acquired or idiopathic forms remain unknown. With the knowledge that the free radical superoxide (O2•-) is able to displace iron from heme-containing proteins, it is hypothesized that the loss of the mitochondrial anti-oxidant enzyme manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, may cause disruption of proper heme synthesis and as such may contribute to a sideroblastic anemia-like phenotype. Methods: Due to the early lethality of the constitutive MnSOD knock-out mouse, a floxed (flanked by loxP) MnSOD mouse was created. Homozygous hematopoietic stem cell specific SOD2 knock-outs were generated by breeding the floxed MnSOD mice with mice expressing Cre-recombinase under the control of the hematopoietic stem cell specific promoter Vav. Results: Mice harboring the hematopoietic knock-out of MnSOD are born viable, and are able to live to maturity. Complete blood count (CBC) analysis of knock-out mice demonstrated a decrease in red blood cells (RBC), thymic white blood cells, and splenic white blood cells. The knock-outs additionally showed a decrease in hemoglobin to 5 g/dL compared to 11 g/dL in controls, a reduction in hematocrit to 32% versus 43.5% in controls, and a reticulocyte count of 31.6% compared to only 7.9% in controls. CBC analysis also revealed many RBC morphology flags including hypochromasia, ansiocytosis, and hematocrit variance. These findings were visualized in blood smears (see below). Finally, electron paramagnetic resonance spectrometry of plasma from the knock-out mice showed a ∼2-fold decrease in transferrin levels compared to controls. Conclusions: The loss of MnSOD in the hematopoietic stem cell population of a mouse creates a viable organism, but with an apparent sideroblastic anemia phenotype. Additionally, the white blood cells within these organisms also may be affected due to the decrease in total cellularity, and thus could contribute to immunodeficiency and/or malignancy later in the animal's life. Overall, this phenotype is very comparable to those observed in human patients, and as such MnSOD may play an undiscovered role in acquired or idiopathic sideroblastic anemia. Future work will focus on further characterization of the animals, examination of human blood samples for MnSOD mutations, and the use of SOD mimetics to potentially reverse the observed phenotype. This work was supported by NIH grants CA73612 and CA115438 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts

The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

Oxidative Stress Causes Heart Failure with Impaired Mitochondrial Respiration

Elderly people insidiously manifest the symptoms of heart failure, such as dyspnea and/or physical disabilities in an age-dependent manner. Although previous studies suggested that oxidative stress plays a pathological role in the development of heart failure, no direct evidence has been documented so far. In order to investigate the pathological significance of oxidative stress in the heart, we generated heart/muscle-specific manganese superoxide dismutase-deficient mice. The mutant mice developed progressive congestive heart failure with specific molecular defects in mitochondrial respiration. In this paper, we showed for the first time that the oxidative stress caused specific morphological changes of mitochondria, excess formation of superoxide (O(2)(*)(-)), reduction of ATP, and transcriptional alterations of genes associated with heart failure in respect to cardiac contractility. Accordingly, administration of a superoxide dismutase mimetic significantly ameliorated the symptoms. These results implied that O(2)(*)(-) generated in mitochondria played a pivotal role in the development and progression of heart failure. We here present a bona fide model for human cardiac failure with oxidative stress valuable for therapeutic interventions.

Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance

Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.

Endothelial Progenitor Cells at Work

Tissue replacement in the adult organism by cell-specific differentiation of autologous stem/progenitor cells has evolved as a fascinating concept in stem cell biology. After organ damage, bone marrow–derived circulating or tissue-resident progenitor cells are thought to differentiate toward the type of cell needed for repair. According to this concept, maturation of these cells would be expected to result at best in a perfect morphological and functional replacement of the injured tissue. However, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , He et al show that endothelial progenitor cells (EPCs) are more than just as capable of in vitro angiogenic tube formation as mature endothelial cells (ECs), but are truly advantageous when it comes to stress tolerance.1 See page 2021 EPCs were originally characterized as cells that are mobilized from the bone marrow and circulate in the peripheral blood and express certain surface membrane markers including the vascular endothelial growth factor receptor (VEGF-R2) KDR and the hematopoietic progenitor cell markers CD34 and CD133.2–4 During ex vivo expansion, these cells develop morphological and functional characteristics typical for ECs, including formation of vascular-like structures in matrigel and other in vitro angiogenesis assays. Most importantly, however, transplanted EPCs exhibit an extraordinary potent in vivo capacity to improve the neovascularization of ischemic tissue in the adult organism.5 In this regard, EPCs were shown to be more effective than mature ECs in animal models of hind limb ischemia,6–8 although mature ECs are well established to exert a potent in vitro angiogenic activity. Thus, the …

Model mice for tissue-specific deletion of the manganese superoxide dismutase (MnSOD) gene

Manganese superoxide dismutase (MnSOD) is the enzyme that converts toxic O 2 − to H 2O 2 in mitochondria. Previous reports showed that a deficiency of MnSOD in mice was neonatal lethal. Therefore, a model mouse was not available for the analysis of the pathological role of O 2 − injuries in adult tissues. To explore an adult-type model mouse, we designed tissue-specific MnSOD conditional knockout mice using a Cre–loxp system. First, we crossbred MnSOD flox mice with transgenic mice expressing Cre recombinase under the control of the chicken actin promoter (CAG). We confirmed that CAG MnSOD knockout mice were completely deficient in MnSOD and died as neonates, validating the use of the Cre–loxp system. Next, we generated liver-specific MnSOD-deficient mice by crossbreeding with Alb–Cre transgenic mice. MnSOD activity and protein were both significantly downregulated in the liver of liver-specific MnSOD knockout mice. However, no obvious morphological abnormality was observed in the liver when biochemical alterations such as lipid peroxidation were not detectable, suggesting a redundant or less important physiological role for MnSOD in the liver than previously thought. In the present study, we successfully generated tissue-specific MnSOD conditional knockout mice that would provide a useful tool for the analysis of various age-associated diseases such as diabetes mellitus, Parkinson's disease, stroke, and heart disease, when crossbred with tissue-specific transgenic Cre mice.

The effect of hyperoxia and N-acetylcysteine on the lungs of neonatal manganese superoxide dismutase knockout mice: [formula omitted], Ting-Ting Huang 2, Hannu Sariola 3, Anna-Liisa Levonen 1, Elaine Carlson 2, Risto Lapatto 1, Charles J. Epstein 2, Kari O. Raivio 1. 1Hospital for Children and Adolescents, Univ. of Helsinki, Helsinki, Finland; 2Department of Pediatrics, Univ. of California, San Francisco, USA;

The effect of hyperoxia and N-acetylcysteine on the lungs of neonatal manganese superoxide dismutase knockout mice: [formula omitted], Ting-Ting Huang 2, Hannu Sariola 3, Anna-Liisa Levonen 1, Elaine Carlson 2, Risto Lapatto 1, Charles J. Epstein 2, Kari O. Raivio 1. 1Hospital for Children and Adolescents, Univ. of Helsinki, Helsinki, Finland; 2Department of Pediatrics, Univ. of California, San Francisco, USA;

Increased Oxidative Damage Is Correlated to Altered Mitochondrial Function in Heterozygous Manganese Superoxide Dismutase Knockout Mice

This study characterizes mitochondria isolated from livers of Sod2(-/+) and Sod2(+/+) mice. A 50% decrease in manganese superoxide dismutase (MnSOD) activity was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice, with no change in the activities of either glutathione peroxidase or copper/zinc superoxide dismutase. However, the level of total glutathione was 30% less in liver mitochondria of the Sod2(-/+) mice. The reduction in MnSOD activity in Sod2(-/+) mice was correlated to an increase in oxidative damage to mitochondria: decreased activities of the Fe-S proteins (aconitase and NADH oxidoreductase), increased carbonyl groups in proteins, and increased levels of 8-hydroxydeoxyguanosine in mitochondrial DNA. In contrast, there were no significant changes in oxidative damage in the cytosolic proteins or nuclear DNA. The increase in oxidative damage in mitochondria was correlated to altered mitochondrial function. A significant decrease in the respiratory control ratio was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice for substrates metabolized by complexes I, II, and III. In addition, mitochondria isolated from Sod2(-/+) mice showed an increased rate of induction of the permeability transition. Therefore, this study provides direct evidence correlating reduced MnSOD activity in vivo to increased oxidative damage in mitochondria and alterations in mitochondrial function.