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The air pollutant ozone induces both airway inflammation and restrictions in lung function. These responses have been proposed to arise as a consequence of the oxidizing nature of ozone, depleting endogenous antioxidant defenses with ensuing tissue injury. In this study we examined the impact of an environmentally relevant ozone challenge on the antioxidant defenses present at the surface of the lung in two groups known to have profound differences in their antioxidant defense network: healthy control (HC) and mild asthmatic (MA) subjects. We hypothesized that baseline differences in antioxidant concentrations within the respiratory tract lining fluid (RTLF), as well as induced responses, would predict the magnitude of individual responsiveness. We observed a significant loss of ascorbate (ASC) from proximal (−45.1%, p < .01) and distal RTLFs (−11.7%, p < .05) in healthy subjects 6 h after the end of the ozone challenge. This was associated (Rs, −0.71, p < .01) with increased glutathione disulphide (GSSG) in these compartments (p = .01 and p < .05). Corresponding responses were not seen in asthmatics, where basal ASC concentrations were significantly lower (p < .01) and associated with elevated concentrations of GSSG (p < .05). In neither group was any evidence of lipid oxidation seen following ozone. Despite differences in antioxidant levels and response, the magnitude of ozone-induced neutrophilia (+20.6%, p < .01 [HC] vs. +15.2%, p = .01 [MA]) and decrements in FEV1 (−8.0%, p < .01 [HC] vs. −3.2%, p < .05 [MA]) did not differ between the two groups. These data demonstrate significant differences between the interaction of ozone with RTLF antioxidants in MA and HC subjects. These responses and variations in basal antioxidant defense were not, however, useful predictive markers of group or individual responsiveness to ozone.

Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E–null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD–null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD–null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet–induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F2αwas related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Oxygen free radicals have been suggested to play important roles in atherogenesis and other pathological processes in the blood vessel wall. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD), which is produced and secreted to the extracellular space by smooth muscle cells. In this study, we investigated the influence of factors regulating tension and proliferation of vascular smooth muscle cells and of some interstitial matrix components on EC-SOD expression. The expression and secretion of EC-SOD were upregulated by histamine, vasopressin, oxytocin, endothelin-1, angiotensin II, serotonin, heparin, and heparan sulfate and were downregulated by platelet-derived growth factors-AA and -BB, acidic and basic fibroblast growth factors, and epidermal growth factor. The responses were slow and developed over several days. The findings suggest that various physiological and pathological conditions might markedly influence EC-SOD expression, significantly altering the susceptibility of the vascular wall to effects of the superoxide radical.

We here evaluate cataract formation in mice lacking the cytosolic copper-zinc superoxide dismutase (CuZn-SOD) in an in vitro model using irradiation with visible light and riboflavin as a photosensitizing agent. Isolated, cultured lenses from wild-type and CuZn-SOD-null mice were irradiated for 1.5 h by a daylight fluorescent light after preincubation with 10 microM riboflavin for 24 h. Cataract formation was evaluated daily with digital image analysis and ocular staging, and after 5 d 86Rb uptake and water contents of the lenses were determined. Basal superoxide concentrations in freshly isolated lenses from wild-type and CuZn-SOD-null mice were determined with lucigenin-derived chemiluminescense, and enzymatic activities of all three SOD isoenzymes in the murine lens were determined with a direct spectrophotometric method. The cytosolic CuZn-SOD accounts for 90% of the total SOD activity of the murine lens. CuZn-SOD-null lenses showed a doubled basal superoxide concentration, and were more prone to develop photochemical cataract in the present model with more opacification, more hydration, and less 86Rb uptake than lenses from wild-type mice. We conclude that CuZn-SOD is an important superoxide scavenger in the lens, and that it may have a protective role against cataract formation.

Oxidative stress has been implicated in human illness such as cardiovascular and neurodegenerative disease. The genetic mechanisms involved are only poorly understood. Here we describe the determination of the allelic frequency and phenotype of a common polymorphism in Se-dependent glutathione peroxidase 1 (GPX1) in Finnish/Swedish populations. A proline/leucine variant occurs at position 197 close to the C-terminus of the protein. The more common allele encoding the Pro variant is present at 59% in a Finnish/Swedish population (n = 66) and at 73% in a Swedish population (n = 315). The genotypes encoding Pro/Pro, Pro/Leu, and Leu/Leu are distributed according to the Hardy–Weinberg relationship. The Swedish population consisted of 101 stroke cases and 214 controls. No significant association between allele frequency and risk to suffer from stroke was evident. Erythrocyte GPX activity was determined in the Finnish/Swedish population and no significant differences were obtained between the genotypes. It can be concluded that the Pro/Leu genetic variation does not appear to compromise the defense against oxidative stress in red blood cells nor to be associated with stroke.

Oxygen free radicals as well as immunological reactions have been suggested to play important roles in atherogenesis and other pathological processes of the blood vessel wall. We have previously shown that the vascular wall contains exceptionally large amounts of extracellular superoxide dismutase (EC-SOD) and that the enzyme is produced and secreted to the extracellular space by the smooth muscle cells. In this work, we studied the influence of inflammatory cytokines on vascular smooth muscle cell expression of EC-SOD, the mitochondrial manganese superoxide dismutase (Mn-SOD) and the cytosolic copper zinc superoxide dismutase (CuZn-SOD). The expression of EC-SOD was up-regulated by interferon-gamma (IFN-gamma) and interleukin 4 (IL-4). and was down-regulated by tumor necrosis factor-alpha (TNF-alpha). The ratio between the maximal stimulation and depression observed was around 20-fold. The responses were slow and developed over periods of several days. The Mn-SOD activity was strongly up-regulated by TNF-alpha and IL-1alpha and moderately by IFN-gamma. The CuZn-SOD activity of the smooth muscle cells was not significantly influenced by any of the cytokines. The findings suggest that large changes in the SOD isoenzymes might occur in vascular diseases, significantly altering the susceptibility of the vascular wall to adverse effects of the superoxide radical.

Progressive myoclonic epilepsies (EPM) are difficult to treat and refractory to most antiepileptic drugs. Besides epilepsy, EPMs also involve continuous neurological deterioration. Oxidative stress is thought to be an important factor in this process. We therefore analyzed a series of antioxidant enzymes in the blood of patients and compared with healthy age matched controls. In addition patients were given high doses of N-acetylcysteine (NAC), a glutathione percursor to determine if symptoms of EPM would improve. Five patients, four with EPM 1 (Unverricht-Lundborg disease) and one patient with EPM2 (Lafora body disease) were treated with 6 g/day of NAC. Before treatment, plasma samples were analyzed for glutathione peroxidase activity, catalase activity, extracellular superoxide dismutase (SOD) and CuZn-SOD and compared with the controls. Erythrocyte CuZn-SOD was significantly lower in the EPM patients compared to controls. NAC improved markedly and stabilized the neurological symptoms in patients with EPM 1 but had a doubtful effect in the patient with EPM 2.

We have previously shown that human beta-cells are resistant to the toxic effects of alloxan. In order to further clarify this characteristic of human islets, we investigated whether these cells might transfer their alloxan resistance to alloxan-sensitive rat or mouse islets. Islets from two species (human-mouse or rat-mouse) were mixed into one graft, which was implanted into the subcapsular kidney space of nude mice. Alloxan or saline was injected intravenously two weeks after implantation and one week thereafter the mice were killed. The number of grafted and endogenous beta-cells were evaluated by a semi-quantitative method after immunohistochemistry. Human islet production of the scavenging enzymes extracellular superoxide dismutase and plasma glutathione peroxidase were analyzed with ELISA-techniques, and mouse and human islet hydrogen peroxide breakdown activity were monitored with a horseradish peroxidase-dependent assay. Mouse beta-cells transplanted together with human islets were protected against alloxan cytotoxicity. Rat islets did not protect mouse beta-cells against alloxan, suggesting that the mixing procedure as such did not impose the protection. Production of extracellular superoxide dismutase and plasma glutathione peroxidase by human islets was very low. Moreover, H2O2 breakdown in vitro, did not differ between human and mouse islets. Alloxan-insensitive human islets protect mouse beta-cells against alloxan-induced lesions, suggesting that yet to be identified extracellular factors are involved in human islet resistance to alloxan toxicity.

Alloxan is a diabetogenic agent which apparently acts through formation of superoxide radicals formed by redox cycling. Superoxide radicals are also formed by a variety of mechanisms in hyperglycemia. We exposed extracellular-superoxide dismutase (EC-SOD) null mutant and wild-type mice to alloxan, and followed up both the initial diabetes induction and the long-term course of the hyperglycemia. The null mutant mice responded with a modestly enhanced hyperglycemia compared to the wild type controls. In the long-term follow-up all mice eventually regained glycemic control, although it took longer for individuals with higher initial hyperglycemia. This delaying effect of the hyperglycemia was much more pronounced in the null mutant mice. These data suggest that the difference in initial diabetes induction between the groups is due to interception by EC-SOD of extracellular superoxide radicals produced by alloxan. The delayed recovery in the null mutant mice suggests that superoxide radicals released as a result of hyperglycemia impair β-cell regeneration and that EC-SOD provides some protection. Mouse islets were found to contain little EC-SOD, whereas the content of the cytosolic Cu- and Zn-containing SOD was very high. This low EC-SOD activity may contribute to the high alloxan susceptibility of β-cells, and may also cause a high susceptibility to superoxide radicals produced by activated inflammatory leukocytes and in hyperglycemia.

Canine leukocyte adhesion deficiency (CLAD) is a fatal immunodeficiency disease found in Irish setters. The clinical manifestations of CLAD are very similar to LAD in humans and BLAD in cattle, which are both caused by mutations in ITGB2 encoding the leukocyte integrin beta-2 subunit (CD18). Sequence analysis of the ITGB2 coding sequence from a CLAD dog and a healthy control revealed a single missense mutation, Cys36Ser. This cysteine residue is conserved among all beta integrins, and the mutation most likely disrupts a disulfide bond. The mutation showed a complete association with CLAD in Irish setters and was not found in a sample of dogs from other breeds. The causative nature of this mutation was confirmed by transduction experiments using retroviral vectors and human LAD EBV B-cells. The normal canine CD18 formed heterodimers with the human CD11 subunit, whereas gene transfer of the mutant CD18 resulted in very low levels of CD11/CD18 expression. The identification of the causative mutation for CLAD now makes it possible to identify carrier animals with a simple diagnostic DNA test, and it forms the basis for using CLAD as a large animal model for the development and evaluation of clinical treatments for human LAD.

A primary male autosomal linkage map of the domestic horse (Equus caballus) has been developed by segregation analysis of 140 genetic markers within eight half-sib families. The family material comprised four Standardbred trotters and four Icelandic horses, with a total of 263 offspring. The marker set included 121 microsatellite markers, eight protein polymorphisms, five RFLPs, three blood group polymorphisms, two PCR–RFLPs, and one single strand conformation polymorphism (SSCP). One hundred markers were arranged into 25 linkage groups, 22 of which could be assigned physically to 18 different chromosomes (ECA1, ECA2, ECA3, ECA4, ECA5, ECA6, ECA7, ECA9, ECA10, ECA11, ECA13, ECA15, ECA16, ECA18, ECA19, ECA21, ECA22, and ECA30). The average distance between linked markers was 12.6 cM and the longest linkage group measured 103 cM. The total map distance contained within linkage groups was 679 cM. If the distances covered outside the ends of linkage groups and by unlinked markers were included, it was estimated that the marker set covered at least 1500 cM, that is, at least 50% of the genome. A comparison of the relationship between genetic and physical distances in anchored linkage groups gave ratios of 0.5–0.8 cM per Mb of DNA. This would suggest that the total male recombinational distance in the horse is 2000 cM; this value is lower than that suggested by chiasma counts. The present map should provide an important framework for future genome mapping in the horse.

Abstract —Oxidative processes play an important role in atherogenesis. Because superoxide anion and nitric oxide (NO) are important mediators in vascular pathology, we studied the expression of extracellular superoxide dismutase (EC-SOD) and inducible nitric oxide synthase (iNOS) in human and rabbit atherosclerotic lesions by using simultaneous in situ hybridization and immunocytochemistry and EC-SOD enzyme activity measurements. We also analyzed the presence in the arterial wall of oxidized lipoproteins and peroxynitrite-modified proteins as indicators of oxidative damage and possible mediators in vascular pathology. EC-SOD and iNOS mRNA and protein were expressed in smooth muscle cells and macrophages in early and advanced lesions. The expression of both enzymes was especially prominent in macrophages. As measured by enzyme activity, EC-SOD was the major SOD isoenzyme in the arterial wall. EC-SOD activity was higher in highly cellular rabbit lesions but lower in advanced, connective tissue–rich human lesions. Despite the abundant expression of EC-SOD, malondialdehyde-lysine and hydroxynonenal-lysine epitopes characteristic of oxidized lipoproteins and nitrotyrosine residues characteristic of peroxynitrite-modified proteins were detected in iNOS-positive, macrophage-rich lesions, thus implying that malondialdehyde, hydroxynonenal, and peroxynitrite are important mediators of oxidative damage. We conclude that EC-SOD, iNOS, and the balance between NO and superoxide anion play important roles in atherogenesis. EC-SOD and iNOS are highly expressed in lesion macrophages. High EC-SOD expression in the arterial wall may be required not only to prevent deleterious effects of superoxide anion but also to preserve NO activity and prevent peroxynitrite formation. Modulation of arterial EC-SOD and iNOS activities could provide means to protect arteries against atherosclerotic vascular disease.

Marklund SL, Nilsson P, Israelsson K, Schampi I, Peltonen M, Asplund K (University Hospital, Umea, Sweden). Two variants of extracellular-superoxide dismutase: relationship to cardiovascular risk factors in an unselected middle-aged population. J Intern Med 1997; 242: 5–14. Objective Description of the distribution of plasma levels of two variants of extracellular-superoxide dismutase (EC-SOD) and their relationship to cardiovascular risk factors in the general population. Design A cross-sectional study. Setting Norrbotten and Vasterbotten counties in northern Sweden. Subjects Four thousand, nine hundred 25–74-year-old randomly selected subjects. Main outcome measures Plasma levels of EC-SOD. Genotyping by an allele-specific PCR method. Results Plasma EC-SOD levels showed a distinct bimodal distribution with the smaller group (3.8%) having a variant of the enzyme with about eight-fold-higher plasma levels. Genotyping was performed in 65 individuals with the high-level variant. All but one were found to carry the same mutation, Arg213Gly, affecting the heparin-binding domain of EC-SOD. Subjects with the high-level variant of EC-SOD had modestly higher body mass index and higher levels of serum cholesterol, serum triglycerides and plasma fibrinogen than those with the common EC-SOD phenotype. Within the population with common EC-SOD, the plasma levels were lower in men than in women and increased with age. Low levels of common phenotype EC-SOD were associated with smoking, high plasma levels of fibrinogen and low activity of tissue plasminogen activator in both univariate and multivariate analyses. Obesity and total serum cholesterol were associated with high common phenotype EC-SOD levels. Conclusions A high-level variant of EC-SOD caused by one and the same mutation and with low tissue binding and high plasma levels is present in approximately four per cent of an unselected middle-aged population in northern Sweden. Plasma levels of EC-SOD may be modulated by lifestyle factors such as smoking and show a complex covariation with many of the conventional cardiovascular risk factors.

Article abstract-Mutations of the gene SOD-1, which encodes the enzyme copper-zinc superoxide dismutase, occur in patients with a familial form of amyotrophic lateral sclerosis (ALS). We investigated 71 families with more than one individual affected by ALS for clinical features and SOD-1 mutations. Mutations were identified in 14 families, indicating the presence of SOD-1 mutations in around 20% of this population. There were 10 different heterozygote missense point mutations in eight different codons, and a novel two-base frameshift insertion (132insTT), which leads to substitution of aspartic acid for glutamic acid at codon 132, and a premature stop codon at 133, with predicted truncation of the protein. SOD enzyme activity was reduced to around 50% of normal in individuals with SOD-1 mutations, and may be a useful predictor for the presence of these mutations. A predilection for disease onset in the lower limbs appears to be a distinguishing feature of familial ALS with SOD-1 mutations, and accords with findings in transgenic mouse models. In general, the finding of an SOD-1 mutation does not accurately predict a prognosis or disease severity.NEUROLOGY 1997;48: 746-751

The development of myringosclerosis has been correlated with increased production of oxygen-derived free radicals. For the present study, we used a null mutant mouse lacking extracellular superoxide dismutase to test the hypothesis that increased production of free radicals can cause the development of myringosclerosis. Null mutant mice and wild-type, control mice were myringotomized and kept in ambient air for 3 weeks. Both groups developed myringosclerosis in the pars flaccida, but not in the pars tensa. The sclerotic lesions were visible in both the light and the electron microscope but not in the otomicroscope. In particular, the localization of the sclerotic deposits was found beneath both the inner and outer epidermal epithelium. No difference concerning the extent or number of sclerotic lesions between the null mutant and the wild-type mice could be distinguished.

A simple, reproducible test was used to quantify muscle weakness in mdx mice, an animal model of Duchenne muscular dystrophy. The effect of bedding on wheat kernels and of dietary supplementation of alpha-tocopherol on the progression of muscle weakness was investigated in mdx mice. When measured during the first 200 d of life, mdx mice developed muscle weakness, irrespective of bedding and diet. When kept on wood shavings and fed a conventional rodent diet, mdx mice showed progressive muscle weakness over the consecutive 200 d, and eventually showed a significant weight loss during the next 200-d observation period. Progression of muscle weakness and weight loss were almost completely prevented in mdx mice that were kept on wheat kernel bedding. In contrast, only incomplete maintenance of muscle strength and body weight was observed in mdx mice kept on wood shavings and fed the alpha-tocopherol-supplemented diet. It is concluded from these experiments that a component of wheat kernels other than alpha-tocopherol is essential to prevent the progression of muscle weakness in mdx mice.

Abstract The levels of the secreted, interstitially located extracellular superoxide dismutase (EC-SOD), the cytosolic copper-and-zinc–containing SOD (CuZn-SOD), and the mitochondrial manganese-containing SOD (Mn-SOD) were measured in the walls of human coronary arteries, proximal thoracic aortas, and saphenous veins. The blood vessel walls, particularly the arteries, were found to contain exceptionally large amounts of EC-SOD, whereas the levels of CuZn-SOD and Mn-SOD were relatively low compared with other tissues. Analysis of EC-SOD by immunohistochemistry indicates an even distribution in the vessel wall, including large amounts in the arterial intima. Arterial smooth muscle cells were found to secrete large amounts of EC-SOD and likely are the principal source of the enzyme in the vascular wall. The EC-SOD concentration in the human arterial wall extracellular space is high enough to efficiently suppress the putative pathological effects of the superoxide radical, such as oxidation of LDL and reaction with nitric oxide to form the deleterious peroxynitrite. The levels of EC-SOD in the aortic wall are found to vary widely among species and were on average 6440 U/g in humans, 4340 U/g in the cow, 2660 U/g in the pig, 160 U/g in the dog, 770 U/g in the cat, 2390 U/g in the rabbit, 90 U/g in the rat, and 3400 U/g in the mouse. There were only moderate differences in the amounts of CuZn-SOD and Mn-SOD. This wide variation in EC-SOD content suggests that the susceptibility to pathologies induced by superoxide radicals in the vascular wall interstitium should vary widely among species.

The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PImt) that two maternally unrelated individuals have, by chance, identical SSCP variants varied between 0.14 and 0.30. We detected no heteroplasmy or deviations from strict and stable maternal inheritance when examining four maternal lineages, each represented by six to eight horses, separated by up to five generations from a common ancestral mare. The study establishes a simple screening method for detecting equine mtDNA types, which can be applied for tracing maternal genealogies and for association studies.

Recent reports have shown heterozygosity for some twenty different mutations in the CuZn–superoxide dismutase (CuZn–SOD) gene in familial amyotrophic lateral sclerosis (FALS), and analysed samples from patients have shown decreased enzymic activity. Here we report homozygosity for an exon 4 mutation, Asp90Ala in fourteen patients among four unrelated ALS families and four apparently sporadic ALS patients from Sweden and Finland. The erythrocyte CuZn–SOD activity is essentially normal. Our findings suggest that this CuZn–SOD mutation causes ALS by a gain of function rather than by loss, and that the Asp90Ala mutation is less detrimental than previously reported mutations.

Copper, zinc superoxide dismutase (SOD1) is involved in neutralizing free radicals within cells, and mutant forms of the enzyme have recently been shown to occur in about 20% of familial cases of amyotrophic lateral sclerosis (ALS). To explore the mechanism of SOD1 involvement in ALS, we have analyzed SOD1 in sporadic ALS using activity assays and immunocytochemistry. Analyses of SOD1 activity in washed erythrocytes revealed no difference between 13 ALS cases and 4 controls. Spinal cord sections from 6 ALS cases, 1 primary lateral sclerosis (PLS) case, and 1 control case were stained using three different antibodies to SOD1. Since astrocytes are closely associated with motor neurons, antibodies to glial fibrillary acidic protein (GFAP) and vimentin were used as independent monitors of astrocytes. The principal findings from localizations are: (1) normal motor neurons do not have higher levels of SOD1 than other neurons, (2) there was no detectable difference in SOD1 levels in motor neurons of ALS cases and controls, (3) ALS spinal cord displayed a reduction or absence of SOD1-reactive astrocytes compared to the control and PLS cases, and (4) examination of GFAP-stained sections and morphometry showed that the normal close association between astrocytic processes and motor neuron somata was decreased in the ALS and PLS cases. These results indicate the disease mechanism in sporadic ALS may involve alterations in spinal cord astrocytes.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.

Progressive retinal atrophy (PRA) in dogs, the canine equivalent of retinitis pigmentosa (RP) in humans, is characterised by vision loss due to degeneration of the photoreceptor cells in the retina, eventually leading to complete blindness. It affects more than 100 dog breeds, and is caused by numerous mutations. RP affects 1 in 4000 people in the Western world and 70% of causal mutations remain unknown. Canine diseases are natural models for the study of human diseases and are becoming increasingly useful for the development of therapies in humans. One variant, prcd-PRA, only accounts for a small proportion of PRA cases in the Golden Retriever (GR) breed. Using genome-wide association with 27 cases and 19 controls we identified a novel PRA locus on CFA37 (p(raw) = 1.94×10(-10), p(genome) = 1.0×10(-5)), where a 644 kb region was homozygous within cases. A frameshift mutation was identified in a solute carrier anion exchanger gene (SLC4A3) located within this region. This variant was present in 56% of PRA cases and 87% of obligate carriers, and displayed a recessive mode of inheritance with full penetrance within those lineages in which it segregated. Allele frequencies are approximately 4% in the UK, 6% in Sweden and 2% in France, but the variant has not been found in GRs from the US. A large proportion of cases (approximately 44%) remain unexplained, indicating that PRA in this breed is genetically heterogeneous and caused by at least three mutations. SLC4A3 is important for retinal function and has not previously been associated with spontaneously occurring retinal degenerations in any other species, including humans.

Twenty-one porcine microsatellite markers were developed by screening DNA libraries and by a computer search of databases. The microsatellites were typed in a large three-generation family established by a cross between the European wild pig and a Swedish Yorkshire breed. Linkage analysis benefited from the fact that due to the divergence between the parental populations, the degree of microsatellite polymorphism was significantly higher in the F1 animals than in either of the parental populations. Parallel typing of a set of 35 restriction fragment length polymorphism, protein, and blood group markers rendered it possible to assign as many as 20 of the microsatellites to the porcine linkage map. Fourteen microsatellites were localized to a chromosome segment, whereas six constituted parts of unassigned linkage groups. Analysis of four microsatellites within genes allowed the assignment of the endoplasmic reticulum Ca2+ transport ATPase locus to chromosome 14, the assignment of the interferon-γ and the diacylglycerol kinase loci to a new linkage group (XI), and the localization of the tumor necrosis factor β locus close to the major histocompatibility complex (SLA) on chromosome 7 to be confirmed. Fluorescence in situ hybridization mapping of two microsatellite-containing cosmids assigned two linkage groups to chromosomes 9 and 12, respectively. In total, 27 new markers were added to the porcine linkage map, thereby almost doubling the number of markers on the map. Linkage groups are now present on 10 of 18 of the pig autosomes. On chromosome 6, the halothane (HAL) linkage group was extended to about 50 cM, and a similar length was established for the linkage group LPLS0089-ATP2-PLAU on chromosome 14. The results illustrate the usefulness of microsatellite markers in gene mapping and suggest that a saturated linkage map of the porcine genome will be feasible in the near future.

Plasma selenium and glutathione peroxidase in erythrocytes were analyzed in a case-control study encompassing 441 cases with breast cancer and 191 controls with benign breast disease. No difference in mean serum selenium level between cases and controls on supplementary selenium intake was seen. If only individuals without supplementary intake, 278 cases and 135 controls, were considered a preventive effect was found increasing with selenium level. This finding was significant among women 50 years old or more with Mantel-Haenszel odds ratio = 0.16 for individuals with serum selenium1.21 mumol/L. Also for subjects with serum selenium in the range 1.00-1.21 mumol/L a significant preventive effect was seen with odds ratio = 0.38. For women under 50 years of age a nonsignificant preventive effect was seen. Glutathione peroxidase in erythrocytes did not correlate well with serum selenium and was not a marker for the risk of breast cancer.

A fundamental property of the secretory tetrameric extracellular superoxide dismutase (EC-SOD) is its affinity for heparin and analogues, in vivo, mediating attachment to heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. EC-SOD is in vivo heterogeneous with regard to heparin affinity and can be divided into subclasses; A which lacks heparin affinity, B with intermediate affinity, and C with strong heparin affinity. The EC-SOD C subunits contain 222 amino acids and among the last 20 carboxyl-terminal amino acids, 10 are positively charged and six of these are located in a cluster in positions 210-215. To analyze if this local accumulation of basic amino acids is responsible for heparin binding we produced three series of recombinant EC-SOD (rEC-SOD) variants, six containing amino acid exchanges in the carboxyl-terminal end, four with truncations, and two with both truncations and substitutions. Exchange of positively or negatively charged amino acids on the carboxyl-terminal side of the cluster results in only minor modifications in heparin affinity, whereas substitution of three of the amino acids in the cluster abrogates the heparin binding. Insertions of stop codons at different positions resulted in either C or A but not B class EC-SOD. In an attempt to produce EC-SODs with intermediate heparin affinities, plasmids defining C and A class EC-SOD were cotransfected into Chinese hamster ovary cells. In addition to the parental A and C class EC-SOD forms, two variants with intermediate heparin affinities were formed. Coincubation of EC-SOD C and A resulted in the appearance of one heterotetramer with intermediate affinity for heparin. We conclude that the cluster of six basic amino acids forms the essential part of the heparin-binding domain and that the composition of the four subunits in the EC-SOD tetramer determines the affinity for heparin. This domain is different from heparin-binding domains of other proteins, and its localization allows the distribution of EC-SOD in vivo to be regulated by proteolytic processing.

The efficacy of human extracellular-superoxide dismutase type C (EC-SOD C) to limit infarct size after ischemia and reperfusion was explored and compared to that of EC-SOD C combined with catalase (CAT) and to that of CAT alone. EC-SOD C binds to heparan sulphate proteoglycan on the cell surfaces. Thirty-two pigs were subjected to 45 min of myocardial ischemia followed by 4 h of reperfusion. Control pigs (group A; n = 8) received 300 mL of saline into the great cardiac vein during a 30-min period started 5 min prior to reperfusion; pigs in group B (EC-SOD C; n = 8) got 16.6 mg of EC-SOD C; pigs in group C (EC-SOD C + CAT; n = 8) got 16.6 mg of EC-SOD C together with 150 mg of CAT. Pigs in group D (CAT; n = 8) received 150 mg of CAT. In groups B, C, and D, the drug was dissolved in saline and infused into the great cardiac. Infarct size expressed as percent of area at risk was smaller in groups B (14.5 +/- 16.7%) and C (40.8 +/- 13.3%) than in groups A (78.8 +/- 8.6%) and D (67.2 +/- 18.6%; p less than .05). Creatine kinase (CK) activity in ischemic myocardium was higher in groups B (1740 +/- 548 U/g) and C (1729 +/- 358 U/g) than in groups A (1184 +/- 237 U/g) and D (1251 +/- 434 U/g; p less than .05). There was an inverse relation (r = -.83) between infarct size and CK content. The EC-SOD C infusions resulted in only minimal increases in plasma SOD activities. In conclusion, the presence of SOD on the cell surfaces is of importance in the prevention of reperfusion injury rather than circulating SOD.

Objective: The aim was to determine if endothelium associated extracellular superoxide dismutase type C (EC-SOD C) exerts any protective effect against cardiac damage induced by ischaemia and reperfusion. Methods: Langendorff perfused rat hearts were subjected to 15 min global ischaemia followed by reperfusion. Prior to the ischaemia the hearts were perfused for 15 min with a buffer containing recombinant human EC-SOD C (rh-EC-SOD C, 20 mg·litre−1), or the corresponding vehicle, followed by extensive perfusion with SOD free medium. Results: In hearts receiving the vehicle, reperfusion was associated with a marked release of creatine kinase into the effluent [28 (SEM 1.5) IU·15 min−1, n=5] and coronary flow measured 15 min after initiation of reperfusion was reduced by 68% compared to preischaemic flow. In hearts pretreated with EC-SOD C but washed with enzyme free buffer before being subjected to ischaemia, the creatine kinase release was significantly smaller, at 14(2.1) IUmin−1, n=5 (p

Extracellular-superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme which, in contrast to the intracellular CuZn SOD, has affinity to the endothelium and a long vascular half-life. In the present study, the effects of EC-SOD C and CuZn SOD on reperfusion-induced myocardial damage were determined in rats subjected to 10 min of left coronary artery ligation followed by 24 h of reperfusion. Recombinant human EC-SOD C (rh-EC-SOD C) or the corresponding volume of the vehicle was administered after completion of the coronary ligation. CuZn SOD was given in two equal doses, the first dose directly after ligation and the second one 6 h later. At the end of the reperfusion period the myocardial damage was quantified by measuring the creatine kinase concentration (CK) in the reperfused part of the left ventricular free wall (LVFW), and expressed as a percentage of the concentration in the non-ischemic septum. In the group given the vehicle, 47 +/- 10 (mean +/- SD) of the CK remained in the reperfused LVFW. In the rats receiving rh-EC-SOD C the corresponding values for each dose: 1.4, 4.2 and 12.6 mg/kg were 55 +/- 12 (ns), 55 +/- 12 (ns) and 65 +/- 12% (p less than 0.05, vs. vehicle, Dunnett's multiple comparison test), respectively. Administration of CuZn SOD (2 x 10 mg/kg) resulted in 58 +/- 16% (ns) CK remaining in the LVFW.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative stress is thought to contribute to diabetes-induced cataract, and the authors have previously demonstrated that lenses from mice lacking the antioxidant enzyme copper-zinc superoxide dismutase (SOD1) show elevated levels of superoxide radicals and are more prone in vitro to develop glucose-induced cataract than are wild-type lenses. In the present study the effect of streptozotocin-induced diabetes mellitus on cataract formation in SOD1-null and wild-type mice in vivo was examined.Eight weeks after diabetes was established by repeated intraperitoneal streptozotocin injections, the mice were killed and the lenses removed and photographed in retroillumination. The cataract was quantified from the photographs by digital image analysis and the lens contents of glutathione (GSH) as well as the lens protein carbonyl contents suggestive of protein oxidation were analyzed.The streptozotocin-induced diabetic SOD1-null mice developed more cataract than the diabetic wild-type mice. Also, lens GSH levels were lower in the diabetic SOD1-null mice than in the nondiabetic SOD1-null mice. However, the protein carbonyls were equally raised in the diabetic mice of both genotypes.The increased cataract formation and the compromised antioxidant capacity found in the diabetic SOD1-null lenses thus emphasize the involvement of superoxide radicals in diabetes-induced cataract.

Levels of selenium and mercury in blood and urine were analysed in 37 male workers exposed to elemental mercury vapour in a chloralkali plant and in 39 unexposed controls of the same age. Mean urinary Hg was 223 nmol l-1 (15 nmol/mmol creatinine) in the exposed group and 26 nmol l-1 (2.0 nmol/mmol creatinine) in the controls. Mean blood and plasma Hg levels were 46 and 36 nmol l-1, respectively, in the exposed group, as compared with 17 and 7 nmol l-1 in the controls. The concentrations of Se in plasma and erythrocytes did not differ between the two groups. Urinary Se levels were, however, slightly but significantly lower in the exposed group (median values 23 vs 29 nmol/mmol creatinine), and there was a negative correlation between urinary Se and plasma Hg in the exposed group. This may be due to a retention of Se in the kidneys. In a subgroup of exposed workers and controls, glutathione peroxidase, superoxide dismutase and catalase were also analysed. No differences were found between the groups with respect to these antioxidative enzymes. The effect on Se status of moderate Hg exposure seems to be of minor clinical importance.

Amalgam restorations were inserted in eight healthy persons, previously unprovided with dental restorations, who had several severe carious lesions. The mean number of surfaces restored were 16.1 (range, 11 to 22). The total mean calculated amount of mercury inserted was 2.9 g (range, 1.5 to 4.3 g). Blood and urinary levels were measured on seven occasions during a 4-month period before and a 3-month period after amalgam placement. One and 3 months after placement, the P-mercury mean values were almost equal to the preplacement values (3.3 nmol/l). After placement U-mercury increased continuously; 3 months after placement a statistically significantly higher (p less than 0.05) mean U-mercury value (0.58 nmol/mmol creatinine) was found compared with the mean preplacement value (0.34 nmol/mmol creatinine). No statistically significant correlation was found between the P- and U-mercury concentrations and the total number of amalgam surfaces. Selenium levels in plasma and urine and erythrocyte glutathione peroxidase showed no systematic change of pattern. The results show that the insertion of amalgam fillings contributed to the U-mercury concentration, but apparently even more extensive amalgam therapy and/or longer exposure periods are needed to affect the P-mercury concentration. No negative effects on the P- and U-selenium or the erythrocyte glutathione peroxidase levels could be found during the 3 months immediately after an extensive amalgam placement. The supplementary blood and urine analyses were not influenced by the insertion of amalgam fillings.

In 10 healthy persons all amalgam fillings were replaced with gold inlays. Blood and urinary levels were measured on 10 occasions during a 4-month period before and a 12-month period after amalgam removal. These variables were also measured three times in 10 healthy controls. A strong statistically significant relation was found between plasma mercury values and both the total number of amalgam surfaces (r = 0.71, p = 0.0006) and the total surface area of the fillings (r = 0.73, p = 0.0004). In the immediate postremoval phase plasma mercury rose three- to four-fold, whereas the urinary and erythrocyte mercury rose about 50%. These peak values declined to the preremoval level at about 1 month. Twelve months after the removal the plasma and urinary mercury levels were significantly reduced to 50% and 25%, respectively, of the initial values for the experimental group. Apart from the significantly lower plasma selenium values 5 and 10 days after removal no significant differences were found with regard to plasma selenium or erythrocyte glutathione peroxidase either within or between the experimental and the control groups. A large number of supplementary biochemical analyses did not show any influence on organ functions or any differences between the groups before or after the amalgam removal. Amalgam fillings considerably contributed to the plasma and urinary mercury levels.

Publisher Summary This chapter discusses the analysis of extracellular superoxide dismutase (EC-SOD) in tissue homogenates and extracellular fluids. The analysis distinguishes between EC-SOD and other SOD isoenzymes. A prominent feature of EC-SOD is its affinity for heparin. On chromatography on heparin–Sepharose, plasma EC-SOD from man, pig, cat, mouse, guinea pig, and rabbit s can be divided into at least three fractions: (1) a fraction without weak heparin affinity, (2) a fraction with weak heparin affinity, and (3) a fraction that elutes relatively late in a NaCl gradient. EC-SOD from tissues is mainly composed of forms with high heparin affinity. In rat plasma, however, only fractions A and B can be demonstrated. The binding to heparin is of electrostatic nature. As EC-SOD carries a net negative charge at neutral pH, the binding to the strongly negatively charged heparin molecule must be mediated by a cluster of positively charged amino acid residues in the enzyme. Such a cluster occurs in the very hydrophilic carboxy-terminal end of EC-SOD 3, which contains three lysines and six arginine residues among the last 20 amino acids. The differences between EC-SOD 1, 2, and 3 probably reside in this region.

Extracellular-superoxide dismutase (EC 1.15.1.1, EC-SOD) is a secretory, tetrameric, Cu and Zn-containing glycoprotein with a subunit molecular weight of about 30 kDa.1,2 EC-SOD is the major SOD isoenzyme in extracellular fluids.3,4 Although it is the least predominant SOD isoenzyme in the tissues, 90 to 99 % of the EC-SOD in the bogy of mammals is located to the extravascular space of tissues.5,6

BACKGROUND: We have developed a formulation of polycationic liposomes that has been shown to be non-toxic, highly stable in the bloodsteam, and very effective in liver gene transfer in mice (Gene Therapy 2003; 10:180–187). Extracellular superoxide dismutase (EC-SOD) inhibits the generation of superoxide anions in the interstitial space of tissues, and may play a role in minimizing oxidative stress in liver injury. The aim of the present study is to investigate whether our polycationic liposome-mediated human EC-SOD gene delivery will protect against lipopolysaccharide (LPS)-induced liver toxicity in D-galactosamine (GalN)-sensitized mice. METHODS: Polycationic liposomes were generated from polycationic lipid (PCL) and cholesterol (Chol). Lipoplexes were formed by complexing liposomes with control plasmid (pEGFP-C1) or EC-SOD plasmid (pEGFP-C1-ECSOD) before use. Mice were injected with thyroid hormone (T3, 4 mg/kg, s.c.) two days before lipoplexes were injected via the portal vein. One day following lipoplex injection, mice were treated with GalN (500 mg/kg, i.p.) plus LPS (25 mg/kg, i.p.). Serum alanine aminotransferase (ALT), SOD activity, liver histology, glutathione (GSH) content and lipid peroxidation were evaluated one day after GalN/LPS exposure. Human EC-SOD gene expression in mouse liver tissue was determined by quantitative RT-PCR two days after lipoplex injection. RESULTS: somes and the control lipoplexes were stable in their size for three months at 4°C. Injection of T3, PCL-Chol liposomes or control lipoplexes did not cause a significant change in serum ALT levels. Real time quantitative RT-PCR analysis showed that human EC-SOD mRNA levels in mouse liver tissue in EC-SOD lipoplex-injected group were 55-fold higher than saline, liposome or control lipoplex controls when mouse -actin was employed as a house-keeping control gene. Serum ALT levels in animals receiving portal vein injections of EC-SOD lipoplexes plus GalN/LPS exposure were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes plus GalN/LPS exposure (62583 vs. 150865, 153194, 1880520 units/ml, p