Formation Of Oxygen-derived Free Radicals Research Articles

Interaction Between Allopurinol and Copper: Possible Role in Myocardial Protection

Allopurinol, a potent inhibitor of xanthine oxidase, is known to effectively protect the heart against damage in patients undergoing cardiac bypass surgery. There is still an ambiguity concerning the presence of xanthine oxidase in the human heart. Thus, the mechanism underlying the protective effect of allopurinol is unclear. Transition metal ions, such as iron and copper, can participate in single-electron reactions and mediate the formation of oxygen-derived free radicals. In this study the interaction between allopurinol and Cu(II) was investigated. Spectrophotometric investigation shows that allopurinol (0-0.8 mM) form a 1:1 complex with Cu(II) ions (0-0.8 mM) with a specific absorbance peak at 364 nm. Also, the rate constant (k) for the copper-catalyzed aerobic oxidation of ascorbate was markedly decreased in the presence of allopurinol (from 0.068 min-1 to 0.014 min-1). Allopurinol substantially reduced the copper-mediated and ascorbate-driven DNA breakage. Spectrophotometric measurements did not indicate a specific interaction between iron ions and allopurinol. It is suggested that the beneficial effects of allopurinol during reperfusion of the heart could stem from its chelation of copper, yielding a complex with low redox activity.

Allopurinol administered prior to hepatic ischaemia in the rat prevents chemiluminescence following restoration of circulation.

Oxygen-derived free radicals produced during reperfusion may be responsible for the disturbed pathology which follows prolonged ischaemia. Measurement of hepatic chemiluminescence (low level light emission resulting from the energy released during chemical reactions of free radicals) allowed determination of whether allopurinol could prevent formation of oxygen-derived free radicals during reperfusion of the ischaemic liver. While control animals demonstrated a burst of light emission shortly after reperfusion, the rats pretreated with allupurinol showed no evidence of chemiluminescence during either ischaemia or reperfusion. It is concluded that allopurinol may modify reperfusion-induced free radical formation and possibly ameliorate the organ damage which can follow ischaemia.

Reduction of canine myocardial infarct size by CI-959, an inhibitor of inflammatory cell activation.

CI-959, a cell-activation inhibitor that prevents the formation of oxygen-derived free radicals by inflammatory cells, was studied to determine its effects on myocardial infarct size and subsequent scar formation in dogs. The left circumflex coronary artery was occluded for 90 min, followed by 6 h of reperfusion. Drug infusion was started 15 min before reperfusion at a loading dose of 8 mg/kg i.v., followed immediately by 2 mg/kg i.v. infused over 80 min. The infarct size, assessed by TTC staining techniques, was significantly reduced in 12 dogs treated with CI-959 (23.3 +/- 3.6% of the area at risk) when compared to 11 vehicle-treated animals (35.5 +/- 4% of the area at risk, p less than 0.05). This reduction in infarct size was not attributed to changes in regional myocardial blood flow, as measured by radioactive microspheres, or to a reduction in myocardial oxygen demand, as estimated by changes in the rate-pressure product. The scar thickness, measured after a 6-week recovery period in 9 animals treated with CI-959, was not significantly reduced in comparison with 11 controls. In vitro, CI-959 effectively inhibited oxygen free radical formation by canine neutrophils. The results of this study show that CI-959 significantly reduces the myocardial infarct size without causing scar thinning, which might lead to ventricular aneurysm, and suggests the most likely mechanism for its beneficial action is the prevention of formation of toxic oxygen radicals.

Interaction of the putative essential nutrient pyrroloquinoline quinone with the N-methyl-D-aspartate receptor redox modulatory site

The putative essential nutrient pyrroloquinoline quinone (PQQ) can efficiently mediate reduction and oxidation reactions in a variety of systems. Therefore, we investigated whether this compound could alter the function of the NMDA receptor via a recently described redox modulatory site. In rat cortical neurons in vitro, 50 microM PQQ could reverse the enhancement of 30 microM NMDA-induced whole-cell ionic currents produced by the reducing agent dithiothreitol (DTT; 2-4 mM). PQQ also depressed native responses in a DTT-reversible fashion. In addition, 50-200 microM PQQ produced a significant degree of neuroprotection in an acute model of NMDA-mediated neurotoxicity in astrocyte-rich cultures of rat cerebral cortex. Under certain conditions, PQQ can lead to the formation of oxygen-derived free radicals, and we have previously observed that these reactive species can oxidize the NMDA receptor. Nevertheless, the enzymatic free radical scavengers superoxide dismutase and catalase (10 micrograms/ml each) did not abolish the actions of PQQ. This observation held true even in astrocyte-poor cortical cultures, where neuronal processes are directly exposed to the extracellular milieu. Therefore, under in vitro conditions in which PQQ is presented without an exogenous electron donor, it appears as if the entire neuroprotective effect of PQQ is attributable to a direct oxidation of the NMDA receptor redox site. These results suggest the possibility of a novel role for PQQ, PQQ-like substances, and quinone-containing proteins in the brain, and may represent a novel therapeutic approach for the amelioration of NMDA receptor-mediated neurotoxic injury.

Repetitive static muscle contractions in humans--a trigger of metabolic and oxidative stress?

Repetitive static exercise (RSE) is a repetitive condition of partial ischaemia/reperfusion and may therefore be connected to the formation of oxygen-derived free radicals and tissue damage. Seven subjects performed two-legged intermittent knee extension exercise repeating at 10 s on and 10 s off at a target force corresponding to about 30% of the maximal voluntary contraction force. The RSE was continued for 80 min (n = 4) or to fatigue (n = 3). Four of the subjects also performed submaximal dynamic exercise (DE) at an intensity of about 60% maximal oxygen uptake (VO2max) for the same period. Whole body oxygen uptake (VO2) increased gradually with time during RSE (P less than 0.05), indicating a decreased mechanical efficiency. This was further supported by a slow increase in leg blood flow (P less than 0.05) and leg oxygen utilization (n.s.) during RSE. In contrast, prolonged RSE had no effect on VO2 during submaximal cycling. Maximal force (measured in six additional subjects) declined gradually during RSE and was not completely restored after 60 min of recovery. After 20 and 80 min (or at fatigue) RSE phosphocreatine (PC) dropped to 74% and 60% of the initial value, respectively. A similar decrease in PC occurred during DE. Muscle and arterial lactate concentrations remained low during both RSE and DE. The three subjects who were unable to continue RSE for 80 min showed no signs of a more severe energy imbalance than the other subjects. A continuous release of K+ occurred during both RSE and DE.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of oxygen radicals in tourniquet-related ischemia-reperfusion injury of human patients

In the current study we evaluated effluent blood from extremities of human patients undergoing reconstructive surgical treatment which is routinely accompanied by upper extremity exsanguination and application of a tourniquet. Following tourniquet release (reperfusion), there were immediate increases in the plasma levels of xanthine oxidase activity, uric acid, and histamine. Xanthine dehydrogenase activity was not detectable. Plasma also contained products consistent with the formation of oxygen-derived free radicals, namely hemoglobin and fluorescent compounds. Our data indicate in humans that ischemia-reperfusion events are associated with the appearance of xanthine oxidase activity and its products in the plasma effluent.

Cardiac reperfusion damage prevented by a nitroxide free radical.

Experimental evidence is presented that directly links ischemia/reperfusion injury to the formation of oxygen-derived free radicals. 2,2,6,6-Tetramethylpiperidine-N-oxyl (TEMPO)--a stable nitroxide radical that disproportionates superoxide radicals and oxidizes reduced metal ions required for OH. formation--was tested for its ability to prevent reperfusion damage in the isolated rat heart subjected to regional ischemia. Severe reperfusion arrhythmia--ventricular fibrillation and ventricular tachycardia--were prominent in control hearts, and their duration was significantly reduced by the presence of 0.4 or 1 mM TEMPO. TEMPO also repressed both postischemic release of lactate dehydrogenase and OH. formation. TEMPO slowed the heart rate, but compensatory pacing did not alter the dramatic effect of the nitroxide on reperfusion arrhythmia. TEMPO was partially protective when introduced at the end of ischemia but had no effect when added 1 min into reperfusion. It was concluded that both reperfusion arrhythmia and cell damage were directly related to oxidative damage incurred during the critical first minute of reperfusion. TEMPO strongly protected against reperfusion injury by preventing the formation of OH. and not by decreasing heart rate or by direct suppression of arrhythmia.

Iron chelation therapy and lung transplantation: Effects of deferoxamine on lung preservation in canine single lung transplantation

Reperfusion injury is a limiting factor in lung transplantation. Deferoxamine is an iron chelator that inhibits the formation of oxygen-derived free radicals. We investigated the effects of deferoxamine on posttransplantation lung function in a canine model of single lung transplantation. Twelve dogs underwent left lung transplantation after 20- to 24-hour hypothermic storage in a modified Euro-Collins solution. In six experiments donor and recipient received a 10 mg/kg dose of deferoxamine before harvest and transplantation, and 10 mg/kg was added to the preservation solution. Arterial oxygen tension, alveolar-arterial oxygen difference, pulmonary vascular resistance, and dynamic lung compliance were measured. Data were recorded for 6 hours after ligation of the native pulmonary artery. At the end of the study the mean arterial oxygen tension was 175.1 mm Hg for the deferoxamine treated group versus 71.1 mm Hg for the control group (p less than 0.001), and the alveolar-arterial oxygen difference was less in the deferoxamine-treated group: 502.3 versus 606.0 mm Hg (p less than 0.001). The mean pulmonary vascular resistance was lower throughout the study, and after 6 hours it was 455.1 dynes/sec/cm(-5) in the deferoxamine-treated group versus 663.7 dynes/sec/cm(-5) in the control group (p less than 0.035). Compliance was similar in both groups. We conclude that deferoxamine improves lung preservation and early posttransplantation function in canine single lung transplantation.

Metabolism of ethanol by rat hepatocytes results in generation of a lipid chemotactic factor: Studies using a cell-free system and role of oxygen-derived free radicals

We have reported that liver cells metabolizing ethanol release a lipid that has chemotactic activity for human polymorphonuclear leukocytes. The purpose of the present study was to investigate the role of ethanol and of oxygen-derived free radicals in generation of this activity. A cell-free, ethanol-metabolizing system consisting of rat liver cytosol was found to generate a polar lipid which possessed a chemotactic activity identical to the activity from intact cells. The importance of acetaldehyde in this process was established by the findings that (i) an inhibitor of the metabolism of ethanol to acetaldehyde, 4-methylpyrazole, blocked production of the chemotactic lipid; (ii) acetaldehyde could substitute for ethanol; and (iii) cytosol alone incubated with ethanol in the absence of mitochondrial acetaldehyde metabolism generated the activity. Acetaldehyde can serve as a substrate for cytosolic oxidases in reactions that generate radicals (e.g., superoxide anion) which have been shown to attack unsaturated lipids to yield lipid hydroperoxides. When scavengers of oxygen-derived free radicals (superoxide dismutase, catalase, and dimethylsulfoxide) were added to the ethanol-metabolizing system they prevented generation of the activity. The data are consistent with a mechanism of chemotactic factor generation by ethanolmetabolizing liver cytosol in which acetaldehyde causes formation of oxygen-derived free radicals that attack unsaturated cytosolic lipid to generate a polar chemotactic compound.

Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damagein vitro: homology with bacterial repair enzymes

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.

Formation of oxygen-derived free radicals during cold injury

Formation of oxygen-derived free radicals during cold injury

The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury

Neutrophilic leukocytes have been implicated as important mediators of ischemic myocardial injury. We investigated the role of neutrophils in skeletal muscle ischemia/reperfusion injury by using the rat hindlimb ischemia model. We rendered Wistar rats neutropenic by administering 750 rad of whole-body radiation (mean white blood cell count, 300 +/- 50/mm3; 3 days after radiation). In anesthetized rats (10 neutropenic and 10 control), 3 hours of ischemia were induced in one hindlimb by application of a tourniquet to the proximal thigh; the contralateral limb served as an internal, nonischemic control. After 1 hour of reperfusion the gastrocnemius and soleus muscles were excised bilaterally and evaluated for ischemic injury by means of a quantitative spectrophotometric assay of triphenyltetrazolium chloride (TTC) reduction. In control rats the reduction of TTC by ischemic muscle averaged 27.0% +/- 7.3% of that by nonischemic muscle; whereas in neutropenic rats the value for ischemic muscle was 65.4 +/- 11.6% (p less than 0.05). To determine if the contribution of the neutrophils to ischemic injury is due to oxygen-derived free radical formation, an additional 10 animals were infused with 5000 units of super-oxide dismutase and 10,000 units of catalase at the time reperfusion was restored. After treatment with free radical scavengers, TTC reduction by ischemic limbs was 25.5% +/- 7.0% of that by nonischemic limbs and did not differ from that in control animals (p greater than 0.05). The results show a protective effect of neutropenia and suggest a significant role of the white cell in the pathophysiology of ischemic skeletal muscle injury.

The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury

Neutrophilic leukocytes have been implicated as important mediators of ischemic myocardial injury. We investigated the role of neutrophils in skeletal muscle ischemia/reperfusion injury by using the rat hindlimb ischemia model. We rendered Wistar rats neutropenic by administering 750 rad of whole-body radiation (mean white blood cell count, 300 ± 50/mm3; 3 days after radiation). In anesthetized rats (10 neutropenic and 10 control), 3 hours of ischemia were induced in one hindlimb by application of a tourniquet to the proximal thigh; the contralateral limb served as an internal, nonischemic control. After 1 hour of reperfusion the gastrocnemius and soleus muscles were excised bilaterally and evaluated for ischemic injury by means of a quantitative spectrophotometric assay of triphenyltetrazolium chloride (TTC) reduction. In control rats the reduction of TTC by ischemic muscle averaged 27.0% ± 7.3% of that by nonischemic muscle; whereas in neutropenic rats the value for ischemic muscle was 65.4 ± 11.6% (p < 0.05). To determine if the contribution of the neutrophils to ischemic injury is due to oxygen-derived free radical formation, an additional 10 animals were infused with 5000 units of superoxide dismutase and 10,000 units of catalase at the time reperfusion was restored. After treatment with free radical scavengers, TTC reduction by ischemic limbs was 25.5% ± 7.0% of that by nonischemic limbs and did not differ from that in control animals (p > 0.05). The results show a protective effect of neutropenia and suggest a significant role of the white cell in the pathophysiology of ischemic skeletal muscle injury.

Investigation of detoxification capacity of rat testicular germ cells and sertoli cells

Isolated pachytene spermatocytes live longer than round spermatids in vitro. Indigenous formation of oxygen-derived free radicals and hydrogen peroxide can cause damage to germ cells. The germ cell antioxidant capacity may play an important role in this respect. In view of this, we have examined the activity and cellular localization of superoxide dismutase (SOD) and glutathione S-transferases (GST) in rat testicular cells. We have found significant differences in the distribution of these enzymatic activities in the germ cells. In addition, this study shows that alpha-tocopherol is found in various amounts in rat testicular cells in the order of: Sertoli cells > pachytene spermatocytes > round spermatids, with a factor of 4 in the alpha-tocopherol content between Sertoli cells and round spermatids.

Ischaemic and metabolic treatment of hepatic tumours.

For treatment of malignancies, physical and metabolic differences between tumour cells and host cells have guided the development of new approaches. In this review, two new approaches to be used in the treatment of liver malignancies are outlined: ischaemic therapy and interferences with the glucose metabolism. Ischaemic therapy of liver malignancies has been used in different forms during the last 20 years: from ligation of the hepatic artery, embolization of the arterial tree, transient occlusion of the hepatic artery to the present day use of temporary, intermittent, transient hepatic arterial occlusion. The beneficial effect of ischaemic therapy on malignancies is supposed to depend on oxygen and nutritional deficiency, formation of oxygen-derived free radicals and loss of function in cellular enzymes. The tumour cells seem thereby to be more sensitive than the host cells. Also, ischaemia might potentiate the effect of cytotoxic drugs. Intereferencies with glucose metabolism might be directed either towards the exaggerated tumour glycolysis, for example by glucose analogues like 2-deoxy-glucose, or towards the exaggerated host gluconeogenesis, for example by hydrazine sulphate. These treatments result in reduction of the glucose availability in the intracellular glucose metabolism in the tumour cells and have experimentally been demonstrated to be correlated to reduced tumour growth. It is concluded that both these approaches, ischaemic therapy and manipulations with the glucose metabolism, seem promising for the future. What is needed now is research to clarify the mechanims behind the effects, to establish their full consequences, and to identify the clinical use of these treatments and their possible combinations.

Free Radicals and Cardioplegia: Allopurinol and Oxypurinol Reduce Myocardial Injury Following Ischemic Arrest

Oxygen-derived free radicals generated by xanthine oxidase may represent a major cause of myocardial injury during ischemia and reperfusion. We have used the isolated working rat heart model of cardiopulmonary bypass and ischemic arrest to assess whether allopurinol or oxypurinol, which should prevent free radical formation through their ability to inhibit xanthine oxidase, can improve postischemic myocardial recovery when the drugs are administered either chronically (pretreatment) or acutely (as an addition to the cardioplegic or reperfusion solution). With normothermic ischemic arrest, both drugs, when given either chronically or acutely, significantly improved postischemic recovery of function. However, under hypothermic conditions, allopurinol conferred no protection when given either as pretreatment or during reperfusion, but it was effective when added to the cardioplegic solution. When administered under the appropriate conditions, both allopurinol and oxypurinol enhanced the protective effect afforded by the St. Thomas' Hospital cardioplegic solution, possibly by inhibiting xanthine oxidase activity and preventing the formation of oxygen-derived free radicals.

Influence of oxygen-derived free radical scavengers on ischemic livers.

It has been hypothesized that the formation of oxygen-derived free radicals may play an important part in ischemically induced tissue injury. Using a canine ischemic liver model, we have assessed the role of two oxygen-free radical scavengers, catalase (CAT) and superoxide dismutase (SOD), used alone and in combination, on the recovery of ischemic livers. Liver ischemia was induced in adult mongrel dogs by cross-clamping of the portal vein and hepatic artery for 40 min. Hepatectomy was then performed, and livers were tested in an isolated perfusion model with 500 ml of an albumin-mannitol solution at 37 degrees C for 3 hr. Liver function tests were performed hourly during the perfusion period. Biopsies and 99m-Tc-HIDA scans were also done at the end of perfusion. Livers in group 1 (n = 6) served as controls and were not pretreated prior to ischemic injury. Livers in group 2 (n = 6), group 3 (n = 6), and group 4 (n = 6) were pretreated, respectively, with CAT (5000 U/kg), SOD (5000 U/kg), and SOD and CAT in combination (5000 U/kg) each. The results indicated that the oxygen-derived free radical scavengers, CAT and SOD, were able to provide partial protection against the free radicals accumulated during ischemic damage. These studies offer some potential avenues for the protection of livers prior to and after transplantation.

Pharmacological protection of reoxygenation damage to in vitro brain slice tissue

Pharmacological protection of central neural function against damage by hypoxia and reoxygenation was studied electrophysiologically and biochemically in hippocampal brain slices. Hypoxia causes a loss of both orthodromically and antidromically evoked potentials in CA1 pyramidal cell neurons. Damage due to hypoxia lasting more than 10 min cannot be restored by reoxygenation. Following pretreatment with methylprednisolone (10 −5 M), indomethacin (10 −5 M) or allopurinol (10 −5 M), reoxygenation after 10 min of hypoxia resulted in complete recovery of the evoked activity. Na +,K +-ATPase activity was not reduced by 10 min of hypoxia, but was reduced by 50% during the first 10 min of subsequent reoxygenation. Allopurinol (10 −5 M) and indomethacin (10 −5 M) protected against loss of this enzyme activity. The protective action by these drugs of both electrophysiological and biochemical aspects of neural function is consistent with the hypothesis that secondary ischemic damage is caused by the formation of oxygen-derived free radicals during reperfusion.