Role of free radicals in primary nonfunction of marginal fatty grafts from rats treated acutely with ethanol

Acute treatment with one large dose of ethanol, which mimics binge drinking, causes marginal fatty liver and decreases survival significantly after liver transplantation in rats, yet mechanisms remain unclear. Therefore, we evaluated the possible role of free radicals in primary nonfunction caused by acute ethanol. Female donor rats were administered ethanol (5 g/kg orally) 20 hr before explantation, and grafts were stored in UW cold storage solution for 24-42 hr before implantation. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone after transplantation, and adducts were detected using electron spin resonance spectrometry. Ethanol increased a carbon-centered radical adduct in bile approximately 2-fold and elevated serum lipid hydroperoxides approximately 4-fold. Ethanol also increased transaminase release 3.7-fold and decreased bile production by 55%. Catechin, a free radical scavenger, minimized the increase in free radicals, blunted transaminase release, and elevated bile production significantly, indicating that free radical production plays an important role in ethanol-induced fatty graft injury. GdCl3 (20 mg/kg intravenously), a selective Kupffer cell toxicant, largely blocked the increases in free radical and lipid hydroperoxide production caused by ethanol. In addition, ethanol nearly doubled white blood cell adhesion after transplantation, leading to increased superoxide production in fatty grafts. GdCl3 largely blocked leukocyte adhesion as well as superoxide production. Allopurinol, an inhibitor of xanthine oxidase, also diminished free radical production, blunted transaminase release, and improved bile production in fatty grafts significantly. Taken together, we conclude that free radical formation increases in ethanol-induced fatty grafts due mainly to activation of Kupffer cells and increased adhesion of white blood cells. Antioxidants can effectively block free radical formation and minimize injury to marginal fatty grafts caused by binge drinking.     

Reactive oxygen species produced in metal-catalyzed oxidation of bis(trifluoromethyl)disulfide and protection by ZE

Bis(trifluoromethyl)disulfide (TFD), used as an industrial fumigant, was found to generate a thiyl free radical as seen by EPR/spin trapping. Oxygen appears to be an absolute requirement for radical production. The results obtained in this investigation implicate the production of thiyl and reactive oxygen species (ROS), superoxide radical anion and hydroxyl radicals, during TFD autoxidation. The rate of production of these free radical intermediates was found to increase in the presence of iron(III) and copper(II). In addition, the metal ion chelator DETAPAC and ROS scavengers ethanol, mannitol, and PEG-SOD/catalase were found to inhibit free radical production. Reactive oxygen species were not formed when a high-potency zinc plus antioxidant, ZE caps, was present. These results provide support for the pro-oxidation of TFD and a protective role for zinc.     

Free radicals and ethanol-induced cytotoxicity in neural crest cells

Associations between ethanol-induced cranial neural crest cell (NCC) damage in mammalian embryos and subsequent malformations as observed in human fetal alcohol syndrome have previously been illustrated. The vulnerability of NCCs to this teratogen may result, at least in part, from their sensitivity to free radical damage. To examine relationships between free radical generation and NCC cytotoxicity, primary culture of mouse NCCs was used. NCC viability was determined in both dose- and time-response studies involving ethanol exposure. After 48 hr of culture, cell viability was significantly diminished at all doses tested (i.e., 50, 100, 150, and 200 mM ethanol). At 100 mM ethanol (a dosage that is teratogenic in vivo and in whole embryo culture), cell viability decreased to approximately 50% of control values over the first 12 hr of culture, and decreased further, to approximately 20% by 48 hr. Using nitroblue tetrazolium as a probe, it was observed that exposure of NCCs to ethanol stimulated the production of superoxide anion radicals. Co-treatment of the ethanol-exposed NCCs with free radical scavengers including 300 units/ml of superoxide dismutase, catalase (500 units/ml), or alpha-tocopherol (300 microM) significantly improved NCC viability. These results suggest that the ethanol-induced NCC injury is mediated, at least in part, through the generation of free radicals. To test this hypothesis further, NCCs were exposed in culture to xanthine/xanthine oxidase. Exogenous free radicals generated by the xanthine/xanthine oxidase system resulted in reduced NCC viability, the severity of which increased in a time and enzyme concentration-related manner. Superoxide dismutase (300 units/ml) and catalase (500 units/ml) significantly reduced the effects of the xanthine/xanthine oxidase-generated free radicals on NCC viability. The similarity between the susceptibility of NCCs to ethanol and their susceptibility to exogenous free radicals in concert with the free radical scavenger-mediated amelioration of ethanol and exogenous free radical-induced NCC death strongly suggest that free radicals play a significant role in ethanol-induced NCC death.     

Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation. It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also stimulated human neutrophils to produce intracellular free radicals.     

Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.     

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.     

Putrescine-modified catalase with preserved enzymatic activity exhibits increased permeability at the blood-nerve and blood-brain barriers

Much evidence exists in support of the hypothesis that free radicals contribute to the pathogenesis of several neurodegenerative disorders and that mechanisms of free radical generation occur both intracellularly and extracellularly. Previous studies in this laboratory have shown that covalent modification of growth factors and antioxidant enzymes with the naturally occurring polyamine, putrescine, increases their permeability at the blood-nerve and blood-brain barriers (BNB and BBB), but does not significantly inhibit bioactivity. Furthermore, putrescine-modified superoxide dismutase (SOD) was shown to reduce neurodegeneration in a rat model of global cerebral ischemia. The purpose of the present study was to modify the antioxidant enzyme, catalase (CAT), with putrescine (PUT) at carboxylic acid groups whose ionization, and hence reactivity, was controlled with pH and investigate the effects on permeability and enzymatic activity. Modification of CAT with PUT increased its permeability 2-3-fold and preserved 67% of its enzymatic activity compared to native CAT and 137% compared to lyophilized CAT. The results of this study indicate that modification of CAT with putrescine increases its permeability while preserving enzymatic activity. PUT-SOD administered in combination with PUT-CAT may eliminate both the superoxide radical and the H2O2 produced from the dismutation of superoxide, respectively, and thus prevent the formation of hydroxyl radicals. This combination may exhibit increased neuroprotective effects, compared to native enzymes, following systemic administration for the treatment of free radical associated neurodegenerative disorders.     

Bystander tumoricidal effect and gap junctional communication in lung cancer cell lines

Calcium dobesilate, a vascular protective agent, was tested in vitro for its scavenging action against oxygen free radicals. Calcium dobesilate was as potent as rutin to scavenge hydroxyl radicals (IC50 = 1.1 vs 0.7 microM, respectively). It was also able to scavenge superoxide radicals, but with 23 times less potency than rutin (IC50 = 682 vs 30 microM, respectively). Calcium dobesilate significantly reduced platelet activating factor (PAF)-induced chemiluminescence in human PMN cells and lipid peroxidation by oxygen free radicals in human erythrocyte membranes, although these actions required calcium dobesilate concentrations > or = 50 microM. Finally, in cultured bovine aortic endothelial cells, magnesium dobesilate reduced the increase in cytosolic free calcium induced by hydrogen peroxide and inhibited phenazine methosulfate-induced cell potassium loss. In conclusion, calcium dobesilate was effective in scavenging hydroxyl radicals in vitro, at therapeutically relevant concentrations. Conversely, higher concentrations of the compound were required to scavenge superoxide radicals or to protect the cells against the deleterious effects of intracellular reactive oxygen species. Further studies in vivo are required to determine if these antioxidant properties of calcium dobesilate can play a role in its vascular protective mechanisms.     

Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical

The role of the beta-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a giso of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the beta-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the beta-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the beta-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.     

Reactive oxygen species in reoxygenation injury of rat brain capillary endothelial cells

OBJECTIVE: To clarify the mechanism of anoxia/reoxygenation (A/R) injury of rat brain capillary endothelial cells (BCEC). METHODS: BCEC isolated from Sprague-Dawley rats by enzymatic treatment and centrifugation were subjected to anoxia (95% N2, 5% CO2) for 20 minutes and then to reoxygenation (95% air, 5% CO2) for 3 hours. Enzyme inhibitors, including oxypurinol, indomethacin, and N(G)-nitro-L-arginine methyl ester, or specific free-radical scavengers, such as superoxide dismutase, catalase, and the ferric iron chelator deferoxamine, were added before A/R injury. The BCEC were incubated in a range of Ca2+ concentrations from 1 to 0.01 mmol/L during A/R injury. Cytotoxicity was assayed by release of intracellular lactate dehydrogenase (LDH). RESULTS: With A/R injury, LDH release from the control group (no protective agents) significantly increased (44.8 +/- 3.3%), compared with a small increase in a normoxic group. BCEC treated with oxypurinol, indomethacin, or N(G)-nitro-L-arginine methyl ester showed suppression of LDH release. LDH release was almost totally suppressed by superoxide dismutase and partially by catalase or deferoxamine. The LDH release was partly dependent on calcium concentration. CONCLUSION: BCEC subjected to A/R become potent generators of free radicals, especially superoxide anion. Free radical production depends on both xanthine oxidase and cyclooxygenase pathways. Peroxynitrite and extracellular Ca2+ both contribute importantly to reoxygenation injury of BCEC.     

Status of free radicals and their scavenging enzymes in pregnancy induced hypertension (PIH)

The levels of lipid peroxidation (malonaldialdehyde), one of the consequence of free radical damage, and the antioxidant enzymes superoxide dismutase and catalase were estimated in the blood samples of fourteen normal and thirteen pregnancy induced hypertensive patients. A marked increase in malonaldialdehyde (p < 0.001) with concomitant decrease in superoxide dismutase (p < 0.001), catalase (p < 0.001) activities were observed in PIH as compared to normal pregnancy, thereby indicating the involvement of free radicals in PIH.     

Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

Protective effect of FK506 on ischemia/reperfusion-induced myocardial damage in canine heart

We investigated the cardioprotective effect of FK506, a newly developed immunosuppressive agent, on ischemia-reperfusion-induced myocardial damage and the inhibitory effect of FK506 on superoxide radical formation by neutrophils. Open-chest anesthetized dogs were divided into two groups: group 1, 2-h occlusion of the coronary artery followed by 1-h reperfusion; and group 2, 2-h occlusion followed by 1-h reperfusion with preadministration of FK506 (0.5 mg/kg). After reperfusion, heart mitochondria were prepared from the normal and reperfused areas and mitochondrial function and mitochondrial GSH (the reduced form of glutathione) and GSSG (the oxidized form of glutathione) concentrations were measured. In addition, neutrophils were collected from normal healthy dogs, and the inhibitory effect of FK506 on superoxide radical formation by neutrophils was also investigated. One-hour reperfusion after 2-h coronary occlusion induced significant mitochondrial dysfunction associated with a marked depletion of mitochondrial GSH concentration. FK506 reduced mitochondrial dysfunction, depletion of mitochondrial GSH concentration, and development of reperfusion arrhythmias. FK506 also reduced stimulant-induced superoxide radical formation by normal neutrophils dose dependently. Radical scavenging activity decreased in association with reperfusion, and FK506 reduced superoxide radical formation by neutrophils, which might contribute to lessening ischemia-reperfusion damage.     

Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin

Meningococcal sodC encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses.     

Distinct subpopulations in HaCaT cells as revealed by the characteristics of intracellular calcium release induced by phosphoinositide-coupled agonists

Both oximes and hydroxylamine (HYAM) are compounds with known oxidative capacity. We tested in vitro whether acetaldoxime (AAO), cyclohexanone oxime (CHO), methyl ethyl ketoxime (MEKO) or HYAM affect haemoglobin oxidation (into HbFe3+), formation of thiobarbituric acid reactive substances (TBARS), and glutathione (GT) depletion in human haemolysate, erythrocytes or blood. All these parameters are known to be related to oxidative stress. Glutathione S-transferase (GST) activity was measured as it may be affected by oxygen radicals. All three oximes caused a low degree of HbFe3+ accumulation in erythrocytes. This was higher in haemolysates indicating that membrane transport may be limiting or that protective mechanisms within erythrocytes are more effective. HbFe3+ accumulation was lower for the oximes than for HYAM. AAO and HYAM caused TBARS formation in blood. For HYAM this was expected as free radicals are known to be generated during HbFe3+ formation. Free radical generation by AAO and HYAM in erythrocytes was confirmed by the inhibition of GST. For the other two oximes (CHO and MEKO) some special effects were found. CHO did inhibit erythrocyte GST while it did not cause TBARS formation. MEKO was the least potent oxime as it caused no TBARS formation, little HbFe3+ accumulation and little GST inhibition in erythrocytes. However, GT depletion was more pronounced for MEKO than for the other oximes, indicating that glutathione conjugation occurs. TBARS formation, GT depletion and GST modulation caused by the oximes and HYAM were also tested in rat hepatocytes. However, no effects were found in hepatocytes. This suggests that a factor present in erythrocytes is necessary for free radical formation. Studies with proposed metabolites of the oximes (i.e. cyclohexanone, acetaldehyde or methylethyl ketone) and addition of rat liver preparations to the erythrocyte incubations with oximes, suggest that metabolism is not a limiting factor in erythrocyte toxicity.     

Influence of a melatonin implant on the free radical load in avian thyroid and its relation with thyroid hormonogenesis

The primary aim of this study was to evaluate the effect of melatonin on the oxiradical load in avian thyroid. The superoxide free radicals have been spin trapped by EPR spectroscopy in the thyroid gland of Indian rock pigeon Columbia livia following melatonin implantation for two weeks. Melatonin implantation resulted in augmentation in the levels of superoxide radical in the thyroid gland of pigeons with a concomitant decrease in the levels of the total superoxide dismutase activity. This was also associated with increased lipid peroxidation. Melatonin implantation caused a significant increase in plasma levels of glucose. Plasma levels of thyroxine (T4) and triiodothyronine (T3) were lower in the melatonin-treated pigeons. However, the T3/T4 ratio was higher following melatonin implantation. Since iodination of tyrosine is an H2O2-dependent phenomenon, the inhibition in the activity of SOD could lead to impaired thyroid hormone synthesis.     

Spin trapping of azidyl and hydroxyl radicals in azide-inhibited rat brain submitochondrial particles

Succinate-driven respiration in azide-inhibited rat brain submitochondrial particles (smps) produces azidyl and hydroxyl radicals that were detected by spin trapping with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Production of radicals required succinate and oxygen and was eliminated by heat denaturation, which indicates that radical production is a result of respiration. The concentrations of both DMPO/.OH and DMPO/.N3 were decreased by addition of catalase to the smps, which indicates that H2O2 is involved in radical production. In the absence of azide anion, DMPO/.OH was not detected in the same system, even after five additions of succinate over a period of 24 h. It is proposed that azide inhibition of cytochrome c oxidase results in increased production of superoxide, which is efficiently converted to hydrogen peroxide by membrane-bound superoxide dismutase. Hydrogen peroxide activates endogenous peroxidase to react with azide anion forming azidyl radical, which damages the peroxidase, resulting in decreased production of azidyl radical with successive additions of succinate. Hydroxyl radical is produced from the hydrogen peroxide that is not removed by peroxidase. The increased production of superoxide in the azide-inhibited system suggests that loss of cytochrome c oxidase activity can lead to increased radical production if other proteins in the respiratory chain remain active. In the azide-inhibited system, reaction of azide anion with H2O2-activated endogenous peroxidase and spin-trapping of the resulting azidyl radical is a convenient monitor of H2O2 production.     

Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study

OBJECTIVES: We sought to demonstrate that direct current (DC) shocks to the heart generate free radicals. BACKGROUND: Although it is a lifesaving maneuver, defibrillation is known to have myocardial toxicity. The mechanism of this toxicity is unknown. If DC shocks generate free radicals, free radicals could be a mechanism of myocardial injury. METHODS: In a canine model, DC shocks of 10 to 100 J were delivered to the epicardium of both beating and fibrillating hearts, and 200-J transthoracic shocks were administered in dogs with beating hearts. Ascorbate free radical (AFR) concentration was measured in arterial blood and blood continuously withdrawn from the coronary sinus. In some dogs, the antioxidant enzymes superoxide dismutase (15,000 U/kg) and catalase (55,000 U/kg) (SOD/Cat) were administered before shocks. RESULTS: Ascorbate free radicals were generated by DC shocks. A peak AFR increase of 14 +/- 2% (mean +/- SEM) was seen 5 to 6 min after 100-J epicardial shocks. A peak AFR increase of 7 +/- 5% occurred after transthoracic shocks. There was a significant linear relation between the shock energy and peak percent AFR increase: %AFR increase = 0.18 (Shock energy) + 2.9 (r = 0.73, p < 0.0001). Shocks delivered to hearts in ventricular fibrillation (30 s) resulted in generation of AFR equal to but not greater than that observed during similar shocks delivered to beating hearts in sinus rhythm. Multiple successive shocks (100 J delivered twice or five times) did not result in a greater AFR increase than single 100-J shocks, indicating that peak, not cumulative, energy is the principal determinant of AFR increase. Animals receiving SOD/Cat before shock administration showed significant attenuation of the AFR increase. CONCLUSIONS: Direct current epicardial and transthoracic shocks generate free radicals; antioxidant enzymes reduce the free radical generation by shocks.     

Prostate cancer in the African American: is this a different disease?

Reactive oxygen species such as superoxides, hydrogen peroxide (H2O2) and hydroxyl radicals have been suggested to be involved in the catalytic action of nitric oxide synthase (NOS) to produce NO from L-arginine. An examination was conducted on the effects of oxygen radical scavengers and oxygen radical-generating systems on the activity of neuronal NOS and guanylate cyclase (GC) in rat brains and NOS from the activated murine macrophage cell line J774. Catalase and superoxide dismutase (SOD) showed no significant effects on NOS or GC activity. Nitroblue tetrazolium (NBT, known as a superoxide radical scavenger) and peroxidase (POD) inhibited NOS, but their inhibitory actions were removed by increasing the concentration of arginine or NADPH respectively, in the reaction mixture. NOS and NO-dependent GC were inactivated by ascorbate/FeSO4 (a metal-catalyzed oxidation system), 2'2'-azobis-amidinopropane (a peroxy radical producer), and xanthine/xanthine oxidase (a superoxide generating system). The effects of oxygen radicals or antioxidants on the two isoforms of NOS were almost similar. However, H2O2 activated GC in a dose-dependent manner from 100 microM to 1 mM without significant effects on NOS. H2O2-induced GC activation was blocked by catalase. These results suggested that oxygen radicals inhibited NOS and GC, but H2O2 could activate GC directly.