Bovine Superoxide Dismutase Research Articles

Possible role of regional superoxide dismutase activity and lipid peroxide levels in cadmium neurotoxicity: in vivo and in vitro studies in growing rats

Cd 2+ (0.4 mg/kg) administration to growing rats (45 ± 5 g) intraperitoneally, daily for 30 days was found to decrease the activity of superoxide dismutase (SOD) in all the brain regions, except hippocampus. The concentration of lipid peroxides were significantly elevated in the cerebellum, cerebral cortex, corpus striatum and midbrain. A 100% inhibition in SOD activity was observed by 14 μM and 50 μM of Cd 2+ in bovine blood and rat brain preparations, respectively. Cadmium-induced strong inhibitory effect on brain and purified bovine blood SOD suggested a direct effect of the metal on enzyme molecule. Furthermore, in vitro addition of a wide range of Cd 2+ (1–100 μM) increased the rate of lipid peroxidation (LPO) reaction in fresh brain homogenate, however, did not affect boiled homogenate. The studies on LPO in reconstituted homogenate resulting from mixing of fresh and/or heated different subcellular fractions indicated the presence of some heat-labile Cd 2+-sensitive factor in 15000 × g pellet fraction. It is suggested that Cd 2+ directly and indirectly through inhibition of SOD, increases the LPO of cell membranes and thus produces damage to the associated physiological functions leading to central nervous dysfunctions.

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.

Administration of bovine superoxide dismutase fails to prevent chronic pulmonary sequelae of neonatal oxygen exposure in the rat

Using a rat model, we assessed the efficacy of varying doses of superoxide dismutase of (SOD) to affect plasma and tissue SOD concentrations and to attenuate dysplastic changes of lung and pulmonary vascular growth, which are chronic sequelae of neonatal oxygen exposure. One hundred forty-three 1-day-old Sprague-Dawley rats were divided into two groups and exposed to hyperoxia (0.96 to 1.0 Fio2) or room air for 8 postnatal days. Each group was subdivided into five treatment groups, which received 6, 20, 100, or 200 mg/kg/d SOD or a placebo, intramuscularly every 12 hours. All rats were then placed in room air; 52 were killed, and lung tissue and blood samples were obtained for measurement of bovine SOD concentration. The remaining rats received routine care until 58 to 60 days of age, when functional and morphologic cardiopulmonary changes were assessed. Bovine SOD concentration of pooled plasma samples increased 26-fold, from 2 to 50 micrograms/mL, between the 6 and 200 mg/kg/d SOD groups, but mean tissue concentration increased only six-fold, from 0.34 to 2.1 micrograms/lung. Cardiovascular and pulmonary changes found in each oxygen group, regardless of SOD dosage, included elevated right ventricular pressure, increased right ventricular weight, decreased number of small pulmonary arteries/mm2, decreased number of alveoli/mm2, and increased volume proportion of lung parenchyma. Thus, high plasma concentrations of bovine SOD failed to prevent the chronic cardiovascular and pulmonary sequelae of neonatal oxygen exposure in the rat, possibly because SOD did not reach the intracellular sites of action.

Comparison of the protective effects by human and bovine superoxide dismutase against ischemia- and reperfusion-induced impairment of kidney function in anesthetized rats

In a rat model of ischemia- and reperfusion-induced kidney damage, the protective effects of human superoxide dismutase produced by genetic technology (hum-SOD) were compared to those of bovine superoxide dismutase (bov-SOD). The intravenous infusion of hum-SOD and bov-SOD, started concomitantly with the kidney reperfusion after a 60-min (or 30-min) period of ischemia, significantly improved the renal function (inulin and p-amino-hippuric acid clearance rates) as compared to the vehicle-treated control group. In contrast, inactive apoenzyme of superoxide dismutase (Apo-SOD) did not improve the impaired renal function after the kidney reperfusion. Therefore, the kidney protection by hum-SOD and bov-SOD may reasonably be ascribed to their specific enzymatic function—scavenging of oxygen radicals. In this respect, hum-SOD proved to be as effective as bov-SOD. To our knowledge this is the first report on a direct pharmacologic comparison of superoxide dismutases from natural and recombinant origin.

Heat stability of superoxide dismutase in cabbage

The occurrence of superoxide dismutase activity (SOD) in extracts of cabbage ( Brassica oleracea var. capitata) was detected by use of the nitroblue tetrazolium assay method for the measurement of superoxide anion (O − 2 ). The SOD activity found in aqueous extracts of cabbage was destroyed at temperatures greater than 45°C, whereas, for commercial preparations of bovine SOD, temperatures in excess of 65°C were required for deactivation of the enzyme. Heat inactivation energies have been calculated. Tests for sensitivity to cyanide have indicated that a substantial proportion of the cabbage SOD activity is due to the CuZn type of enzyme. As shown by isoelectric focusing, three SOD isoenzymes were present in the aqueous extracts of cabbage.

Effects of liposomal superoxide dismutase on human neutrophil activity.

Study of the effects of liposomal bovine copper superoxide dismutase on human polymorphonuclear neutrophils with respect to production of active oxygen species, chemotaxis and random migration, or bacterial killing show that no significant interference with neutrophil function is observed at levels far exceeding the clinical doses used in the treatment of various pathologies.

Treatment of radiofibrosis with liposomal superoxide dismutase. Preliminary results of 50 cases.

Well and long established radio-fibroses have been treated successfully with a liposomal encapsulated bovine copper superoxide dismutase. After a short treatment (three weeks intramuscular injection of 5 mg twice a week) regression of the fibrosis is stable. The average size is reduced by one third and significant softening occurs in 82% of the cases. Efficiency is independent of the time between radiotherapy (origin of the fibrosis) and treatment with liposomal SOD. Complete regression even after this limited treatment is seen in cases of chronic prefibrotic inflammatory syndromes and prophylactic action in cases where the probability of fibrosis formation is certain appears to be successful. The roles of superoxide and superoxide dismutase are discussed.

Oxidant-mediated lung disease in newborn infants

High concentrations of oxygen are administered with increased airway pressure to most preterm neonates with respiratory distress syndrome (RDS). Among 20% to 30% of survivors a form of chronic lung disease, bronchopulmonary dysplasia (BPD), develops. Its pathogenesis may include tissue damage caused by the superoxide anion (O 2 −) and other free oxygen radicals. Animal experiments and other data suggested a rationale for superoxide dismutase (SOD) administration in an effort to prevent or ameliorate BPD. Our preliminary studies in 19 prematures with RDS demonstrated its safety in human newborns and permitted measurement of its plasma levels. No adverse clinical findings occurred, and laboratory parameters were unchanged. Subcutaneous administration (0.25 mg/kg) of bovine SOD led to detectable levels at 1 1/2 h (mean 0.22 μg/ml), with a slight rise to a higher peak at 2 1/2–4 h and a plateau over the remainder of the 12-h interval. Following doses 2-5, peak levels of 0.64 μg/ml occurred at 4–8 h. With this background, a prospective double-blind controlled study of 45 neonates (mean gestational age, 29 weeks; birth weight, 1,100 g) showed a statistically significant reduction in prevalence of clinical and X-ray signs of BPD with fewer days of continuous positive airway pressure required. The safety and pharmacokinetics of bovine SOD were confirmed.

Structural analyses of various Cu 2+, Zn 2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

The thermal denaturation profile of the Cu 2+, Zn 2+ metalloenzyme, bovine Superoxide dismutase, consists of two primary components, the major component denatures irreversibly at T m = 104 °C with a total enthalpy (Δ H cal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers T m to 96 °C and Δ H cal to 6.96 cal/ g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 °C with an enthalpy only one-half that of the holo-form. The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme. Reconstitution with Zn 2+, Cu 2+, or Zn 2+ and Cu 2+ raises T m to 80, 89, or 102 °C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu 2+ alone is greater than that of Zn 2+, although both are needed for full stability. Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization. The value of T m of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II). The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly. Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site.

Primary structure of copper-zinc superoxide dismutase from Neurospora crassa.

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.

Serum lipid peroxide level and blood superoxide dismutase activity in workers with occupational exposure to lead.

We studied whether lead exposure increased the serum lipid peroxide (LPO) level and inhibited blood superoxide dismutase (SOD) activity in workers with occupational exposure to lead and rats injected with lead. We examined the following subjects: (1) manual workers (712 males) from 18 to 59-years-old in steel production with occupational exposure to lead, (2) office workers (155 males) without exposure to lead, (3) rats subcutaneously injected with lead in concentrations of 10 or 20 mg/kg as lead acetate. The nutritional intakes of manual workers and office workers were approximately equal. Serum LPO and high-density lipoprotein cholesterol (HDL-CL) levels in manual workers (LPO: 4.4 +/- 1.9 nmol/ml, HDL-CL: 55.6 +/- 14.2 mg/dl) were significantly higher than those in office workers (LPO: 4.0 +/- 1.4 nmol/ml, HDL-CL: 53.0 +/- 13.9 mg/dl). Serum LPO level in the manual workers increased with an increase of the lead concentration in the blood, while blood SOD activity decreased. Similar phenomena were observed in rats subcutaneously injected with lead acetate. Furthermore, the addition of lead at higher than 20-microM concentrations to non-treated rats liver microsomes increased NADPH-dependent liquid peroxidation, and these lead concentrations inhibited bovine erythrocyte SOD activity in vitro assay system. In conclusion, the present results seem to indicate that the increase of serum LPO level in workers with occupational exposure to lead is due not only to the stimulation of lipid peroxidation, but also to the inhibition of SOD activity by exposure to lead in the manufacturing processes.

Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome. by rosenfeld w, evans h, concepcion l, jhaveri r, schaeffer h, and friedman a. j pediatr 1984; 105:781–785

Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome. by rosenfeld w, evans h, concepcion l, jhaveri r, schaeffer h, and friedman a. j pediatr 1984; 105:781–785

Prevention of bronchopulmonary dysplasia by administration of bovine superoxide dismutase in preterm infants with respiratory distress syndrome

The effectiveness of bovine superoxide dismutase (SOD) in the prevention of bronchopulmonary dysplasia was evaluated in a prospective double-blind controlled study in 45 neonates (mean gestational age 28.7 weeks, mean weight 1154 gm) with severe respiratory distress syndrome. All were ventilator dependent with FiO2 greater than or equal to 0.7 at 24 hours of age. Either bovine SOD (0.25 mg/kg) or saline solution was administered subcutaneously every 12 hours according to random selection until patients could be maintained in room air without ventilatory or continuous positive airway pressure (CPAP) support. SOD levels were detected in all patients given treatment. Mean peak values at 4 hours after dose ranged from 0.15 micrograms/ml (dose 1) to 0.45 micrograms/ml (dose 10). The drug was well tolerated, and no side effects were detected. Among the 31 survivors (SOD 14, placebo 17) radiologic evidence of BPD was significantly less in patients given SOD (3/14 vs 12/17, P = 0.008). Clinical signs of bronchopulmonary dysplasia (wheezing, pneumonia) were less in patients given SOD (3/14 vs 11/17, P = 0.019). Patients given SOD required fewer days of CPAP (P less than 0.003). There were no differences in days of O2 therapy, intermittent positive pressure breathing, or incidence and severity of patent ductus arteriosus or intraventricular hemorrhage. This preliminary study suggests that SOD may be helpful in reducing the severity of bronchopulmonary dysplasia in infants with respiratory distress syndrome.

Pharmacokinetic and anti-inflammatory properties in the rat of superoxide dismutases (Cu SODs and Mn SOD) from various species

The comparison of the anti-inflammatory properties (carrageenan paw edema in the rat) of exogenous Cu SODs from various species and human Mn SOD administered at doses corresponding to clinical schedules, shows that human and bovine Cu SOD are fully active, whereas rat Cu SOD and Mn SOD are inefficient. This difference does not correspond to the similarity of pharmacokinetic properties (blood levels, subcellar location in kidney cortex) of the Cu proteins or to the increased circulating life time of Mn SOD. The pharmacological activity of exogenous SOD is limited to heterologous Cu SODs in the rat. A similar problem may occur in man. The true mechanism of action Cu SOD remains to be elucidated.

An extended-X-ray-absorption-fine-structure study of bovine erythrocyte superoxide dismutase in aqueous solution. Direct evidence for three-co-ordinate Cu(I) in reduced enzyme.

Copper and zinc K-edge e.x.a.f.s. (extended X-ray-absorption fine structures) were measured for the metal sites of oxidized and reduced bovine superoxide dismutase in aqueous solution. Detailed analysis of the spectra indicates that the copper site of the enzyme changes on reduction and is most probably co-ordinated to three imidazole groups at a shorter distance Cu-N(alpha) = 0.194 nm (1.94 A) in the reduced form compared with a co-ordination of four imidazole groups at 0.199 nm (1.99 A) and an oxygen atom from solvent water at 0.224 nm (2.24 A) in the oxidized form. Examination of the edge, near-edge structure and e.x.a.f.s. of the zinc sites indicates that the stereochemical changes at copper that accompany reduction introduce minimal perturbation on the stereochemistry at zinc.

PREVENTION OF BRONCHOPULMONARY DYSPLASIA (BPD) BY ADMINISTRATION SUPEROXDIE (SOD)

The effectiveness of bovine SOD in the prevention of BPD was evaluated in a prospective double blind controlled study of 45 neonates (m GA 28.7wks; m b.wt. 1154gms) with severe IRDS. All were ventilator dependent with FiO2 ≥ 0.7 at 24 hours of age. Either bovine SOD (0.25mg/kg) or placebo (saline) was administered SC q12h according to random selection until patients could be maintained in room air without ventilatory or CPAP support.Twenty-one patients (m GA 28.8wks; m b.wt. 1156gms) received SOD and 24 (m GA 28.5wks; m b.wt. 1153gms) received placebo. 14/21 SOD and 17/24 placebo patients survived (p=NS). SOD levels were detected in all treated cases. Mean peak values at 4 hrs. p dose ranged from 0.15ugm/ml (dose #1) to 0.45ugm/ml (dose #10).Among survivors evidence of BPD on xray (hyperaeration and/or atelectasis) was significantly less in SOD patients (3/14 vs 12/17; p < 0.02). Clinical signs of BPD (wheezing, pneumonia) were less in SOD patients (3/14 vs 12/17; p < 0.02). SOD patients required fewer days of CPAP (p < 0.03). There were no differences in days of O2, IPPB, incidence and severity of PDA and IVH. SOD administration was safe and well tolerated.SOD reduced the severity of BPD using clinical and radiologic criteria. This extends our earlier observations (SPR 407A, 1983) and suggests the need to investigate variation in dosage schedule, and combined treatment with other antioxidant agents.

Investigation of zinc-deprived bovine superoxide dismutase

Zinc-deprived bovine superoxide dismutase and its adducts with azide and thiocyanate ions have been investigated through water 1H NMR relaxation measurements. The affinity constants of the anions for the modified protein have been determined and compared with those for the native enzyme. The results suggest that a histidine different from the bridging one is displaced upon anion coordination.

Pharmacology of free radicals; Recent views on their relation to inflammatory mechanisms

Production of free radicals from molecular oxygen during the inflammatory process can exhibit beneficial effects against the phlogistic stimulus and may act as a defence mechanism. Nevertheless, in many cases this production is associated with toxic reactions related to inflammatory response. Many compounds including bovine superoxide dismutase, non steroidal anti-inflammatory drugs, radical scavengers (e.g. acetaminophen), corticoids etc., have been shown to counteract this phenomenon. Their beneficial effects and mechanism of action are reviewed.

Erratum: Malaria Parasites Adopt Host Cell Superoxide Dismutase

In the report "Malaria parasites adopt host cell superoxide dismutase" by A. S. Fairchild et al. (19 Aug., p. 764), the caption for figure 2 on page 765 was incorrect and should read as follows: "Comparison of host and parasite SOD's ( 9 ) by (A) polyacrylamide gel electrophoresis ( 8 ) and (B) isoelectric focusing ( 7 ). Gels were stained for SOD activity ( 10 ), and bovine erythrocyte SOD (Sigma, type 1) was used as a reference [ p I = 4.95 ( 13 )]. Lane 1, rat-derived P. berghei SOD; lane 2, rat erythrocyte SOD ( p I = 5.1); lane 3, mouse-derived P. berghei SOD; lane 4, mouse erythrocyte SOD ( p I = 5.7); lane 5, bovine erythrocyte SOD." The p I's of mouse and rat erythrocyte SOD's cited in the text should also be corrected to the values given above.

Dissolution of Kroc Foundation

In the report "Malaria parasites adopt host cell superoxide dismutase" by A. S. Fairchild et al. (19 Aug., p. 764), the caption for figure 2 on page 765 was incorrect and should read as follows: "Comparison of host and parasite SOD's (9) by (A) polyacrylamide gel electrophoresis (8) and (B) isoelecteric focusing (7). Gels were stained for SOD activity (10), and bovine erythrocyte SOD (Sigma, type 1) was used as a reference [pI = 4.95 (13)]. Lane 1, rat-derived P. berghei SOD; lane 2, rat erythrocyte SOD (pI = 5.1); lane 3, mouse-derived P. berghei SOD; lane 4, mouse erythrocyte SOD (pl = 5.7); lane 5, bovine erythrocyte SOD." The pI's of mouse and rat erythrocyte SOD's cited in the text should also be corrected to the values given above.