Mice overexpressing extracellular superoxide dismutase have increased resistance to focal cerebral ischemia

Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n = 21) and wild-type (n = 19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type =37+/-14 mm3, transgenic = 27+/-13 mm3, P=0.03; subcortex: wild type = 33+/-14 mm3, transgenic = 23+/-10 mm3, P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was <25 ml/100g/min) was similar between groups (cortex: wild type = 51+/-15 mm3, transgenic = 47+/-9 mm3, P=0.65; subcortex: wild type = 39+/-16 mm3, transgenic= 37+/-17 mm3, P=0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.     

Gene transfer of extracellular superoxide dismutase to atherosclerotic mice

Clinical and epidemiological studies have provided circumstantial evidence that oxidized low-density lipoprotein (LDL) and antioxidants are involved in the pathogenesis of atherosclerosis. Superoxide dismutases (SODs) have been shown in vitro to protect LDL from deleterious effects of superoxide anions. In the present study, we have used adenoviral gene transfer to determine effect of extracellular SOD (EC-SOD) on atherogenesis in LDL receptor -/- mice. Intravenous administration of EC-SOD adenovirus (2 x 10(9) plaque forming units) into tail vein targeted transgene mainly to liver and induced a 3.5- to sevenfold increase in plasma total SOD activity. EC-SOD was secreted into circulation for 2-3 weeks mostly in a truncated B-form, suggesting that endogenous proteolytic mechanisms control the level and distribution of the enzyme. Therapeutic potential was determined by measuring plasma resistance against copper oxidation and analyzing atherosclerotic lesion areas in aortas of LDL receptor -/- mice. Mice were kept on a cholesterol diet for 10 weeks before gene transfer and 3 or 6 weeks after the gene transfer. Results showed a tendency for a reduction in the overall lesion area after EC-SOD gene transfer as compared with LacZ transduced control mice, but the difference did not reach statistical significance. It is concluded that short-term overexpression of EC-SOD in vivo does not affect atherogenesis in LDL receptor -/- mice.     

Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid perxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47); P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 ± 0.71 μmole MDA/g tissue) thatn at 2 months (5.69 ± 0.90) and significantly lower than 28 months (5.13 ± 0.33) than at 12 months.Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.     

Status of hepatic heme and heme oxygenase during Plasmodium yoelii nigeriensis infection in mice

Plasmodium yoelii nigeriensis infection in albino mice caused a significant increase in hepatic heme level, the increase being concomitant with a rise in parasitemia. This elevated heme was found to be associated with all the subcellular fractions except the cytosol, where its content remained unaltered. Activity of heme oxygenase, the key enzyme responsible for catabolism of heme, also increased progressively with rise in parasitemia. Treatment of normal mice with cobalt chloride [60 mg (kg body wt)-1; subcutaneously] brought about a 150% increase in the level of heme oxygenase; similar treatment of infected mice at low parasitemia could induce the enzyme activity while at high parasitemia the enzymic activity remained unaltered as compared to untreated infected mice. In spite of an increased level of heme oxygenase in the cobalt-treated mice, the level of heme did not show any noticeable change. Oral administration of chloroquine [64 mg (kg body wt)-1 x 4 days] brought about a 56% reduction in the level of heme oxygenase of normal animals but there was no change in infected animals when compared with the corresponding untreated infected mice. However, the amount of chloroquine present in livers of normal and infected animals was not significantly different.     

Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells

BACKGROUND: The present study was undertaken to investigate the cAMP-dependent regulation of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) by ovarian steroids in human endometrial stromal cells (ESC). METHODS and RESULTS: To examine the effect of cAMP on SOD expression, ESC were incubated with dibutyryl-cAMP (db-cAMP, 0.5 mmol/l), forskolin (25 micromol/l), or estradiol (E(2), 10(-8) mol/l) + medroxyprogesterone acetate (MPA, 10(-6) mol/l), for 18 days. E(2) + MPA significantly increased Cu,Zn-SOD activity and mRNA concentrations, whereas db-cAMP and forskolin had no effect. On the other hand, Mn-SOD activity and mRNA concentration were significantly increased by all of these treatments. Insulin-like growth factor-binding protein-1, a marker of decidualization, was clearly induced by db-cAMP, forskolin or E(2) + MPA, accompanied by morphological changes characteristic of decidualization. To study whether the increase in Mn-SOD by db-cAMP or E(2) + MPA was mediated by cAMP-dependent protein kinase A (PKA), ESC were incubated with protein kinase inhibitor (PKI) (10 microg/ml), an inhibitor of PKA, in the presence of db-cAMP or E(2) + MPA. The increase in Mn-SOD activity following db-cAMP or E(2) + MPA was completely inhibited by PKI. CONCLUSIONS: In the process of decidualization, E(2) + MPA increases Mn-SOD expression via a cAMP-dependent pathway. Cu,Zn-SOD is also up-regulated by E(2) + MPA, but via a different pathway from that involving cAMP.     

Hippocampal memory and plasticity in superoxide dismutase mutant mice

The reactive oxygen species (ROS) superoxide is well known for its role in disease mechanisms. Mounting evidence indicates, however, that superoxide also is generated for useful purposes and contributes to normal physiologic function. Studies with transgenic mice that overexpress superoxide scavengers show that certain types of memory function and underlying neuronal processes are impaired under conditions of severely reduced superoxide signaling. These findings have implications for the use of antioxidant treatments as well as for our understanding of the signaling events involved in cognition.     

Hemozoin stability and dormant induction of heme oxygenase in hemozoin-fed human monocytes.

Human monocytes avidly ingest malarial pigment, hemozoin. Phagocytosed hemozoin persists in the monocyte for a long time and modifies important monocyte functions. Stability of phagocytosed hemozoin may depend on modifications of the hemozoin heme moiety or reduced ability to express heme-inducible heme oxygenase. We show here that the spectral characteristics of alkali-solubilized hemozoin were identical to those of authentic heme, although hemozoin was solubilized by alkali much more slowly than authentic heme. Alkali-solubilized hemozoin was a substrate of microsomal rat heme oxygenase and bilirubin reductase, with bilirubin as the main final product. Hemozoin feeding to human monocytes did not induce heme oxygenase, but authentic heme and alkali-solubilized hemozoin supplemented to hemozoin-fed monocytes induced heme oxygenase and were degraded normally. Lysosomes isolated from hemozoin-fed monocytes released only traces of heme while lysosomes from erythrocyte-fed monocytes liberated considerable quantities of heme. Lack of heme release from hemozoin did not depend on proteolysis-resistant, heme-binding proteins, since lysosomal proteases fully degraded hemozoin-associated proteins but did not solubilize hemozoin. In conclusion, our data indicate that lack of induction of HO1 is due to the intrinsic structural characteristics of hemozoin and not to hemozoin-mediated impairment of the mechanism of HO1 induction.     

Failed induction of heme oxygenase 1 in endothelial cells exposed to the hemoglobin based oxygen carrier Oxyglobin

We investigated the effect of the bovine hemoglobin based oxygen carrier HBOC-200 (Oxyglobin) in rat aortic endothelial cells (RAEC) on the activation of heme oxygenase 1 (HO-1). RAEC were incubated in the presence of 75 microM (G1) or 225 microM (G2) HBOC-200. The positive control (G3) was performed by incubation with 50 microM Hemin. For negative control (G4) cells were incubated with medium alone. G2 and G3 reached a significant increase of bilirubin concentration compared to G4. A positive HO-1 signal in the Western Blot was seen in G3 12 and 24 h after incubation. The Western Blot of G1, G2 and G4 showed no HO-1 signal. These data suggest that HBOC-200 in the applied dosage cannot induce HO-1 expression in RAEC, and may be degraded by isoenzymes at a lower level.     

Mechanisms of hyporeactivity in mice after injection of lysed erythrocyte antigen

A state of specific hyporeactivity to sheep's red cells (SRBC) was induced in mice by injection of hemolyzed SRBC. Blocking serum factor in these mice was shown not to be identical in the character of its action with antired-cell antibodies, but to be probably an antigen-antibody complex. After combined injection of hemolyzed SRBC and cyclophosphamide (CP) into mice production of blocking serum factor was suppressed but the reactivity of the mice to SRBC was considerably reduced. It is suggested that in this case inactivation of the immunocompetent cells took place through the combined action of CP and SRBC antigen in a nonimmunogenic form.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 253–256, March, 1979.     

Interdiction of the diabetic state in NOD mice by sustained induction of heme oxygenase: possible role of carbon monoxide and bilirubin

The aims of the present study were to assess whether sustained HO-1 expression could moderate or prevent diabetes in an animal model of the disease and, if so, to examine the possible mechanisms involved. Our results showed that HO-1 expression and HO activity were upregulated in the pancreas of non-obese diabetic (NOD) mice by the weekly administration of cobalt protoporphyrin (CoPP). Blood glucose levels in CoPPtreated mice decreased to normal, but continuously increased in untreated controls. Beta-cell numbers were preserved in the islets of CoPP-treated mice, whereas no beta cells were found in untreated diabetic mice. The number of CD11c(+) dendritic cells was significantly decreased in the pancreas of CoPP-treated NOD mice, but this effect was reversed by the inhibition of HO activity. Increased levels of HO-1 produced a new pancreatic phenotype, as reflected by increases in phosphorylated AKT, BcL-xL and RSK levels, and decreases in O(2)- and 3-NT levels. These novel findings provide a link between the increase in HO-1 activity, with its concurrent enhanced production of carbon monoxide (CO) and bilirubin, a decrease in infiltrated CD11c(+) dendritic cells and an increase in anti-apoptotic proteins, including RSK and BcL-xL, in the interdiction of the diabetic state.     

Extracellular superoxide dismutase overexpression protects against aging-induced cognitive impairment in mice

Extracellular superoxide dismutase (EC-SOD) controls the availability of extracellular superoxide and appears to play a role in controlling oxidative stress and intercellular signaling. Whether EC-SOD overexpression would help or hinder neurobehavioral function appears to depend on the age of the individual. In young adult mice, we have found that EC-SOD overexpression can interfere with learning on the radial-arm maze, possibly by reducing control over nitric oxide neurotransmission. In aged mice, we found, in the current study, that EC-SOD overexpression greatly improves learning on the radial-arm maze. Control (N = 17) and EC-SOD overexpressing mice (N = 13) acquired the 8-arm radial maze over 21 sessions of training. The EC-SOD overexpressing mice had significantly better choice accuracy than the control mice (p < 0.005). The EC-SOD overexpressing mice averaged 6.34 ± 0.22 correct arm entries before an error (entries to repeat) during the acquisition phase, while the control mice averaged 5.18 ± 0.22 entries to repeat. EC-SOD genotype did not cause a main effect on response latency. The advantage held by the EC-SOD overexpressing mice persisted during the eight-session post-acquisition phase of testing (p < 0.01). When there was a shift from high to low levels of motivation by reducing the period of food restriction before testing, the EC-SOD overexpression-induced improvement was reduced slightly, but it was still significant compared with the wild-type controls (p < 0.025). Then, after 4 months of no testing, the mice were tested for retention and reacquisition of performance on the radial-arm maze. The EC-SOD overexpressing mice maintained their significantly better choice accuracy (p < 0.05). Enhancement of EC-SOD activity appears to improve learning and memory performance, specifically in aging mice. EC-SOD mimetic treatment during the course of aging may hold promise for aging-induced cognitive impairment.     

Genetic modifiers of the phenotype of mice deficient in mitochondrial superoxide dismutase

Sod2-/- mice, which are deficient in the mitochondrial form of superoxide dismutase (MnSOD), have a short survival time that is strongly affected by genetic background. This suggests the existence of genetic modifiers that are capable of modulating the degree of mitochondrial oxidative damage caused by the MnSOD deficiency, thereby altering longevity. To identify these modifier(s), we generated recombinant congenic mice with quantitative trait loci (QTL) containing the putative genetic modifiers on the short-lived C57BL/6J genetic background. MnSOD deficient C57BL/6J mice with a QTL from the distal region of chromosome 13 from DBA/2J were able to survive for as long as those generated on the long-lived DBA/2J background. Within this region, the gene encoding nicotinamide nucleotide transhydrogenase (Nnt) was found to be defective in C57BL/6J mice, and no mature NNT protein could be detected. The forward reaction of NNT, a nuclear-encoded mitochondrial inner membrane protein, couples the generation of NADPH to proton transport and provides NADPH for the regeneration of two important antioxidant compounds, glutathione and thioredoxin, in the mitochondria. This action of NNT could explain its putative protective role in MnSOD-deficient mice.     

Effects of a common human gene variant of extracellular superoxide dismutase on endothelial function after endotoxin in mice

A common gene variant in the heparin-binding domain (HBD) of extracellular superoxide dismutase (ECSOD) may predispose human carriers to ischaemic heart disease. We have demonstrated that the HBD of ECSOD is important for ECSOD to restore vascular dysfunction produced by endotoxin. The purpose of this study was to determine whether the gene variant in the HBD of ECSOD (ECSOD_R213G) protects against endothelial dysfunction in a model of inflammation. We constructed a recombinant adenovirus that expresses ECSOD_R213G. Adenoviral vectors expressing ECSOD, ECSOD_R213G or β-galactosidase ( LacZ , a control) were injected i.v. in mice. After 3 days, at which time the plasma SOD activity is maximal, vehicle or endotoxin (lipopolysaccharide or LPS, 40 mg kg⁻¹) was injected i.p. Vasomotor function of aorta in vitro was examined 1 day later. Maximal relaxation to sodium nitroprusside was similar in aorta from normal and LPS-treated mice. Maximal relaxation to acetylcholine (10⁻⁵) was impaired after LPS and LacZ (63 ± 3%, mean ± s.e.m. ) compared to normal vessels (83 ± 3%) ( P < 0.05). Gene transfer of ECSOD improved ( P < 0.05) relaxation in response to acetylcholine (76 ± 5%) after LPS, whereas gene transfer of ECSOD_R213G had no effect (65 ± 4%). Superoxide was increased in aorta (measured using lucigenin and hydroethidine) after LPS, and levels of superoxide were significantly reduced following ECSOD but not ECSOD_R213G. Thus, ECSOD reduces superoxide and improves relaxation to acetylcholine in the aorta after LPS, while the ECSOD variant R213G had minimal effect. These findings suggest that, in contrast to ECSOD, the common human gene variant of ECSOD fails to protect against endothelial dysfunction produced by an inflammatory stimulus.     

Changes in superoxide dismutase activity during stimulation of peripheral blood polymorphonuclear leukocytes

No. 4 Department of Biophysics and Internal Medicine, N. I. Pirogov Second Moscow Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 4, pp. 334–336, April, 1990.     

Altered long-term corticostriatal synaptic plasticity in transgenic mice overexpressing human CU/ZN superoxide dismutase (GLY(93)-->ALA) mutation

Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.     

The neurotoxin MPTP does not affect striatal superoxide dismutase activity in mice

Intraneuronal superoxide generation may be a possible mechanism of the dopaminergic (DA) neurotoxicity of MPTP. In such case, MPTP might theoretically affect superoxide dismutase (SOD) activity. We determined SOD activity and DA levels in striata of mice at 0.5, 1, 4, 16, 24 h or 7 days after an acute single injection of MPTP (40 mg/kg, s.c.). MPTP produced marked striatal DA depletions from 4 h post-treatment but SOD activity remained unaltered and similar to controls at all time points. Intrastriatal injections of purified SOD 15 min prior to systemic administration of MPTP did not attenuate the MPTP-induced striatal DA depletions in mice at 7 days post-treatment. Combined administration of MPTP with the SOD inhibitor diethyldithiocarbamate markedly enhanced striatal DA decreases produced by MPTP alone. Findings suggest that MPTP does not act via inhibition of SOD. Therefore, potentiation of MPTP toxicity by diethyldithiocarbamate may be due to interference with other enzymatic systems.     

Impaired tolerance to repetitive hypoxia in hippocampal slices of Cu,Zn superoxide dismutase transgenic mice

Inhibition of the subthalamic nucleus (STN) has been shown to suppress seizures in different animal models of epilepsy. The aim of this study was to examine the role of the pallidal inputs to the STN in the control of absence seizures in a genetic model in the rat. Disinhibition of the globus pallidus or the ventral pallidum, by local injections of a GABA(A) antagonist, suppressed absence seizures. Conversely, inhibition of the ventral pallidum by a GABA(A) agonist aggravated absence seizures. Furthermore, the antiepileptic effects of intrapallidal injections of a GABA(A) antagonist were correlated with a decrease of extracellular levels of glutamate in the substantia nigra. Our results show that both the globus pallidus and the ventral pallidum exert a modulatory influence on absence seizures and suggest that these effects are mediated through the STN.     

溃疡性结肠炎患者血清超氧化物歧化酶及脂质过氧化物的变化

用黄嘌呤氧化法及硫代巴比妥酸反应产物比色法分别对120例溃疡性结肠炎患者血清超氧化物歧化酶（SOD）及脂质过氧化物（LPO）含量进行检测。结果表明：溃疡性结肠炎患者SOD明显低于对照组（P＜0．001）;LPO明显高于对照组（P＜0．001）。其中单纯湿热内蕴型或兼胃虚弱型或兼脾虚气滞血瘀型或兼阴虚气滞血瘀型血清SOD含量均明显低于对照组（P＜0．001）;而血清LPO含量均明显高于对照组（P＜0．001）。结果提示：以湿热内蕴型为主的溃疡性结肠炎患者清除氧自由基,保护细胞免受损伤的SOD降低,而间接反映细胞损伤的LPO升高。当伴有虚证时,尤以脾胃虚弱型或脾虚气滞血瘀型的溃疡性结肠炎患者SOD含量明显降低和LPO含量明显增高。     

Expression of heme oxygenase in human airway epithelial cells

Elevated levels of carbon monoxide (CO) are found in the exhaled breath of patients with inflammatory diseases such as asthma and cystic fibrosis. Endogenous CO is derived from heme oxygenase (HO) (EC 1.14.99.3), which catabolizes heme-producing CO and biliverdin. There are three isoforms of HO: HO-1 is inducible by inflammatory cytokines and oxidants, including nitric oxide (NO), whereas HO-2 and HO-3 are expressed constitutively. Primary airway epithelial cells were treated with either 50 ng/ml interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma (cytomix), or the NO donor NOC-18 for up to 24 h. Cytomix-induced HO-1 expression peaked at 4 h, returning to baseline by 24 h, whereas HO-2 expression remained unchanged. This increase in HO-1 expression could not be explained by an increase in NO production as inducible NO synthase expression increased between 12 and 24 h. However, the NO donor NOC-18 (500 microM) increased HO-1 expression twofold and HO activity 25-fold, whereas cytomix treatment increased HO activity eightfold. NO induction of HO-1 was not mediated via guanylyl cyclase and was not attenuated by 1 microM dexamethasone, although dexamethasone increased HO-2 protein. Therefore, airway epithelial cells express HO-2 and can express HO-1; thus, the epithelium may be a source of increased CO in airway diseases.     

Activity and electrophoretic multiplicity of molecular forms of superoxide dismutase in human blood cells

The level of superoxide dismutase (SOD) activity calculated relative to protein in the various human blood cells falls in the following order: platelet > erythrocyte > lymphocyte > granulocyte. During electrophoresis in homogeneous polyacrylamide gel of homogenates of granulocytes, lymphocytes, and platelets three zones of SOD activity were identified. Two fractions of the enzyme, disappearing after treatment with cyanide, were found in lysates of erythrocytes after removal of the hemoglobin. Of the two SOD fractions of platelets, lymphocytes, and granulocytes migrating rapidly toward the anode, the first corresponds in its ability to be inhibited by cyanide or organic solvents to the cytosol isozyme, the second to the mitochondrial isozyme. The third cathode fraction was not identified. The functional role of SOD in specialized blood cells and the cause of heterogeneity of the enzyme are discussed.Department of Hyperbaric Oxygenation, All-Union Scientific-Research Institute of Clinical and Experimental Surgery, Moscow. Department of Biochemistry, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 166–168, August, 1977.