Catalase Treatment Research Articles

Genotoxic effects of paraquat and diquat evaluated by sister-chromatid exchange, chromosomal aberration and cell-cycle rate

We studied the induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations in cultured mammalian cells exposed to paraquat or diquat in order to elucidate the genotoxic effects of these two common herbicides. The effects of paraquat and diquat were studied at various concentrations with special attention given to concentrations that are thought to be non-toxic. With increasing concentrations of paraquat and diquat (0.08–20.0 μ m), increasing frequencies of SCEs were found in Chinese hamster lung cells. The effects of the herbicides on the cell-cycle rate were also studied: the cycle rate was stimulated at low concentrations but inhibited at higher concentrations. There was no significant induction of chromosomal aberrations at herbicide concentrations that stimulated the cell-cycle rate. However, at concentrations found to inhibit the cell-cycle rate, an increased number of chromatid gaps and breaks were observed, especially after long incubation periods, although no chromatid exchanges were noted during our experiments. These results show that the genotoxic effects of paraquat and diquat occur not only at high concentrations, but also at concentrations low enough to stimulate the cell-cycle rate. The role of superoxide anions and/or hydrogen peroxide, produced by herbicide action, in the induction of sister-chromatid exchanges and chromosomal aberrations was demonstrated by further studies using catalase and/or superoxide dismutase treatment.

"Reperfusion injury" by oxygen-derived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow.

Do oxygen-derived free radicals, generated at the time of reperfusion, lethally injure viable, previously ischemic myocardium, damage vascular endothelium, and impair recovery of postischemic contractile function? To address these issues, 23 anesthetized open-chest dogs underwent 2 hours of left anterior descending coronary artery occlusion followed by 4 hours of reperfusion. Immediately prior to reflow, each dog was randomized to receive either the free radical scavenging agents superoxide dismutase (SOD) + catalase, or saline alone. SOD + catalase had no significant beneficial effect on infarct size measured by triphenyltetrazolium staining: area of necrosis averaged 38.5 +/- 6.1% vs. 46.3 +/- 6.2% of the area at risk in treated compared with control animals respectively (p = NS). Furthermore, infusion of SOD + catalase did not alter contractile function of the viable subepicardium: mean segment shortening (measured using sonomicrometry) at 4 hours postreperfusion was -23 +/- 5% of baseline, preocclusion values in controls dogs and -24 +/- 9% of preocclusion values in animals that received the scavenging agents. However, SOD + catalase treatment preserved the endocardial microvasculature (assessed by semiquantitative electron microscopic analysis) and enhanced regional myocardial blood flow after reperfusion. Specifically, mean score for microvascular injury was 0.41 +/- 0.14 vs. 0.10 +/- 0.08 (p less than 0.05) in control compared with SOD + catalase treated groups, and blood flow averaged 0.56 +/- 0.11 vs. 1.27 +/- 0.33 ml/min/g tissue (p less than 0.05), respectively, in the previously ischemic endocardium at 2 hours postreflow. Thus, SOD + catalase given at the time of reperfusion had no acute beneficial effect on either the extent of myocyte necrosis or postischemic contractile function in this canine model. SOD + catalase did, however, attenuate both endocardial vascular injury and the "low reflow" phenomenon. These data suggest that microvascular injury and low reflow following prolonged (2 hour) but transient coronary occlusion may be mediated by oxygen-derived free radicals generated at the time of reperfusion.

Acute hemorrhage and oxygen free radicals.

Oxygen free radicals are known to produce cellular injury. There are various mechanisms during hemorrhagic shock that can lead to an increase in the oxygen free radicals, which would produce a loss of membrane integrity and a decrease in cardiac function and cardiac contractility. The authors studied the effect of acute hemorrhage and reperfusion on the hemodynamics, blood lactate, and oxygen free radicals. Dogs were divided into two groups, group I, hemorrhagic shock and reinfusion; group II, hemorrhagic shock and reinfusion with superoxide dismutase (SOD) and catalase treatment. The dogs were bled (50% of the estimated blood volume) over a period of five to twelve minutes. After an hour of bleeding, the shed blood was transfused over twenty to twenty-five minutes. Group II received SOD (0.7 mg/kg, IV) and catalase (0.7 mg/kg, IV) within ten minutes of bleeding. The hemodynamic measurements and collection of blood samples for measurement of oxygen free radicals and lactate were made before hemorrhage and at fifteen, thirty, and sixty minutes of hemorrhage and at fifteen, thirty, and sixty minutes after reperfusion of shed blood. There was a decrease in the cardiac function and index of myocardial contractility associated with an increase in the blood lactate after hemorrhage in group I. Similar but less marked changes in these parameters were observed in group II. Recovery of hemodynamic parameters with reinfusion was better in group II as compared with that in group I. There was an increase in blood oxygen free radicals (superoxide anions) in group I, especially after reinfusion. No such changes were observed in group II.(ABSTRACT TRUNCATED AT 250 WORDS)

Parenteral Superoxide Dismutase Plus Catalase Diminishes Pancreatic Edema in Sodium Taurocholate-Induced Pancreatitis in the Rat

Scavengers of toxic oxygen reduction products have been reported to reduce the inflammatory reaction in some models of pancreatitis. In a blinded study, the effect of parenteral pretreatment with superoxide dismutase plus catalase was compared with placebo on pancreatitis induced in rats by infusion of 0.25% or 2% sodium taurocholate into the hepatopancreatic duct. The degree of inflammation was assessed by macroscopic examination of the pancreas, dry/wet weight ratios of pancreatic specimens, amylase activity in plasma and peritoneal exudate, the weight of the exudate, and its content of total protein. All parameters were indicative of a more severe inflammation in rats given the higher concentration of sodium taurocholate. The only significant effect of the superoxide dismutase plus catalase treatment was a moderate reduction of the dry/wet weight ratio, i.e., pancreatic edema, in rats given 2% sodium taurocholate. Our results indicate that toxic oxygen reduction products, available for interception by parenterally administered superoxide dismutase plus catalase, are of only minor importance in the pathogenesis of sodium taurocholate-induced pancreatitis in the rat.

Further evaluation of the effects of laminin, testosterone, ganglioside GM 1, and catalase on early growth in rat nerve regeneration chambers

We present here the results of an extended series of experiments on the effects of exogenous biochemical agents on nerve regeneration. They were applied by multiple injection into in vivo nerve regeneration chambers developed in this laboratory for rat sciatic nerve. Previously, a mixture of laminin, testosterone, ganglioside GM 1, and catalase at a defined concentration was shown to substantially advance the progress and maturation of regeneration in 16-day chambers, out of a variety of numerous other agents screened. In this study, an attempt is made to further clarify the contribution of the single ingredients used in combination. None of the agents proved to be able to mimick the previously observed effects by themselves (to their full extent). The best results were obtained by application of laminin; catalase treatment revealed results apparently worse than those in controls. Therefore, a redefined combination of the described agents, catalase now being omitted, was also tested. These results appeared to be grossly similar to those we recently reported.

Apparent Effect of Catalase on Airway Edema in Guinea Pigs

The airway edema that develops in guinea pigs after exposure to toluene diisocyanate (TDI) requires the presence of polymorphonuclear leukocytes (PMN). To determine whether this airway edema is mediated by the release of hydrogen peroxide from PMN, we treated animals intravenously with catalase bound to polyethylene glycol and examined the extravasation of Evans blue dye into the tracheal wall after exposure to air or 3 ppm TDI for 1 h. Catalase (25,000, 100,000, and 300,000 IU/kg) caused a dose-dependent inhibition of the TDI-induced increase in dye extravasation. However, treatment with catalase, inactivated at the peroxide binding site with 3-aminotriazole, inhibited dye extravasation after exposure to TDI as effectively as the equimolar 100,000 IU/kg dose of active catalase. Injection of polyethylene glycol alone was without effect. Dose-dependent decreases in extravascular migration of PMN and in circulating PMN also were noted after catalase treatment. These results suggest that the catalase preparations used in these studies inhibited the PMN-dependent airway edema by an effect other than hydrogen peroxide scavenging. Examination of this and other commercially available catalase preparations revealed trace concentrations of endotoxin at levels that could be responsible for the observed effects on PMN function. Treatment of animals with doses of Escherichia coli endotoxin similar to those inadvertantly administered to the catalase-treated groups (0.1 ng/kg to 100 ng/kg, intravenously) inhibited TDI-induced extravasation of Evans blue dye in a dose-dependent manner. These results suggest that contaminating endotoxin may contribute to some of the protective effects of preparations of catalase observed in previous studies of vascular permeability.

Pathophysiology of superoxide radical as potential mediator of reperfusion injury in pig heart.

The role of oxygen-derived free radicals in myocardial reperfusion injury was studied using the isolated in situ pig heart model. The free radical scavengers, superoxide dismutase (SOD) and catalase, protected the ischemic pig heart subjected to one hour of normothermic regional ischemia followed by one hour of global hypothermic arrest and one hour normothermic reperfusion. A significant increase in thiobarbituric acid reactive material and oxidized glutathione appeared in the perfusate demonstrating free radical-mediated lipid peroxidation during reperfusion, and this was prevented by the addition of SOD plus catalase. The values of three important antioxidative enzymes, SOD, catalase, and glutathione peroxidase, showed reduced activities after 2 hours of ischemia. These values did not change significantly after 60 minutes of reperfusion following the 2 hours ischemic insult. The concentrations of high-energy phosphate compounds including creatine phosphate (CP), adenosine triphosphate (ATP), and total adenine nucleotide were reduced significantly during ischemia and reperfusion in hearts which were not protected by SOD and catalase. The plasma creatine phosphokinase levels were lowered appreciably as a result of SOD and catalase treatment. It may be concluded from these experiments that oxygen-derived free radicals are present during reperfusion and SOD and catalase play a significant role in the protection of ischemic myocardium from reperfusion injury.

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

Antioxidant modulation of phacoanaphylactic endophthalmitis.

Catalase treatment reduces the expected inflammation in experimental phacoanaphylactic endophthalmitis. Catalase is a scavenger of hydrogen peroxide and presumably protects against the cytolytic effects of reactive oxygen metabolites. Recent work indicates that oxygen-derived free radicals may play a critical role in the early tissue damage in immune complex disease.

Oxygen free radicals in ischemic acute renal failure in the rat.

During renal ischemia, ATP is degraded to hypoxanthine. When xanthine oxidase converts hypoxanthine to xanthine in the presence of molecular oxygen, superoxide radical (O-2) is generated. We studied the role of O-2 and its reduction product OH X in mediating renal injury after ischemia. Male Sprague-Dawley rats underwent right nephrectomy followed by 60 min of occlusion of the left renal artery. The O-2 scavenger superoxide dismutase (SOD) was given 8 min before clamping and before release of the renal artery clamp. Control rats received 5% dextrose instead. Plasma creatinine was lower in SOD treated rats: 1.5, 1.0, and 0.8 mg/dl vs. 2.5, 2.5, and 2.1 mg/dl at 24, 48, and 72 h postischemia. 24 h after ischemia inulin clearance was higher in SOD treated rats than in controls (399 vs. 185 microliter/min). Renal blood flow, measured after ischemia plus 15 min of reflow, was also greater in SOD treated than in control rats. Furthermore, tubular injury, judged histologically in perfusion fixed specimens, was less in SOD treated rats. Rats given SOD inactivated by prior incubation with diethyldithiocarbamate had plasma creatinine values no different from those of control rats. The OH X scavenger dimethylthiourea (DMTU) was given before renal artery occlusion. DMTU treated rats had lower plasma creatinine than did controls: 1.7, 1.7, and 1.3 mg/dl vs. 3.2, 2.2, and 2.4 mg/dl at 24, 48, and 72 h postischemia. Neither SOD nor DMTU caused an increase in renal blood flow, urine flow rate, or solute excretion in normal rats. The xanthine oxidase inhibitor allopurinol was given before ischemia to prevent the generation of oxygen free radicals. Plasma creatinine was lower in allopurinol treated rats: 2.7, 2.2, and 1.4 mg/dl vs. 3.6, 3.5, and 2.3 mg/dl at 24, 48, and 72 h postischemia. Catalase treatment did not protect against renal ischemia, perhaps because its large size limits glomerular filtration and access to the tubular lumen. Superoxide-mediated lipid peroxidation was studied after renal ischemia. 60 min of ischemia did not increase the renal content of the lipid peroxide malondialdehyde, whereas ischemia plus 15 min reflow resulted in a large increase in kidney lipid peroxides. Treatment with SOD before renal ischemia prevented the reflow-induced increase in lipid peroxidation in renal cortical mitochondria but not in crude cortical homogenates. In summary, the oxygen free radical scavengers SOD and DMTU, and allopurinol, which inhibits free radical generation, protected renal function after ischemia. Reperfusion after ischemia resulted in lipid peroxidation; SOD decreased lipid peroxidation in cortical mitochondria after renal ischemia and reflow. We concluded that restoration of oxygen supply to ischemic kidney results in the production of oxygen free radicals, which causes renal injury by lipid peroxidation.

Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages.

This study demonstrates that the promastigote form of virulent Leishmania donovani and Leishmania tropica are both deficient in endogenous enzymatic scavengers of H(2)0(2) (catalase, glutathione peroxidase) and susceptible to low fluxes of H(2)O(2) in a cell-free model. In addition, the killing of promastigotes by H(2)0(2) is markedly enhanced in the presence of a peroxidase and halide. Promastigotes also readily trigger the macrophage oxidative burst including the generation of H(2)0(2), and most intracellular promastigotes are killed within 18 h by unstimulated normal resident cells. Catalase, but not scavengers or quenchers of O(2)(-), OHx, or (1)O(2), protected promastigotes in a cell-free xanthine oxidase microbicidal system, and catalase also partially inhibited the leishmanicidal activity of resident macrophages. Thus, amongst various oxygen intermediates, H(2)0(2) alone appeared to be both necessary and sufficient for promastigote killing. Depriving macrophages of exogenous glucose, which inhibits the generation of oxygen intermediates, achieved effects similar to catalase treatment. These observations directly contrast with the intracellular parasite, T. gondii which is richly endowed with catalase and glutathione peroxidase, highly resistant to H(2)0(2), and requires products of O(2)(-)-H(2)0(2) interaction for effective oxidative killing. Toxoplasmas also fail to trigger the respiratory burst of normal macrophages, and readily multiply within these cells (1-5). Macrophages first activated by in vivo or in vitro immunologic stimuli, however, display an enhanced capacity to generate oxygen intermediates beyond O(2)(-) and H(2)0(2), and are able to kill toxoplasmas or inhibit their intracellular replication (1, 2). These studies illustrate the wide spectrum of susceptibility to oxidative products which appears to exist for virulent intracellular protozoans, and indicate that such differences may be reflected in contrasting fates of parasites within cell-free oxidative environments and the cytoplasm of normal resident macrophages. In addition, these observations also demonstrate that nonactivated phagocytes may display effective microbicidal activity against certain intracellular pathogens utilizing an oxygen-dependent mechanism.

Beneficial effect of catalase treatment on growth of Clostridium perfringens

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.

Dissociation of beef liver catalase in its subunits

Earlier results from other laboratories have shown that beef liver catalase is built up of subunits. However there was no integral relationship between the number of subunits and the number of active sites (heme groups). To clarify the question how the heme groups are distributed among the subunits, the quaternary structure of catalase was studied under different denaturing conditions. 1 5 M guanidine-HCl, in the presence of 0.1 M β-mercaptoethanol, caused dissociation of catalase into subunits each with a molecular weight of 59,000 (s°20, w= 2.10 S; D°20, w= 3.20 F). In the absence of β-mercaptoethanol the molecular weight in 5 M guanidine-HCl was found to be about 140,000 (s°20, w= 3.50; D°20, w= 2.17 F). 2 Succinylation of native catalase with succinic anhydride at neutral pH yielded subunits each with a molecular weight of 65,000 (s°20, w= 3.22 S; D°20, w= 4.43 F). 3 After short incubation (up to six hours) at pH 12.5–12.8 catalase dissociated into subunits each with a sedimentation coefficient (s°20, w) of 2.89 S. The molecular weight of these subunits was estimated to be 54,000 and 67,000 respectively. The first value was obtained by combining sedimentation and viscosity measurements ([η] = 17.78 ml/g) from the relation deduced by Scheraga and Mandelkern using a β-value of 2.5 × 106. The principle of Archibald was used for the estimation of the second value. Longer incubation (e.g. twenty hours) caused splitting into several components, the main component after sixty hours possesses a sedimentation coefficient of 1.7 S. Probably this splitting is due to hydrolysis of peptide bonds. For this reason it was not possible to estimate the diffusion coefficient of the S2.9-component which needs a dialyzed solution. 4 During the incubation of native catalase in the presence of 7.3–14.6 mM sodium dodecyl sulfate an incomplete dissociation into a component with s°20, w= 3.4 S was observed. Separation of the dissociation product by means of a separation cell yielded a single component with a molecular weight of about 80,000 (s°20, w= 3.3 S; D°20, w= 3.3 F). The significance of this component is not clear, because the molecular weight disagrees with the value of 61,000 obtained by all other methods. Treatment of catalase or apocatalase with formic acid followed by incubation with sodium dodecyl sulfate caused dissociation into subunits with a sedimentation coefficient of 3.8 S (D°20, w= 5.1 F) and 4 S respectively. The molecular weight of the S3.8-component was found to be 60,000. 5 8 M urea resulted in a dissociation into subunits each with a molecular weight of 133,000 (s°20, w= 3.3 S; D°20, w= 2.23 F). About the same molecular weight (120,000) was obtained by Samejima and Yang at pH 3. By comparison with the molecular weight of the native enzyme (240,000–250,000) it was concluded that beef liver catalase consists of four subunits of equal size (molecular weight 60,000–65,000). This conclusion was confirmed by electron microscopic studies. In 5 M guanidine-HCl, or in 8 M urea, or at pH 3 the catalase molecule dissociated into two subunits only, whereas all other denaturing conditions used gave rise to four subunits. Together with the reported renaturation experiments the dissociation scheme of beef liver catalase is shown in the following equation: As mentioned above guanidine-HCl (and probably urea), in the presence of β-mercaptoethanol, caused dissociation into four subunits, ẃhereas in the absence of β-mercaptoethanol only two subunits were obtained. It is assumed that the reason for this difference might be the oxidation of SH groups to disulfide bridges during the denaturation linking subunits together. This process is prevented in the presence of β-mercaptoethanol. In the native enzyme molecule the four subunits are held together only by noncovalent bonds. It was concluded that each of the four subunits contains one active center.