Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

pH dependence of lipid peroxidation and albumin oxidative modification: possible implications to the pH paradox

The hydrogen ion concentration may have an important influence in biological free radical reactions. We studied the effect of an acidic pH on two models of free radical-mediated damage: copper-induced lipid peroxidation in plasma and copper/hydrogen peroxide-induced oxidative modification of albumin. A reduction of pH from 7·4 to 6·6 decreased diene conjugation by 32%, thiobarbituric acid-reactive substances formation by 25% and fluorescence generation by 53% in plasma exposed to cupric chloride. At pH values lower than 6·6 an even greater inhibition of lipid peroxidation in plasma was obtained. Visible fluorescence development in albumin by exposure to site-specific generation of free radicals was also increasingly reduced by decreasing pH values. From pH 7·4 to 6·6 there was a 50% fluorescence generation inhibition. The observed partial protection of lipids and proteins against oxidative damage by an acidic pH alerts to the need for rigorously controlling the pH values when assaying compounds for antioxidant properties in vitro. It may also contribute to the explanation for the protective effect of an acidic pH against anoxic cell injury and for cell death that is precipitated by a rapid return to a normal pH following reperfusion (the ‘pH paradox’).     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

Studies on the Salicylic Acid Inducted Lipid Peroxidation and Defense Gene Expression in Tobacco Cell Culture

Salicylic acid (SA) could inhibit catalase activity, induce rapid lipid peroxidation and PR-1 gene expression of the tobacco ( Nicotiana tabacum L. ) cell culture which was incubated with exogenous SA. Ρ-ihydroxybenzene and H2O2 could also induce lipid peroxidation and PR-1 gene expression at different level, but they were not able to inhibit the catalase activity of tobacco cells. Inhi0itors of mRNA and protein-synthesis (a-amanitine and cycloheximide, respectively) could not induce both lipid peroxidation and PR-1 gene expression of tobacco cell culture. However, coordinated action with SA respectively, a-amanitine or cycloheximide was able to induce lipid peroxidation effectively, but strongly blocked the activation of PR-1 gene expression by SA in tobacco cell culture. These results suggested that the generation of reactive metabolites or free radicals, which were induced by SA or other inducers through reaction with catalase or other compounds, initiated lipid peroxidation, subsequently activated pathogen-resistance genes expression. Obviously the lipid peroxidation molecule played an important role in SA signal transduction in tobacco.     

Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N ^ω-nitro- l -arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO₂ and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na⁺,K⁺-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

Effects of free radicals and oxidants on myocardial cellular injury

Harmful effects of some biochemically generated free radicals and oxidants, including superoxide anions (O2-), hydroxyl radical (OH.), hydrogen peroxide, hypochlorous acid, and hypohalite radical, on isolated cardiac myocytes were compared in an attempt to identify the exact nature of the free radicals/oxidants responsible for myocardial ischemic-reperfusion injury. All of these free radicals/oxidants, with the exception of O2-, caused significant injury to the myocytes as evidenced by the enhanced lactate dehydrogenase and creatine kinase release as well as by morphologic examinations, simultaneously causing lipid peroxidation and oxidized glutathione release from the cells, OH. being the most detrimental of all.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Oxidative Processes Induced by <Emphasis Type="Italic">tert</Emphasis>-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 +/- 6.4 kJ/mol, and that of oxyHb oxidation was 108 +/- 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.     

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.     

Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes

Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation.     

Synthesis of acylated SOD derivatives which bind to the biomembrane lipid surface and dismutate extracellular superoxide radicals

Involvement of oxygen radicals in the pathogenesis of various inflammatory diseases has been the focus of recent attention. Since lipid peroxidation of cell membranes is postulated to be one of the major reasons for radical-induced tissue injury, inhibition of oxygen toxicity at or near plasma membranes is important. To metabolize extracellular superoxide radicals effectively at or near cell membranes, we synthesized amphipathic superoxide dismutase (SOD) derivatives (AC-SOD) by covalently linking hydrophobic fatty acids with different chain lengths, such as caprylic acid, capric acid, lauric acid and myristic acid, to the lysyl amino groups of the enzyme. When incubated with erythrocytes or polymorphonuclear leukocytes (PMNs), AC-SOD, but not SOD, bound to plasma membranes of these cells. When topically instilled to the eye, AC-SOD also bound to corneal epithelial cell surface. Upon activation by phorbolmyristyl acetate, extracellular cytochrome c was rapidly reduced by PMNs which were pretreated with SOD. In contrast, PMNs preincubated with AC-SOD failed to catalyze the reduction of cytochrome c under the same experimental conditions. These results suggested that AC-SOD bound to cell membranes and effectively dismutated superoxide radicals at or on the outer surface of plasma membranes.     

Effect of cryopreservation on lipid peroxidation in chick cornea.

A mechanism suggested to cause injury to the preserved organs in vitro is the generation of oxygen free radicals either during preservation or after transplantation due to reperfusion. Methods to suppress generation of oxygen free radicals may lead to improved methods of organ preservation. In this study, increase in the levels of lipid peroxidation in chick cornea after cryopreservation is reported. Addition of fetal bovine serum (FBS) in cryopreservation medium was found to prevent lipid peroxidation. Addition of FBS was also found to be protective towards corneal viability during cryopreservation.     

Ethanol-Induced Cell Death by Lipid Peroxidation in PC12 Cells

Free radical generation is hypothesized to be the cause of alcohol-induced tissue injury. Using fluorescent cis-parinaric acid and TBARS, lipid peroxidation was shown to be increased in the presence of trace amounts of free ferrous ion in PC12 cells. This increase in lipid peroxidation was enhanced by ethanol in a dose dependent manner and also correlated with loss of cell viability, as measured by increased release of lactate dehydrogenase (LDH). Resveratrol, a potent antioxidant, had a protective effect against lipid peroxidation and cell death. These findings strongly suggest that ethanol-induced tissue injury and cell death is a free radical mediated process, and may be important in alcohol-related premature aging and other degenerative diseases.     

Free radicals and their biological significance: present and future]

Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.     

Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max)

Inhibition of root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI₃) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the root tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe²⁺ (FeSO₄). A close relationship existed between lipid peroxidation and inhibition of root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of root tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

Measurement of doxorubicin-induced lipid peroxidation under the conditions that determine cytotoxicity in cultured tumor cells.

We have investigated doxorubicin-induced lipid peroxidation by the measure of malondialdehyde (MDA) formation in rat glioblastoma cells and human breast carcinoma cells in culture. There was a significant production of MDA when the cells were incubated with pharmacologically relevant doxorubicin concentrations, i.e., concentrations that produce a significant cytotoxicity (0.1 micrograms/ml). At equitoxic doses, vincristine provided no lipid peroxidation, indicating that MDA formation is not a consequence of cell death. Doxorubicin-induced lipid peroxidation was maximal 24 h after incubation of the cells with doxorubicin, indicating that a delay was necessary for the free radical-mediated membrane damage induced by doxorubicin. In the presence of alpha-tocopherol in the culture medium, the doxorubicin-induced MDA formation was inhibited. The development of this method will help in defining the role of free radicals and lipid peroxidation in the cytotoxicity of doxorubicin.     

Evidence of direct toxic effects of free radicals on the myocardium

The hypothesis that oxygen-derived free radicals do indeed play a role in myocardial ischemic and reperfusion injury has received a lot of support. Experimental results have shown that free radical scavengers can protect against certain aspects of myocardial ischemic injury and that on reperfusion the heart approaches a level that is more normal than those hearts not receiving additional scavenging agents. Superoxide dismutase, catalase, glutathione peroxidase, hydroxyl radical scavengers and iron chelators such as desferrioxamine have proven successful in providing an increased level of recovery. These results indicate, as would be expected, that superoxide, hydrogen peroxide and hydroxyl radicals may all, at some point, either contribute to the injury or be important in generating a subsequent radical which causes damage. In addition, solutions capable of generating free radicals have been shown to cause damage to myocardial cells and the vascular endothelium that is similar to the damage observed during myocardial ischemic and reperfusion injury. Alterations in function, structure, flow, and membrane biochemistry have been documented and compared to ischemic injury. The continued investigation of the role of free radicals in ischemic injury is warranted in the hope of further elucidating the mechanisms involved in free radical injury, the sources of their generation, and in defining a treatment that will provide significant protection against this particular aspect of ischemic damage.