Superoxide formed from cigarette smoke impairs polymorphonuclear leukocyte active oxygen generation activity.

Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, O2-. was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated O2-. was an initiating factor in the impairment and OH. generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as alpha-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) O2-. is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated O2-. can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as alpha-tocopherol or DHLA might be successful in preventing these deleterious effects.     

Effect of stobadine on oxygen free radical generation in stimulated human polymorphonuclear leukocytes

The generation both superoxide and a mixture of reactive oxygen species was recorded in a suspension of human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. While stobadine dose-dependently decreased chemiluminescence, only its highest concentration used reduced significantly superoxide generation. The results suggest that stobadine is a more effective scavenger of free radicals rather than a quencher of superoxide anion.     

Aging and acute exercise enhance free radical generation in rat skeletal muscle.

Reactive oxygen species (ROS) are implicated in the mechanism of biological aging and exercise-induced oxidative damage. The present study examined the effect of an acute bout of exercise on intracellular ROS production, lipid and protein peroxidation, and GSH status in the skeletal muscle of young adult (8 mo, n = 24) and old (24 mo, n = 24) female Fischer 344 rats. Young rats ran on a treadmill at 25 m/min and 5% grade until exhaustion (55.4 +/- 2.7 min), whereas old rats ran at 15 m/min and 5% grade until exhaustion (58.0 +/- 2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of ROS and other intracellular oxidants production in the homogenate of deep vastus lateralis, was 77% (P < 0.01) higher in rested old vs. young rats. Exercise increased DCFH oxidation by 38% (P < 0.09) and 50% (P < 0.01) in the young and old rats, respectively. DCFH oxidation in isolated deep vastus lateralis mitochondria with site 1 substrates was elevated by 57% (P < 0.01) in old vs. young rats but was unaltered with exercise. Significantly higher DCFH oxidation rate was also found in aged-muscle mitochondria (P < 0.01), but not in homogenates, when ADP, NADPH, and Fe(3+) were included in the assay medium without substrates. Lipid peroxidation in muscle measured by malondialdehyde content showed no age effect, but was increased by 20% (P < 0.05) with exercise in both young and old rats. Muscle protein carbonyl formation was unaffected by either age or exercise. Mitochondrial GSH/ GSSG ratio was significantly higher in aged vs. young rats (P < 0.05), whereas exercise increased GSSG content and decreased GSH/GSSG in both age groups (P < 0.05). These data provided direct evidence that oxidant production in skeletal muscle is increased in old age and during prolonged exercise, with both mitochondrial respiratory chain and NADPH oxidase as potential sources. The alterations of muscle lipid peroxidation and mitochondrial GSH status were consistent with these conclusions.     

The effect of free radical derived hydroxyicosatetraenoic acids on hexose transport in the human polymorphonuclear leukocyte

The following racemic hydroxyicosatetraenoic acids were prepared and assayed for their ability to stimulate hexose transport in human polymorphonuclear leukocytes: 15-, 12-, 11-, 9-, 8-, and 5-hydroxyicosatetraenoic acids. The compounds were isolated from reduced, autoxidized arachidonic. The results demonstrate that only the 12-, and 5-hydroxyicosatetraenoic acids are biologically active, inducing half-maximal responses at 820 and 176 nM, respectively. Thus, the bioactions of hydroxicosatetraenoates are crucially dependent upon the position of the hydroxy residue. Response to both hydroxyicosatetraenoates was effectively blocked by two inhibitors of arachidonic acid metabolism: nordihydroguaiaretic acid and indomethacin. A third arachidonic acid antimetabolite, 5,8,11,14-eicosatetraynoic acid, completely inhibited the response to 12-HETE but caused only partial inhibition of the response to 5-HETE.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Effect of galactose on free radical reactions of polymorphonuclear leukocytes.

To account for impaired bactericidal activity of polymorphonuclear leukocytes during galactosemia, we have investigated the effects of galactose upon free radical reactions associated with the oxygen-dependent killing mechanism of guinea pig polymorphonuclear leukocytes. Millimolar levels of galactose, which are encountered in the circulation of infants with galactosemia, did not affect the chemical reaction of Superoxide anion with either ferricytochrome c or nitroblue tetrazolium. Galactose did, however, significantly inhibit the reaction of hydroxyl radical with methional, and we propose that such hydroxyl radical removal would be deleterious to normal bactericidal activity. Additional experiments demonstrated that polymorphonuclear leukocytes incubated in the presence of galactose reduced less ferricytochrome c than polymorphonuclear leukocytes incubated without galactose. These results imply that galactose impairs the cellular release of Superoxide anion, which would further disable bactericidal activity.     

Cytoprotective effects of carvedilol against oxygen free radical generation in rat liver

The protective effects of carvedilol, an antihypertensive agent, against oxidative injury caused by acetaminophen were studied in rat liver. Male Wistar rats (250 +/- 30 g) were pre-treated with carvedilol (3.6 mg/kg, p.o.) for 10 days and on the 11th day received an overdose of acetaminophen (800 mg/kg, p.o.). Four hours after acetaminophen administration, blood was collected to determine serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). After that, rats were killed and the livers were excised to determine reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and carbonyl protein contents, and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST), and also the DNA damage index. Acetaminophen significantly increased the levels of TBARS, the DNA damage and SOD, AST and ALT activities. Carvedilol was able to prevent lipid peroxidation, protein carbonilation and DNA fragmentation caused by acetaminophen. Moreover, this drug prevented increases in SOD, AST and ALT activities. These results show that carvedilol exerts cytoprotective effects against oxidative injury caused by acetaminophen in rat liver. These effects are probably related to the O2*- scavenging property of carvedilol or its metabolites.     

Electromagnetic pulse reduces free radical generation in rat liver mitochondria in vitro

Abstract

Non-ionizing radiation electromagnetic pulse (EMP) is generally recorded to induce the generation of free radicals in vivo. Though mitochondria are the primary site to produce free radicals, a rare report is designed to directly investigate the EMP effects on free radical generation at mitochondrial level. Thus the present work was designed to study how EMP induces free radical generation in rat liver mitochondria in vitro using electron paramagnetic resonance technique. Surprisingly, our data suggest that EMP prevents free radical generation by activating antioxidant enzyme activity and reducing oxygen consumption and therefore free radical generation. Electron spin resonance measurements clearly demonstrate that disordering of mitochondrial lipid fluidity and membrane proteins mobility are the underlying contributors to this decreased oxygen consumption. Therefore, our results suggest that EMP might hold the potentiality to be developed as a non-invasive means to benefit certain diseases.     

Analysis of age-associated changes in mitochondrial free radical generation by rat testis

Throughout spermatogenesis, mitochondria undergo a morphological and functional differentiation. Mitochondria are involved in the production of reactive oxygen species (ROS), considered one of the mediators of ageing. Particularly, lipid peroxidation is regarded as a major phenomenon by which ROS can impair cellular function. In the present study, we examined the production of superoxide anion, superoxide dismutase activity and the effect of Fe²⁺/ascorbate induced-lipid peroxidation on the respiratory chain activities of testis mitochondria throughout the process of spermatogenesis and ageing. Mitochondria from rat testes generated superoxide anion, mainly using NADH as substrate, which increased according to age. The activity of SOD is age-dependent and greatly stimulated during the first wave of spermatogenesis, but decreases in adulthood and old age. TBARS concentration was also markedly increased by ageing. The activity of mitochondrial respiratory chain complexes is differentially affected by oxidative stress induced by iron/ascorbate, succinate-dehydrogenase activity being less vulnerable than that of NADH-dehydrogenase and cytochrome c oxidase. The data suggest that ageing is accompanied by reduced activity of SOD, leading to excessive oxidative stress and enhanced lipid peroxidation that compromises the functionality of the electron transport chain. The data support the concept that mitochondrial function is an important determinant in ageing.     

Role of oxygen-derived free radicals on rat splanchnic eicosanoid production during acute hemorrhage.

The effect of superoxide dismutase (SOD), an oxygen-derived free radical scavenger, on rat splanchnic eicosanoid synthesis was examined following hemorrhagic shock. Anesthetized male rats were hemorrhaged to 30 mm Hg for 30 minutes (Shock), killed, or treated with the shed blood (Shock plus reperfusion). The Shock plus reperfusion group was treated with saline vehicle or SOD (2500, 5000, 7500, 10,000 or 15,000 U/Kg, i.v.) 15 minutes prior to the reperfusion of the shed blood. The superior mesenteric artery was removed in continuity with the end organ intestine (SV+SI) and perfused in vitro with oxygenated Krebs-Henseleit buffer (3 ml/min at 37 degrees C). Venous effluent was measured for basal release of 6-keto-PGF1 alpha, PGE2 and thromboxane B2 at 15, 30, 60 and 90 minutes of perfusion. The SV+SI compensated for acute shock by increased release of 6-keto-PGF1 alpha (300%) (and not PGE2 or thromboxane B2) which was abolished by reperfusion of the shed blood following shock. Prior treatment of the Shock plus reperfusion group with 7500 U/Kg or more of SOD restored the increased release of SV+SI 6-keto-PGF1 alpha found following shock alone (p less than 0.05). These data provided indirect evidence that ODFRs contributed to endogenous SV+SI regulation of PGI2 synthesis and release during hemorrhagic shock and reperfusion of shed blood.     

Ascorbate both activates and inactivates bleomycin by free radical generation.

The chemotherapeutic agent bleomycin (BLM) is activated by reducing agents to break isolated DNA. Paradoxically, these same reducing agents protect cellular DNA from BLM damage. To resolve this paradox, we have examined the reaction of FeIIIBLM with DNA in the presence of ascorbate. As expected, ascorbate augments FeIIIBLM-induced DNA damage. However, when ascorbate is added to FeIIIBLM prior to exposure to DNA, a redox-inactive BLM is produced in a reaction that generates the ascorbyl radical. This reaction occurs in both ascorbate-supplemented buffer and unsupplemented plasma. In buffered solution, this reaction was found to be stoichiometric; for each mole of BLM present, 6.9 mol of ascorbate was oxidized and 4.7 mol of oxygen was consumed. Iron was found to serve only as a catalyst for the reaction. These data suggest that both activation of BLM and the generation of redox-inactive BLM occur via the same reaction and that BLM-induced DNA damage depends upon BLM reaching DNA prior to its interaction with reducing agents.     

Macrophages suppress CTL generation in rat mixed leukocyte cultures.

The generation of CTL in rat MLC was actively suppressed by a cell population present in spleen cell preparations from normal rats. These suppressor cells were characterized by using a variety of cell fractionation techniques. Suppressor cells were removed by passage of spleen cells through nylon wool columns or by treatment with carbonyl iron. Suppressive activity was present in the mononuclear cell fraction of spleen cells obtained by Ficoll-Hypaque density gradient centrifugation. After velocity sedimentation at unit gravity, enrichment of suppressive activity was demonstrated in the fractions containing large cells as compared to the fractions containing small cells. Populations rich in macrophages were shown to have similar suppressive activity upon CTL induction in MLC. These studies suggest that macrophages present in normal rat spleen cell preparations account for the difficulty in generating CTL in MLC prepared with rat cells.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Mitochondrial respiratory chain and free radical generation in stroke

Being the second most common cause of death in the industrial countries and one of the major causes of death and disability, stroke has a great effect on public health and is the neurological disease which accounts for the largest number of hospitalizations. In order to develop new treatments, biochemical mechanisms involved in brain damage have been investigated. Among them, oxidant species generated during stroke have been implicated as critical mediators of neuronal injury in this condition, although neuroprotective roles have also been demonstrated. This review is focused on the role of the mitochondrial respiratory chain as both source and target of reactive oxygen and nitrogen species such as nitric oxide, superoxide and peroxynitrite produced in cerebral ischemia. The neuroprotective role of antioxidants or other molecules acting on the mitochondrial respiratory chain and ATP synthesis in the setting of cerebral ischemia is discussed.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Purification and N-terminal amino-acid sequence analysis of rat polymorphonuclear leukocyte cathepsin G

Cathepsin G was purified by single-step cation-exchange chromatography from rat polymorphonuclear leukocytes, obtained from the peritoneal cavity after induction of a mild peritonitis. The 26 N-terminal amino acids were determined and showed 73% identity to those of human cathepsin G. Total amino-acid composition demonstrated a high degree of basic amino acids in accordance with its high affinity for the cationic-exchange gel medium. The protein was found to be a glycoprotein with a glucosamine content of 7.4% of the calculated Mr28,900. On SDS/polyacrylamide-gel electrophoresis the protein showed a Mr of 28,400. It migrated as two bands in a gradient SDS/polyacrylamide-gel indicating isoforms. The pH optimum for the proteinase was determined to be 8.0-8.5 using Suc-Ala-Ala-Pro-Phe-Nan as substrate (Suc = 3-carboxypropionyl; Nan = 4-nitroanilide). Km and Kcat/Km values for Suc-Ala-Ala-Pro-Phe-Nan were 0.86mM and 280M-1S-1 and for Suc-Phe-Leu-Phe-Nan 0.24mM and 3600M-1S-1, respectively.     

Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants

Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H₂O₂), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H₂O₂ and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H₂O₂ in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.     

Vitamin E protects porcine but not bovine cultured aortic endothelial cells from oxygen-derived free radical injury due to hydrogen peroxide

The antioxidative effects of vitamin E (VE) are well known and have been demonstrated in in vitro studies. Since we previously observed that dextran sulfate was markedly more protective of porcine versus bovine aortic endothelial cells when damaged by hydrogen peroxide (H2O2), our objectives were to determine if a similar species difference could be observed with VE. The effects of VE or Trolox (a more water-soluble VE) against oxygen-derived free radical (OFR) injury produced by H2O2 was studied in porcine aortic endothelium (PAE) vs. bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). VE or Trolox was added to culture medium for at least 24 h prior or immediately prior to H2O2 addition. In PAE, pretreatment with VE dissolved in either ethanol (VE-EtOH) or Tween 20 (VE-Tween 20), or Trolox dissolved in DMSO (Trolox-DMSO) was protective, shown by increased percent viable cells and reduced lactate dehydrogenase (LDH) release. EtOH, Tween 20 or DMSO alone was protective in PAE although DMSO or Tween 20 alone was less effective than when added with VE. VE-Tween 20 or Trolox-DMSO protected PAE when added just prior to H2O2 injury, but protection was significantly less than with pretreatment. DMSO immediately prior to H2O2 injury had no protective effect. Tween 20 immediately prior resulted in complete cell death. In BAE and BBME, pretreatment with VE-EtOH, EtOH, Trolox-DMSO, or DMSO alone had little or no protective effect. Pretreatment with VE-Tween 20 or Tween-20 alone was protective of BAE with Tween 20 being more effective than VE-Tween 20 suggesting that Tween 20 was the protective agent. These studies show that the protective effects of VE and Trolox as well as DMSO, EtOH, and Tween-20 are species dependent.