Generation Of Oxygen-derived Free Radicals Research Articles

Evidence of cytosolic iron release during post-ischaemic reperfusion of isolated rat hearts Influence on spin-trapping experiments with DMPO

Previous studies of oxygen-derived free radical generation based on spin-trapping methods have shown a signal formed of six bands (sextet) using electron spin resonance spectrometry (ESR) or coronary effluents collected during post-ischaemic reperfusion of isolated hearts perfused with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The origin of this signal has recently become controversial. In the present study we show that, in the rat, this sextet and cytosolic iron release occur simultaneously, and that this signal can be inhibited by the iron chelator desferrioxamine. It also appears that the iron release is not protein bound, and could therefore have a marked catalytic activity. This may be responsible for the production or an artefactural signal observed as the sextet.

Alterations of energetic metabolite levels by free radicals during optic nerve ischemia.

An experimental model of optic nerve ischemia was designed in the rabbit to determine early biochemical alterations, i.e.--changes of high energy phosphate metabolites (ATP and phosphocreatine)--in occlusive and peri-occlusive areas. Vascular occlusion provoked a rapid fall of ATP and phosphocreatine in the optic nerve. Free radicals scavengers, superoxide dismutase plus catalase or dimethylthiourea were able to counteract the drop of phosphate metabolites in the peri-occlusive area. These results show that hypoxia leads to oxygen-derived free radical generation which can be responsible for cell damage and emphasize the role of free radicals in the pathogenesis of ocular diseases related to vascular dysfunction.

The role of oxygen-derived free radicals in two models of experimental acute pancreatitis: Effects of catalase, superoxide dismutase, dimethylsulfoxide, and allopurinol

The role of oxygen-derived free radicals was evaluated in two models of experimental acute pancreatitis by testing the effects of agents which either reduce oxygen-derived free radical generation or scavenge those free radicals. Those agents (catalase, superoxide dismutase, polyethylene glycol-superoxide dismutase, dimethylsulfoxide, and allopurinol) were evaluated using the choline-deficient ethionine-supplemented diet-induced model of acute hemorrhagic pancreatic necrosis and the supramaximal caerulein stimulation model of acute interstitial edematous pancreatitis. In both models, the only effect associated with administration of the test agents was a reduction in the degree of pancreatic edema. These results suggest that oxygen-derived free radicals may play an important role in the development of pancreatic edema during pancreatitis but that those free radicals do not play an important role in the development of acinar cell injury.

Decreased stability of digestive gland lysosomes from the common musselMytilus edulis L. byin vitro generation of oxygen-free radicals

A consequence of xenobiotic exposure is oxidative stress resulting from one electron metabolism with the concomitant generation of oxygen-derived free radicals. The growing interest in molluscs as sentinels of environmental pollution has prompted studies on the responsiveness of lysosomal membranes from the common marine mussel,Mytilus edulis L. to oxidative stress by the oxyradicalgenerating hypoxanthine/xanthine oxidase reaction. We report that oxyradicals generated external to the lysosomal membrane cause a decrease in the stability of the lysosomal membrane, thereby resulting in leakage of enzymes from this organelle. This leakage can be prevented by catalase but not by hydroxyl radical scavenging agents. Superoxide dismutase appears to be a major enzyme exhibiting latent activity upon attack of lysosomes by oxyradicals but does not afford sufficient protection to prevent enzyme leakage under the conditions studied. Further, non-fluorescent dihydrorhodamine 123 is converted to highly fluorescent products after uptake into the lysosomal compartment of digestive gland cells suggesting that oxyradicals may be produced within the lysosomal compartment in association with the pinocytotic activity of the digestive cells. Thus, a consequence of xenobiotic-mediated proliferation of oxyradicals might be the release of potent hydrolytic enzymes into the cytosol with ensuing enzyme degradation and damage to other organelles.

Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25–2 μm) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.

The Superoxide-forming Enzymatic System of Phagocytes

The generation of superoxide and oxygen-derived free radicals by the phagocytes (neutrophils, monocytes and macrophages) is catalyzed by a membrane-bound NADPH oxidase system which is dormant in resting cells and becomes activated during phagocytosis or by stimulating cells with suitable soluble stimulants. This oxidase system has been thought to be an electrontransport chain consisting of a flavoprotein, a b-type cytochrome (cytochrome b-558: cyt b-558) and cytosolic components (p47-phox, p67-phox and unknown factor). When activated, these components seem to act synergistically to form superoxide, but the roles of these components in the electron transport system are not fully elucidated.

Enhanced chemiluminescence as a measure of oxygen-derived free radical generation during ischemia and reperfusion.

It has been suggested that oxygen-derived free radicals may contribute to the myocardial injury associated with ischemia and reperfusion. As the presence of enhanced free radical generation is a prerequisite for such damage, several techniques have been used to provide evidence of increased oxygen free radical production during reperfusion; however, all such techniques have substantial limitations. In this study, we used enhanced chemiluminescence to evaluate oxygen free radical generation during ischemia and reperfusion in the isolated Langendorff-perfused rat heart. The chemiluminescent technique, which has high sensitivity and can monitor radical generation continuously, avoids some of the limitations of earlier methods. Chemiluminescence (expressed as counts per second) decreased from 219 +/- 11 at baseline to 142 +/- 9 during ischemia and markedly increased to a peak of 476 +/- 36 during the first 3-5 minutes of reperfusion. This was followed by a slow decline over 11-16 minutes to a steady-state level of 253 +/- 14 (each sequential change in chemiluminescence was highly significant; p less than 0.001). Superoxide dismutase (2,000 units/min) significantly decreased peak reperfusion chemiluminescence to 316 +/- 17 (p less than 0.01). Hearts subjected to a second period of ischemia and reperfusion had a higher peak chemiluminescence (626 +/- 62), which also was significantly attenuated by 1,000 units/min superoxide dismutase (398 +/- 16; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic antiallergic drug on the generation of oxygen-derived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC30 = 3.9 microM) and AM (IC30 = 9.8 microM). In AM zymosan-induced CL was inhibited 21.7% by 10 microM azelastine, whereas in PMNL it remained unchanged up to 10 microM azelastine. Azelastine has a significantly stronger inhibitory effect (IC30 = 4.2 microM) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibits oxygen-derived free radical generation by interaction with protein kinase C.

The effects of an iron chelator on cellular injury induced by vascular stasis caused by hypothermia

Rewarming of a cooled rabbit leg was associated with the generation of oxygen-derived free radicals, shown to be instrumental for tissue injury occurring during rewarming. The present study used a compound that, by its ability to bind with free iron, can remove hydroxyl radical (OH·) from tissue. Deferoxamine reduced tissue injury during cooling and rewarming, as evidenced by its ability to decrease tissue release of lactic acid dehydrogenase and creatine kinase. Deferoxamine also reduced the formation of OH· and lipid peroxidation during the rewarming phase. This compound did not have any effect on the arterial blood flow pattern, which uniformly decreased during cooling and was restored during rewarming. The results of this study indicate the efficacy of deferoxamine in reducing cellular injury associated with cold and rewarming and further suggest a role for oxygen—derived free radicals in the pathophysiology of cold-rewarming injury.

Microvascular Changes during the Early Phase of Experimental Bacterial Meningitis

We investigated the temporal profile of the changes in regional CBF (rCBF) and intracranial pressure (ICP) during the early phase of pneumococcal meningitis in the rat. rCBF, as measured by laser-Doppler flowmetry, and ICP were continuously monitored during 6 h post infection (p.i.). Brain edema formation was assessed by brain water content determinations. Meningitis was induced by intracisternal injection of 75 microliters of 10(7) colony-forming units/ml pneumococci (n = 7). In control animals (n = 6), saline was injected. There was no change in the rCBF or ICP of controls throughout the experiment. However, there was a dramatic increase in rCBF and ICP associated with brain edema formation in untreated meningitis animals. rCBF increased to 135.3 +/- 33.8% (mean +/- SD) in the untreated animals at 1 h p.i. and reached 211.1 +/- 40.5% at 6 h p.i. (p less than 0.05 compared with controls). ICP increased from 2.9 +/- 1.4 to 10.4 +/- 4.7 mm Hg at 6 h p.i. (p less than 0.05 compared with controls). Brain water content was significantly elevated (79.69 +/- 0.24 compared with 78.94 +/- 0.16% in the control group, p less than 0.05). We investigated the effect of dexamethasone (3 mg/kg i.p.), which was given prior to the induction of meningitis (n = 3) or at 2 h after pneumococcal injection (n = 5), indomethacin (10 mg/kg i.v., n = 5), and superoxide dismutase (SOD; 132,000 U/kg i.v. per 6 h, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory Effects of the Bone-Derived Growth Factors Osteoinductive Factor and Transforming Growth Factor-β on Isolated Osteoclasts*

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Detection of oxygen-derived free radical generation in the canine postischemic heart during late phase of reperfusion.

To define the relation between oxygen-derived free radical (oxy-radical) generation in the reperfused ischemic myocardium and the progression of myocardial damage, we measured oxy-radical generation in the ischemic myocardium and the propagating infarct size in a model of canine coronary occlusion (90 minutes) and reperfusion. We used electron paramagnetic resonance spin-trapping techniques (5,5-dimethyl-1-pyrroline N-oxide [DMPO]) to detect oxy-radicals in the rapidly frozen myocardial samples taken by needle biopsy. There was no detectable generation of DMPO adducts in the normal myocardium before or after reperfusion. In the reperfused ischemic myocardium, electron paramagnetic resonance signals of DMPO-OOH (superoxide anion) and DMPO-OH (hydroxyl radical) were detected, with peak concentrations at 1 hour after reperfusion for DMPO-OOH and at 3 hours after reperfusion for DMPO-OH, respectively. These DMPO adducts were also detected during the early phase (15 seconds) of reperfusion, but the concentrations of these signals were much less than those during the late phase of reperfusion. Treatment with human recombinant superoxide dismutase (2.5 mg/kg/hr) and catalase (2.5 mg/kg/hr) during the course of experiments abolished DMPO-OOH formation but had little effect on DMPO-OH formation. Infarct size (percent of risk area infarcted), quantified by a dual staining method with Evans blue dye and triphenyltetrazolium chloride, was 18.3 +/- 4.8% (mean +/- SEM) at 90 minutes of occlusion. After 5 hours of reperfusion, infarct size increased to 43.6 +/- 7.2%. These results indicate that a greater magnitude of oxy-radical generation was sustained in the ischemic myocardial tissue during the late phase (1-3 hours) of reperfusion, associated with the progression of myocardial infarction. The concurrent appearance of oxy-radicals and progressive infarction may support the view that a chain reaction of oxy-radicals contributes to the propagation of myocardial cell damage in the postischemic heart.

Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo.

The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.

Pulmonary reperfusion injury: evidence for oxygen-derived free radical mediated damage and effects of different free radical scavengers

Blood granulocyte-mediated reactions involving generation of oxygen-derived free radicals have recently been shown to be capable of causing injury to the lungs. These findings suggest a similar mechanism also to be involved in the development of pulmonary ischemia/reperfusion injury. In the present study, therefore, the effects of three oxygen-derived free radical scavengers, superoxide dismutase (SOD; 1 mg/kg), catalase (20,000 IU/kg) and allopurinol (45 mg/kg), were evaluated during reperfusion in a rabbit model after 2 h normothermic ischemia of the lung. During reperfusion, ischemic lungs were found to have an elevated pulmonary vascular resistance, increased total and extravascular lung water content, and decreased arterial oxygen tension (PaO2) compared to control animals. SOD and catalase, but not allopurinol, were able to reduce pulmonary injury by lowering the pulmonary vascular resistance, but could not prevent pulmonary damage as shown by total lung water (TLW) or PaO2. It is concluded that oxygen-derived free radicals such as hydrogen peroxide and the superoxide anion may play an important role in precipitating pulmonary injury after ischemia. The failure of xanthine oxidase inhibition (allopurinol) to exert protective effects may suggest that oxygen-derived free radical generation following pulmonary ischemia occurs predominantly via leukocyte-mediated reactions.

A rapid, widely applicable screen for drugs that suppress free radical formation in ischemia/reperfusion

Substantial injury can occur during reoxygenation of previously ischemie tissue in many experimental models, as the result of the generation of oxygen-derived free radicals. To test the antiradical activity of potentially protective compounds in this setting, we developed a simple screening system, applicable to fresh biopsy specimens, in which warm ischemia and reoxygenation of excised tissue are performed in vitro. Tissue production of malondialdehyde (MDA) equivalents is used as a nonspecific-but-sensitive marker of oxygen radical damage. Test compounds with putative antiradical activity are added prior to the reoxygenation phase, and their ability to suppress MDA production is an index of activity in preventing reoxygenation injury. Comparison with ischemie but not reoxygenated controls confirms the oxygen-dependent nature of the effect. Standard positive controls of known effective agents, such as butylated hydroxytoluene or deferoxamine, provide a reference for the activity of the test compound. The method is applicable to surgical biopsy specimens in veterinary and human medicine.

Gastric mucosal lesions induced by hemorrhagic shock in baboons. Role of oxygen-derived free radicals.

In this study we sought to define the role of oxygen-derived free radicals during ischemia and reperfusion in the production of acute damage to the gastric mucosa of baboons. The protective effect of the xanthine oxidase inhibitor, allopurinol, the superoxide scavenger, superoxide dismutase (SOD), and a long-acting SOD-albumin was determined. Mucosal damage was evaluated using light and scanning electron microscopy. Evidence for oxidative insult to the gastric mucosa was sought by measuring tissue concentrations of reduced (GSH) and oxidized (GSSG) glutathione. Gastric mucosal blood flow was estimated using the microsphere technique. A similar pattern of tissue damage was found at the end of ischemia in all three groups. Thirty minutes after reperfusion, severe mucosal damage (grade 3) increased only in the untreated control. In the two treated groups, grade 3 damage remained unchanged during reperfusion and a decrease in the percentage of moderate damage (grade 2) was seen. Both GSH and GSSG tissue concentrations were lower in the untreated controls as compared to the scavenger-treated groups, making it questionable whether GSH/GSSG tissue levels adequately reflect oxidative stress. We conclude that in our ischemia-reperfusion model the generation of oxygen-derived free radicals produces mucosal damage and prevents the restitution of moderate mucosal damage during reperfusion. In ischemia, factors other than free radicals seem to be responsible for mucosal damage. The protective effect of allopurinol and SOD was not mediated by changes in gastric mucosal blood flow.

The role of iron in injury of endothelial cells in vitro and in vivo.

Injury of cells and organs resulting from the generation of oxygen-derived free radicals is now a well-accepted observation (reviewed 1). Both the in vitro as well as the in vivo generation of oxygen radicals from activated phagocytic cells (neutrophils, eosinophils, monocytes and macrophages) via activation of NADPH oxidase results in injury of eukaryotic and prokaryotic cells (reviewed 1). All our studies have focused on acute injury of the rat lung and in vitro destruction of endothelial cells in an attempt to define the mechanisms of cell injury. What will be reviewed in this communication is the evidence for the critical role of iron in oxygen-radical-mediated cell injury and a linkage between these reactions and the appearance of products of lipid peroxidation.

Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

We examined the effects of α-thrombin (the native enzyme) on neutrophil activation as assessed by the measurement of chemiluminescence. α-Thrombin in physiological concentrations (10 −9 − 10 −8 M) did not induce neutrophil chemiluminescenee. However, when neutrophils were coincubated with opsonized zymosan and α-thrombin, the chemiluminescence response to opsonized zymosan was enhanced in a concentration-dependent manner. The neutrophil chemiluminescence responses to opsonized zymosan and to opsonized zymosan plus α-thrombin were dependent on the generation of oxygen-derived free radicals since the chemiluminescence was inhibited by superoxide dismutase. The results indicate that thrombin per se does not induce neutrophil chemiluminescence. However, thrombin enhances the chemiluminescence response to opsonized zymosan suggesting an interaction between thrombin and complement receptors in inducing neutrophil activation. The chemiluminescence response to thrombin and opsonized zymosan is the result of oxygen-derived free radicals.

Identification of free radical species in the myocardium.

With the increasing interest in the role of oxygen-derived free radical generation in various diseases, several attempts to directly evaluate free radicals in tissue have been performed using EPR.1–8 However, none of the previous studies has fully determined the species of the radicals and none has deliberated on the possible existence of artifactual radicals which are likely to be generated during sample handling. The purposes of this study were 1) to establish an artifact-free sampling method for the quantification of freeze-trapped free radicals in myocardium by EPR, 2) to identify artifact radicals and the radicals native to the myocardium and 3) to demonstrate the relationship of these radicals to reperfusion injury.

Relaxation of canine coronary artery to electrical stimulation: limited role of free radicals.

Electrical stimulation induces tetrodotoxin-insensitive relaxation of the canine coronary artery. The present study was designed to verify whether this relaxation involves the production of oxygen-derived free radicals. Isolated rings of canine coronary arteries were suspended for isometric tension recording in organ chambers filled with Krebs-Ringer bicarbonate solution. They were stimulated electrically (9 V, 3 Hz, 2 ms for 2 min) by means of two platinum electrodes during contractions evoked by various vasoactive agonists. Under control conditions, electrical stimulation caused rapid, reversible relaxations. Superoxide dismutase in association with catalase or mannitol, sodium ascorbate, dimethyl sulfoxide, and glutathione did not inhibit the relaxation caused by stimulation applied for only 2 min; neither did the removal of chloride ions from the salt solution nor the association of Cl-free solution in the presence of mannitol, superoxide dismutase, and catalase. Prolonging the electrical stimulation (9 V, 3 Hz, 2 ms) for up to 20 min produced a secondary relaxation. This second phase was inhibited by sodium ascorbate. These experiments indicate that the rapid relaxation induced by short-lasting electrical stimulation is probably not due to the generation of oxygen-derived free radicals. However, prolonged stimulation causes the production of such radicals, which then evoke irreversible inhibition of the vascular smooth muscle of the canine coronary artery.