Iron-catalyzed Haber-Weiss Reaction Research Articles

Enhanced autotrophic astaxanthin production from Haematococcus pluvialis under high temperature via heat stress-driven Haber–Weiss reaction

High temperatures (30-36 °C) inhibited astaxanthin accumulation in Haematococcus pluvialis under photoautotrophic conditions. The depression of carotenogenesis was primarily attributed to excess intracellular less reactive oxygen species (LROS; O2 (-) and H2O2) levels generated under high temperature conditions. Here, we show that the heat stress-driven inefficient astaxanthin production was improved by accelerating the iron-catalyzed Haber-Weiss reaction to convert LROS into more reactive oxygen species (MROS; O2 and OH·), thereby facilitating lipid peroxidation. As a result, during 18 days of photoautotrophic induction, the astaxanthin concentration of cells cultured in high temperatures in the presence of iron (450 μM) was dramatically increased by 75 % (30 °C) and 133 % (36 °C) compared to that of cells exposed to heat stress alone. The heat stress-driven Haber-Weiss reaction will be useful for economically producing astaxanthin by reducing energy cost and enhancing photoautotrophic astaxanthin production, particularly outdoors utilizing natural solar radiation including heat and light for photo-induction of H. pluvialis.

The time course of hydroxyl radical formation following spinal cord injury: the possible role of the iron-catalyzed Haber-Weiss reaction.

This study explores whether the hydroxyl radical (*OH)-one of the most destructive reactive oxygen species-plays a role in secondary spinal cord injury (SCI). First, we measured the time course of *OH formation in rat spinal tissue after impact SCI by administering salicylate as a trapping agent into the intrathecal space of the cord and measuring the hydroxylation products of salicylate, 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA) by HPLC. The 2,3-DHBA concentration was significantly higher in injured spinal tissue than in sham controls at 5 min, 1 and 3 h, but not at 5 h post-injury. Second, we generated *OH by administering H(2)O(2) and FeCl(2)/EDTA (Fenton's reagents) at the concentrations produced by SCI into the gray matter of the cord for 4 h and found that it induced significant cell loss at 24 h post-*OH exposure. Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP)-a broad spectrum reactive species scavenger-significantly reduced *OH-induced cell death. Finally, we generated superoxide and administered FeCl(3)/EDTA in the intrathecal space of the cord at the concentration produced by SCI and measured extracellular *OH formation in the gray matter of the cord by microdialysis sampling. We found that the levels of *OH significantly increased compared to the pre-administration level, indicating that *OH can be produced in vivo by the iron-catalyzed Haber-Weiss reaction. All together, we demonstrated that *OH is an endogenous secondary damaging agent following SCI and the metal-catalyzed Haber-Weiss reaction may contribute to early *OH formation after SCI.

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species.

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.

Iron and gallium increase iron uptake from transferrin by human melanoma cells: further examination of the ferric ammonium citrate-activated iron uptake process

Previously we showed that preincubation of cells with ferric ammonium citrate (FAC) resulted in a marked increase in Fe uptake from both 59Fe-transferrin (Tf) and 59Fe-citrate (D.R. Richardson, E. Baker, J. Biol. Chem. 267 (1992) 13972–13979; D.R. Richardson, P. Ponka, Biochim. Biophys. Acta 1269 (1995) 105–114). This Fe uptake process was independent of the transferrin receptor and appeared to be activated by free radicals generated via the iron-catalysed Haber-Weiss reaction. To further understand this process, the present investigation was performed. In these experiments, cells were preincubated for 3 h at 37°C with FAC or metal ion solutions and then labelled for 3 h at 37°C with 59Fe-Tf. Exposure of cells to FAC resulted in Fe uptake from 59Fe-citrate that became saturated at an Fe concentration of 2.5 μM, while FAC-activated Fe uptake from Tf was not saturable up to 25 μM. In addition, the extent of FAC-activated Fe uptake from citrate was far greater than that from Tf. These results suggest a mechanism where FAC-activated Fe uptake from citrate may result from direct interaction with the transporter, while Fe uptake from Tf appears indirect and less efficient. Preincubation of cells with FAC at 4°C instead of 37°C prevented its effect at stimulating 59Fe uptake from 59Fe-Tf, suggesting that an active process was involved. Previous studies by others have shown that FAC can increase ferrireductase activity that may enhance 59Fe uptake from 59Fe-Tf. However, there was no difference in the ability of FAC-treated cells compared to controls to reduce ferricyanide to ferrocyanide, suggesting no change in oxidoreductase activity. To examine if activation of this Fe uptake mechanism could occur by incubation with a range of metal ions, cells were preincubated with either FAC, ferric chloride, ferrous sulphate, ferrous ammonium sulphate, gallium nitrate, copper chloride, zinc chloride, or cobalt chloride. Stimulation of 59Fe uptake from Tf was shown (in order of potency) with ferric chloride, ferrous sulphate, ferrous ammonium sulphate, and gallium nitrate. The other metal ions examined decreased 59Fe uptake from Tf. The fact that redox-active Cu(II) ion did not stimulate Fe uptake while redox-inactive Ga(III) did, suggests a mechanism of transporter activation not solely dependent on free radical generation. Indeed, the activation of Fe uptake appears dependent on the presence of the Fe atom itself or a metal ion with atomic similarities to Fe (e.g. Ga).

The Haber–Weiss reaction and mechanisms of toxicity

The concept that the highly reactive hydroxyl radical (HO ) could be generated from an interaction between superoxide (O 2 − ) and hydrogen peroxide (H 2O 2) was proposed (with Joseph Weiss) in Professor Haber’s final paper published in 1934. Until it was recognized that free radicals are produced in biological systems, this finding seemed to have no relevance to biology. However, following the discovery that O 2 − was a normal cellular metabolite, it was quickly recognized that the Haber–Weiss reaction (O 2 − +H 2O 2 → HO +O 2+HO −) might provide a means to generate more toxic radicals. Although the basic reaction has a second order rate constant of zero in aqueous solution and thus cannot occur in biological systems, the ability of iron salts to serve as catalysts was discussed by these authors. Because transition metal ions, particularly iron, are present at low levels in biological systems, this pathway (commonly referred to as the iron-catalyzed Haber–Weiss reaction) has been widely postulated to account for the in vivo generation of the highly reactive HO . Recent data documenting the importance of redox regulation of various cellular signaling pathways makes it clear that free radicals are essential for normal cellular function. However, this also makes it obvious that disruptions of free radical production or defenses at many different levels can lead to adverse effects on cells. While the generation of HO , which is by far the most reactive oxygen species, is generally indicative of an overtly toxic event, it is through studies at this level that we have reached a better understanding of free radicals as both signaling molecules and toxic species.

Alternative RNA Splicing Generates a Glycosylphosphatidylinositol-anchored Form of Ceruloplasmin in Mammalian Brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.

Iron and infection

Intravenous iron therapy maintains iron stores and decreases erythropoietin demand in patients undergoing regular dialysis therapy. Microbiology studies show a close relationship between the availability of iron and bacterial virulence. Iron is also an essential requirement of bacteria for multiplication in the host. Therefore, clinical conditions associated with iron excess in the host may increase the risk for infection. Parenteral iron has already been shown in human and animal studies to be harmful when administered during infection. There is now convincing evidence that hydroxyl radicals, produced either by the Fenton reaction or by the iron-catalyzed Haber-Weiss reaction, are species responsible for the damaging effects of iron. The unavailability of iron limits microbial growth but also impairs host resistance. In end-stage renal disease, patients' overtreatment with iron enhances the pre-existing risk for infectious complications caused by dialysis procedure per se, malnutrition, increased intracellular calcium, as well as low and high molecular weight uremic toxins. Intravenous iron therapy may not only adversely affect phagocytes in end-stage renal disease patients, but also T and B lymphocytes.

Alterations in phosphatidylcholine metabolism of stretch-injured cultured rat astrocytes.

The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 microM unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [3H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretreating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (±)- myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (±)- myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri- O-benzoyl- myo-inositol and (±)-3,4,5,6-tetra- O-benzyl- myo-inositol were phosphorylated with dibenzyl N, N-di-isopropylphosphoramidite in the presence of 1 H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (±)-1- O-( tert-butyldiphenylsilyl)-2,3- O-cyclohexylidene- myo-inositol and (±)-4,5,6-tri- O-benzoyl-1- O-( tert-butyldiphenylsilyl)-2,3- O-cyclohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (±)- myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO . production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (±)- myo-inositol 1,2-bisphosphate potentiated HO . production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Oxidative stress increases glyceraldehyde-3-phosphate dehydrogenase mRNA levels in isolated rabbit aorta.

We have recently shown that inhibition of endogenous Cu,Zn superoxide dismutase (SOD) by diethyldithiocarbamate (DDC) increased superoxide anion levels in isolated rabbit aortic rings, describing a useful experimental model to examine the effects of oxidative stress on the vessel wall. The present study examined the effects of oxidative stress on the steady-state mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Aortic rings were incubated in physiological salt solution at 37 degrees C for up to 6 h. DDC (2 mM) decreased total SOD activity to < 5% of control levels and increased superoxide anion level ninefold. Steady-state mRNA levels of GAPDH were increased under comparable conditions. Although decreased biological activity of endothelium-derived nitric oxide was indicated by lower basal guanosine 3',5'-cyclic monophosphate levels in aortic rings treated with DDC compared with those in control rings (1.2 +/- 0.1 vs. 1.9 +/- 0.3 fmol/microgram protein, P < 0.05), neither endothelium denudation nor NG-nitro-L-arginine methyl ester had any effects on the steady-state mRNA levels of GAPDH. The cell.permeable iron chelator 1,10-phenanthroline completely prevented the increases in GAPDH mRNA levels induced by DDC. These results suggest that oxidative stress resulting from inhibition of endogenous Cu,Zn SOD causes induction of GAPDH gene expression and that the hydroxyl radical, produced through the iron-catalyzed Haber-Weiss reaction, is the intracellular reactive oxygen species responsible for the DDC-stimulated increase in GAPDH mRNA.

Identification of a mechanism of iron uptake by cells which is stimulated by hydroxyl radicals generated via the iron-catalysed Haber-Weiss reaction

Recent studies have demonstrated that preincubation of SK-Mel-28 melanoma cells with ferric ammonium citrate (FAC) resulted in marked stimulation of 59Fe uptake from 59Fe- 125I-transferrin (Tf), but only at Tf concentrations above that required for saturation of the Tf receptor (Richardson and Baker (1992) J. Biol. Chem. 267, 13972–13979). The mechanism responsible for this stimulation was unknown and is the subject of the present report. Preincubation of cells with FAC (25 μg/ml), followed by a 2 h incubation with 59Fe- 125I-Tf (0.1 mg/ml; 1.25 μM), resulted in temperature-dependent 59Fe uptake to approx. 200% of the control value. Furthermore, the effect was not specific for melanoma cells and was also observed in other normal and neoplastic cells. Preincubation of melanoma cells with FAC also stimulated 59Fe uptake from 59Fe-citrate, but to a far greater extent than that observed with 59Fe- 125I-Tf (viz., > 20-fold that seen for the control). Interestingly, neither receptor-mediated endocytosis nor the postulated diferric Tf reductase were involved in the FAC-activated Fe uptake process from Tf. However, the addition of free radical scavengers to FAC such as catalase, superoxide dismutase, ceruloplasmin, Hepes, mannitol and high concentrations of BSA or ascorbate, markedly depressed FAC-activated 59Fe uptake from 59Fe- 125I-Tf and 59Fe-citrate. These agents when added to control cells had no effect on 59Fe uptake. The addition of superoxide generating agents and hydrogen peroxide to minimum essential medium (MEM) containing FAC but not to MEM alone, also stimulated 59Fe uptake. These data suggest that the initial activation of the FAC-stimulated Fe uptake system was caused by the production of hydroxyl radicals via the Fe-catalysed Haber-Weiss reaction. We propose that this Fe uptake process represents an important cellular defense mechanism against oxidant stress generated in the presence of low-molecular-weight Fe complexes.

Action of Reactive Oxygen Species and Their Antagonists on Twitch Tension of the Rat Phrenic Nerve‐Diaphragm

Reactive oxygen species have been implicated in normal and pathological processes of many tissues, including skeletal muscle. I extended previous studies by examining the effect of these intermediates and eight of their antagonists (superoxide dismutase, catalase, deferoxamine, [Cu(II)]2(3,5-diisopropylsalicylate)4, 1,2-dimethyl-3-hydroxy-pyridone, 1,3-dimethyl-2-thiourea, N-(2-mercaptopropionyl)-glycine, vitamin E) on indirectly stimulated twitch tension of an in vitro neuroskeletomuscular preparation, the phrenic nerve-diaphragm of the rat. In the absence of exogenous reactive oxygen species, none of the antagonists potentiated twitch tension, and all but one (N-[2-mercaptopropionyl]-glycine) of the membrane-permeant antagonists attenuated twitch tension. The reactive oxygen intermediate-generating system of purine plus xanthine oxidase reduced indirectly stimulated twitch tension by 36% while having no effect on directly stimulated twitch tension. Catalase (but not superoxide dismutase or deferoxamine) eliminated the reduction in twitch tension, indicating that hydrogen peroxide played a role in the reduction. The membrane-permeant antagonists [Cu(II)]2(3,5-diisopropylsalicylate)4 and 1,2-dimethyl-3-hydroxy-pyridone also eliminated the reduction in twitch tension caused by reactive oxygen species, suggesting that hydrogen peroxide could have acted intracellularly through an iron-catalyzed Haber-Weiss reaction to produce hydroxyl radical, which in turn reacted with intracellular components, thereby reducing twitch tension.

Nitrofurantoin-Stimulated Reactive Oxygen Species Production and Genotoxicity in Digestive Gland Microsomes and Cytosol of the Common Mussel (Mytilus edulis L.)

The ability of nitrofurantoin (NF) to produce reactive oxygen species (ROS) was investigated in subcellular fractions of digestive gland of the mussel Mytilus edulis in terms of oxygen consumption and the formation of superoxide anion radical (O2-) (measured as SOD-sensitive cytochrome c reduction or SOD-sensitive sensitive .OH production), H2O2 (effect of catalase), and hydroxyl radical (.OH) (iron/EDTA-mediated oxidation of KMBA to ethylene). Additionally, the genotoxic effects of NF were examined using the Salmonella typhimurium umu mutagenicity assay. Microsomal NAD(P)H-dependent oxygen consumption was stimulated by NF, leading to the formation of H2O2. Stimulation of microsomal O2- production by NF was evident for NADH but not NADPH, confirming redox cycling at least with the former coenzyme. No stimulation of O2- production was obvious for cytosolic fraction with either coenzyme. NF stimulated microsomal NAD(P)H-dependent .OH production; the rates of .OH production were greater for NADH than NADPH; and the .OH was indicated to be formed, at least in part, by an iron-catalyzed Haber-Weiss reaction. A role was indicated for a free radical driven Fenton reaction in the NF-stimulated microsomal production of .OH from NADPH. The production of mutagenic species from NF was observed for cytosol but not for microsomes, and the former effects were greater for NADH than NADPH. Overall, the NAD(P)H-dependent microsomal generation of ROS, and the lack of correlation of ROS production with mutagenicity, are considered indicative of the potential of digestive gland to metabolize NF by both one-electron and two-electron reductive pathways. From this and other studies, enhanced ROS production by NF and other redox cycling xenobiotics is indicated to be a widespread phenomenon in aquatic organisms and a potential mechanism of pollutant-mediated toxicity.

Free radical induction in the brain and liver by products of toluene catabolism

Toluene and its metabolites have been studied with respect to their reactive oxygen species-enhancing potential in isolated systems and in vivo. The induction of reactive oxygen species (ROS) production was assayed using the probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA). Intraperitoneal injection of toluene, benzyl alcohol or benzaldehyde caused a significant elevation in the rate of ROS formation within hepatic mitochondrial fractions (P2). In the brain, only toluene induced ROS formation, while benzyl alcohol and benzaldehyde did not have any effect. Glutathione (GSH) levels were depressed in liver and brain regions from toluene-treated rats. However, no such depression was evident in brains treated with toluene metabolites. P2 fractions from phenobarbital-pretreated rats exhibited a heightened ROS response when challenged with toluene, in vitro. Pretreatment of rats in vivo with 4-methylpyrazole, an alcohol dehydrogenase inhibitor, or sodium cyanamide, an aldehyde dehydrogenase inhibitor, prior to exposure to toluene, caused a significant decrease and increase, respectively, in toluene-stimulated rates of ROS generation in the CNS and liver. Electron spin resonance spectroscopy, employing the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was conducted. Incubation of the spin trap with P2 fractions and toluene or benzaldehyde elicited a spectrum corresponding to the hydroxyl radical. Incubation of benzaldehyde with aldehyde dehydrogenase produced a strong signal that was blocked completely by Superoxide dismutase and inhibited partially by catalase, suggesting the presence of Superoxide radicals and the involvement of the iron-catalyzed Haber-Weiss reaction leading to the production of hydroxyl radicals. Thus, ROS generation during toluene catabolism may occur at two steps: cytochrome P450 oxidation and aldehyde dehydrogenase oxidation. In addition, GSH may play an important role in protection against the induction of ROS generation in the CNS and liver following exposure to toluene.

Oxygen radicals in cerebral ischemia.

Superoxide production was measured as the superoxide dismutase (SOD)-inhibitable portion of nitro blue tetrazolium (NBT) reduction after cerebral ischemia-reperfusion in anesthetized cats equipped with cranial windows. Significant superoxide production was found in the early reperfusion period and continued for more than 1 h after ischemia. Superoxide was not detected in control animals not subjected to ischemia, during ischemia, and at 120 min of reperfusion. After ischemia, the vasoconstrictor response to arterial hypocapnia was reduced. This effect was prevented by pretreatment with SOD plus catalase or by deferoxamine. The response to topical acetylcholine was converted to vasoconstriction after ischemia. The normal vasodilator response reappeared spontaneously at 120 min of reperfusion. The vasodilator response to acetylcholine was preserved in animals pretreated with SOD plus catalase. Blood-brain barrier permeability to labeled albumin and horseradish peroxidase was increased after ischemia. These effects were minimized by pretreatment with SOD and catalase. We conclude that superoxide generation occurs during reperfusion after cerebral ischemia for a fairly long period and that superoxide and its derivatives are responsible at least in part for the vasodilation and the abnormal reactivity as well as for the increase in blood-brain barrier permeability to macromolecules seen after ischemia. Furthermore, the findings suggest that the agent responsible for the vascular abnormalities is hydroxyl radical generated via the iron-catalyzed Haber-Weiss reaction.

Generation of Semiquinone and Oxygen Radicals by the Reaction of Menadione with Reduced Glutathione at Various pH.

Semiquinone radicals of menadione were generated during the reaction of menadione with reduced glutathione (GSH), dependent upon the pH. Under aerobic conditions, cytochrome c was reduced during the reaction, and superoxide dismutase (SOD) inhibited the cytochrome c reduction. The inhibitory effect of SOD was greater at a high pH than at a low pH. In the presence of Fe3+ or ethylenediaminetetraacetic acid (EDTA)-Fe3+, deoxyribose was degraded during the reaction of menadione with GSH, dependent upon the pH. Greater amounts of deoxyribose were degraded at a low pH than at a high pH. The reduction of Fe3+ of EDTA-Fe3+ also depended on the pH, and SOD strongly inhibited the Fe3+ reduction, indicating that Fe3+ or Fe3+-EDTA was reduced by superoxide. SOD, catalase, mennitol and benzoate inhibited the deoxyribose degradation at various pH values. These results indicate that the menadione semiquinone radical is readily formed at an alkali pH but a hydroxyl radical is predominantly produced near a neutral pH. A hydroxyl radical may be generated via an iron-catalyzed Haber-Weiss reaction.

Protective Effects of Antioxidants on Paraquat-Induced Acute Renal Failure in Mice

The protective effects of antioxidants against elevated blood urea nitrogen (BUN) were studied in newly developed acute renal failure in mice induced by paraquat. The administration of paraquat caused marked azotemia accompanied by a decreased glomerular filtration rate. In contrast, elevated BUN was significantly reduced by the preadministration of either dimethylthiourea, deferoxamine or α-tocopherol. These data suggest that acute renal failure induced by paraquat is mainly related to the hydroxyl radicals produced via the iron-catalyzed Haber-Weiss reaction.

Electron spin resonance spectroscopy of oxygen radicals generated by synthetic fecapentaene-12 and reduction of fecapentaene mutagenicity to Salmonella typhimurium by hydroxyl radical scavenging.

Fecapentaenes form a class of potent fecal mutagens and have been suggested to play an initiating role in colon carcinogenesis. Although several indications have been found that fecapentaenes may induce oxidative DNA damage as well as DNA alkylation, the mechanism of genotoxicity remains unknown. In this study, electron spin resonance spectroscopy with several spin traps has been used in order to determine whether reactive oxygen species can be formed by fecapentaene-12 (FP-12). No specific conditions could be defined that resulted in the direct formation of oxygen radicals from FP-12. However, peroxidation of FP-12 by various peroxidative enzymes has been shown to result in the formation of superoxide adducts of the spin traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of superoxide dismutase resulted in a decreased spectrum intensity, whereas the hydroxyl radical scavenger t-butyl alcohol (tBA) appeared of no influence on the signal, both confirming the formation of superoxide. The formation of hydroxyl radical spin adducts has been demonstrated after peroxidation of FP-12 in incubations with the spin-trapping agent 2,2,6,6-tetramethyl-piperidine (TMP). Further, the effects of scavenging hydroxyl radicals with respect to the genotoxic potential of FP-12 in the Salmonella mutagenicity assay has been investigated. It was clearly shown that radical scavenging reduced the number of revertants in Salmonella strains TA100, TA102 and TA104. This mutagenicity-reducing effect was more convincing using both spin traps DMPO and TMP as compared to the effect of hydroxyl radical scavengers tBA and DMSO. Based on these findings, a reaction scheme is proposed that suggests the formation of superoxide after peroxidation of FP-12, which is subsequently converted to hydroxyl radicals by the iron-catalysed Haber-Weiss reaction.

Reactive oxygen metabolites in endotoxin-induced acute renal failure in rats

Based on recent reports that reactive oxygen metabolites may play a role in endotoxin-induced injury in other tissues, we postulated that reactive oxygen metabolites may be important mediators of endotoxin-induced acute renal failure. Superoxide dismutase, a scavenger of superoxide, or catalase, which destroys hydrogen peroxide, did not protect against endotoxin-induced renal failure. Similarly, neither the hydroxyl radical scavenger dimethylthiourea nor the iron chelator deferoxamine (which presumably would act by preventing the generation of hydroxyl radical via the iron-catalyzed Haber-Weiss reaction) prevented the endotoxin-induced fall in renal function. In separate experiments, we found no increase in renal cortical lipid peroxidation (a marker of reactive oxygen metabolite-mediated tissue injury) in endotoxin-treated rats, providing further evidence against a role for reactive oxygen metabolites in endotoxin-induced renal injury. Finally, using the aminotriazole-induced inhibition of catalase (a measure of in vivo changes in the hydrogen peroxide generation) we found no evidence of enhanced hydrogen peroxide generation in the renal cortex in endotoxin-treated rats. Taken together, the data from these three separate experimental approaches suggest that reactive oxygen metabolites are not important mediators of endotoxin-induced acute renal failure.