Amelioration of antigen-induced arthritis in rats by transfer of extracellular superoxide dismutase and catalase genes.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), while antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD) and catalase, block radical-induced events. The present study tested if the ex vivo transfer of EC-SOD and catalase genes alone or in combination in the knee joint of rats with monoarticular antigen-induced arthritis (AIA) was anti-inflammatory, and examined the potential mechanisms involved. Synoviocytes isolated from female Wistar rats were immortalized with a retroviral vector SUV19.5. These cells were permanently transfected with an EC-SOD expression plasmid (pEC-SODZeo) or a catalase expression plasmid (pCatalaseZeo) to create cells overexpressing EC-SOD or catalase, as measured by RT-PCR and Western blots. The cells were engrafted in knee joints of animals at the time of the induction of AIA. Three gene transfer groups, an EC-SOD group, a catalase group and a combined therapy group (EC-SOD and catalase) were included in these experiments. Animals in the control group were engrafted with synoviocytes transfected with the plasmid pZeoSV2 without an insert. Clinical and histological assessments were performed, as well as tissue measurements of SOD, catalase and gelatinase activities. Ex vivo gene transfer of EC-SOD and catalase into rat knee joints produced about a six- to seven-fold increase in EC-SOD activity and a two- to three-fold increase in catalase activity compared with the control animals. Rats treated with cells overexpressing EC-SOD, catalase or a combination of EC-SOD and catalase showed significant suppression of knee joint swelling, decreased infiltration of inflammatory cells within the synovial membrane and reduced gelatinase activity in knee joints, compared with animals receiving cells transfected with the plasmid alone. No statistically significant difference was found between the groups treated with cells overexpressing EC-SOD, catalase or a combination of both. Gene therapy involving the local intra-articular overexpression of two antioxidant enzymes, EC-SOD and catalase, was anti-inflammatory in AIA. One mechanism appears to be the suppression of gelatinase activities by both EC-SOD and catalase.

Chronic cobalt exposure affects antioxidants and ATP production in rat myocardium.

Chronic cobalt exposure is characterized by severe cardiac insufficiency. Since the mechanisms of cobalt toxicity are not yet clear, we analysed the effects of chronic cobalt exposure on antioxidant enzyme activities and myocardial mitochondrial ATP production rate in a rat model. One group of rats was fed a conventional diet and another a cobalt supplemented diet for 24 weeks. The manganese-superoxide dismutase activity was markedly reduced in the cobalt rats (18+/-4.7 U/mg protein) compared to the control rats (100+/-22 U/mg protein; p <0.001). Activity in the respiratory chain enzymes succinate-cytochrome c reductase, NADH-cytochrome c reductase and cytochrome c oxidase was also reduced in the cobalt rats (p<0.01). Glutamate dehydrogenase activity, located in the mitochondrial matrix, was unchanged. The mitochondrial ATP production rate in relation to myocardial mass was lower in the cobalt rats for all substrates tested except palmitoyl-l-carnitine + malate. In conclusion, 24 weeks of chronic cobalt exposure induces a marked decrease in manganese-superoxide dismutase activity, a moderate decrease in mitochondrial ATP production rate and a general reduction in the capacity of the respiratory chain. The impairment in mitochondrial ATP production might be secondary to the decreased manganese-superoxide dismutase activity, causing inactivation of mitochondrial factors susceptible to superoxide radicals.

Wheat kernel ingestion protects from progression of muscle weakness in mdx mice, an animal model of Duchenne muscular dystrophy.

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.

Levels of selenium in plasma and glutathione peroxidase in erythrocytes in patients with prostate cancer or benign hyperplasia.

Plasma selenium and glutathione peroxidase in erythrocytes were analysed in a case-control study encompassing 164 cases with prostate cancer and 152 controls with benign prostate hyperplasia. Plasma selenium levels were divided into three groups; I > 1.17, II 1.00-1.17 and III < 1.00 mumol/l. For the 124 cases with no supplementary intake of selenium pills, the mean plasma selenium level was 0.99 (range 0.27-1.47) and for the corresponding 121 controls 1.08 (range 0.52-1.50) mumol/l, a difference which was significant (P = 0.0007). The three categories of selenium levels had odds ratios (OR) of 0.3 and a 95% confidence interval (CI) of 0.1-0.7 for group I, an OR of 0.6 and a CI of 0.3-1.1 for group II, and group III was used as the reference entity. No significant differences in levels of glutathione peroxidase in erythrocytes were found between cases and controls.

Pharmacokinetics of extracellular-superoxide dismutase in the vascular system.

Extracellular-superoxide dismutase C (EC-SOD C) is a secretory tetrameric Cu- and Zn-containing glycoprotein which has high affinity for heparin and heparan sulfate. Upon intravenous injection into rabbits, recombinant human (rh) EC-SOD C was found to be rapidly 97-98% sequestered to the vascular wall, forming an equilibrium with the plasma phase. Recombinant EC-SOD truncation variants with reduced, T216, and without, T213, heparin affinity were found to be sequestered to a reduced extent and not at all, respectively, establishing the importance of the heparin affinity for this behaviour. The halflife of rhEC-SOD C in the vasculature was of the order of 20 h. Injection of large doses resulted in saturation of the binding of rhEC-SOD C to the vascular wall. Scatchard analysis revealed a heterogeneity in affinity of the ligands on the vascular wall. The maximal binding capacity was very high. The equilibration of rhEC-SOD C to the vascular wall of an organ, clamped during enzyme injection, and the primary equilibration phase was studied by comparing binding to a clamped and reperfused kidney with binding to the contralateral control kidney. rhEC-SOD C injected in a low dose was found to equilibrate very slowly to the reperfused kidney with a halftime of about 2 h. With higher rhEC-SOD C doses, at which evidence for saturation is seen, and with the variant rhEC-SOD with reduced heparin affinity. T216, very rapid equilibrations were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of selenium in plasma and glutathione peroxidase in erythrocytes and the risk of breast cancer. A case-control study.

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 selenium > 1.21 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.

Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.

Levels of selenium and antioxidative enzymes following occupational exposure to inorganic mercury.

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.

Effects of variation in glutathione peroxidase activity on DNA damage and cell survival in human cells exposed to hydrogen peroxide and t-butyl hydroperoxide.

The selenium-dependent glutathione peroxidase activities of two human cell lines, the colon carcinoma HT29 and the mesothelioma P31, cultured in medium containing 2% serum, increased from 195 to 541 and from 94 to 361 units/mg of protein respectively after supplementation with 100 nM-selenite. The catalase activity remained unchanged by this treatment. The effects of the obtained variation in glutathione peroxidase activities were investigated by exposing cells to H2O2 and t-butyl hydroperoxide. Selenite supplementation resulted in a decrease in H2O2-induced DNA single-strand breaks in both HT29 and P31 cells. A small, but significant, decrease in the number of DNA single-strand breaks for low doses (10-50 microM) of t-butyl hydroperoxide was found only in P31 cells and not in HT29 cells. We could detect neither induction of double-strand breaks (detection limit approx. 1000 breaks per cell) nor DNA-protein cross-links after exposing the cells to the two peroxides. In spite of the apparent protective effect of increased glutathione peroxidase activity on DNA single-strand break formation, there were no differences between selenite-supplemented and non-supplemented cells in cell survival after exposure to peroxide.

The influence of dental amalgam placement on mercury, selenium, and glutathione peroxidase in man.

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.

Mercury, selenium, and glutathione peroxidase before and after amalgam removal in man.

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.

Mercury, selenium, and glutathione peroxidase in dental personnel.

Eighteen persons, dentists and nurses, with urinary mercury levels higher than the group median value of all dental personnel in the country of Västerbotten were compared with a group consisting of 15 persons with low urinary mercury levels working in the same clinics. A statistically significant difference between the high urinary mercury group and the low urinary mercury group could be seen in the plasma mercury level. In each group a statistically significant relation could be seen between the plasma mercury level and the total number of amalgam surfaces. The two groups did not differ with regard to the levels of plasma selenium and erythrocyte glutathione peroxidase, and no correlation between these two variables and the plasma mercury levels could be found. To evaluate organ functions, a large number of supplementary analyses were performed. These analyses did not indicate any influence on organ functions. Although the persons in the present study were occupationally exposed to mercury, none of the biologic variables analyzed seemed to be affected. Even among dental personnel who handle amalgam professionally the number of amalgam surfaces is a major contributory factor to the P-mercury level.

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage.

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.

Some aspects of antioxidant status in blood from alcoholics.

The effect of ethanol consumption on serum concentration of alpha-tocopherol, erythrocyte activities of superoxide dismutase, glutathione peroxidase, and catalase were studied in 34 male alcoholics and 35 age-matched controls. Serum concentration of alpha-tocopherol was 30% lower in the alcoholics as compared to the controls (p less than 0.001). No significant difference was found in erythrocyte activities of Cu-Zn-containing superoxide dismutase, glutathione peroxidase, or catalase between the groups. Of the 12 alcoholics with subnormal serum alpha-tocopherol, 50% had concomitant neurological clinical scores and cerebellar atrophy, and their neurological scores were significantly higher (82%) than for alcoholics with normal alpha-tocopherol levels (p less than 0.03). However, no significant correlation was observed between levels of alpha-tocopherol and neurological clinical scores or cerebellar atrophy. When entering the study, alcoholics and controls were each randomized into two separate groups, receiving vitamin E supplementation (100 mg/day) or placebo capsules for 10 days, respectively. In the four subgroups, alpha-tocopherol levels increased only in alcoholics receiving vitamin E supplementation (23%) (p less than 0.001). The reduced serum levels of alpha-tocopherol in alcoholics may be normalized by vitamin E supplementation.

Heparin-, dextran sulfate- and protamine-induced release of extracellular-superoxide dismutase to plasma in pigs.

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomena in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. When given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.

Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product.

A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1), has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.

Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation.

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300-1000 nM. Growth-retarding effects of selenite became apparent at 300-2000 nM, showing a large cell line variation. Supplementation with 50-100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.