In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: An ESR spin-trapping investigation

Abstract

In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a^N =14.86±0.03 G and a^H_β =2.48±0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl₃ (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2^?/^?), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.     

Sexual dimorphism in the acute effects of secondhand smoke on thyroid hormone secretion, inflammatory markers and vascular function

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.     

Separable detection of lipophilic- and hydrophilic-phase free radicals from the ESR spectrum of nitroxyl radical in transient MCAO mice

Free radicals are believed to be key factors that promote ischemia reperfusion injury in the brain. This study used the characteristic spectrum of methoxycarbonyl-PROXYL to detect free radical reactions in hydrophilic and lipophilic compartments in a transient middle cerebral artery occlusion (MCAO) mouse model. Methoxycarbonyl-PROXYL, which has a high water/octanol partition coefficient, allows the detection of nitroxyl radical in both compartments simultaneously. Free radicals generation was analysed from the enhanced ESR signal decay rate of methoxycarbonyl-PROXYL. The signal decay rate in the lipidic compartment was significantly enhanced 1 h after reperfusion following MCAO. The enhanced signal decay rate was significantly suppressed by Trolox. The accumulation of lipid peroxidation products increased by 6 h post-reperfusion and was suppressed by methoxycarbonyl-PROXYL or Trolox. These results demonstrate that information pertaining to different sites of free radical generation in vivo can be obtained simultaneously and that lipid-derived radicals are generated in transient MCAO mice.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: Application of profluorescent nitroxide switch

Abstract

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical–radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical-radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Spin trapping of lipid-derived radicals in liposomes

Electron-spin resonance-spin trapping has been used to detect lipid-derived radicals in liposomes. Using the lipid-soluble spin trap 2-methyl-nitrosopropane (MNP), we have detected both the lipid and hydrogen-atom spin adducts in liposomes composed of a fully saturated phospholipid (dimyristoylphosphatidylcholine, DMPC) with various mol fractions of unsaturated phospholipid (1-palmitoyl-2-arachidonoylphosphatidylcholine, PAPC) or fatty acid (arachidonic acid, AA). The lipid-derived spin adduct formed during autoxidation of liposomes was separated by thin-layer chromatography and found to co-migrate with the product(s) formed by direct addition of MNP to the corresponding unsaturated lipid or fatty acid. Both the MNP-PAPC and MNP-AA spin adducts showed some restriction of rotational motion when in the liposome bilayer (rotational correlation times 0.72 and 0.69.10(-9) s, respectively), and nitrogen hyperfine coupling constants (14.94-14.96 G) consistent with a hydrophobic localization. Radical versus non-radical mechanisms of spin adduct formation during liposome autoxidation were separated using alpha-tocopherol as a radical scavenger. The utility of nitroso spin traps in trapping of radicals in liposomes is discussed.     

Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.     

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.     

Mechanisms involved in gastric protection of melatonin against oxidant stress by ischemia-reperfusion in rats

The generation of oxygen-derived free radicals has been suggested to be significantly responsible for ischemia-reperfusion injury in gastrointestinal tissues. Biochemical mechanisms include the xanthine-oxidase-derived oxidants mainly the superoxide anion. Both in vitro and in vivo studies have demonstrated that the pineal hormone melatonin possesses free radical scavenging and antioxidant properties. The indolamine has been effective in reducing the induced-oxidative damage in several tissues and biological systems. The aim of this study was to elucidate additional antioxidant mechanisms responsible for the gastroprotection afforded by the indolamine in ischemia-reperfusion gastric injury. Therefore, changes of related enzymes such as xanthine-oxidase, superoxide dismutase, glutathione reductase and total glutathione were investigated. Our results showed that treatment with 5, 10 or 20 mg kg(-1) of melatonin, administered i.p., clearly diminished the percentage of damage to 49.56 +/- 17.20, 37.54 +/- 11.40 and 26.70 +/- 8.12 respectively. Histologically there was a reduction of exfoliation of superficial cells and blood cell infiltration. These protective effects were related to a significant reduction of xanthine-oxidase activity (2.23 +/- 0.38 U/mg prot x 10(-4) with the highest tested dose of melatonin) and significant increases in superoxide dismutase reaching a value of 6.20 +/- 0.56 U/mg prot with 25 mg/Kg of melatonin and glutation reductase activities (417.44 +/- 29.72 and 649.43 +/- 81.11 nmol/min/mg prot with 10 and 20 mg/Kg of melatonin). We conclude that the free radical scavenger properties of melatonin mainly of the superoxide anion, probably derived via the xanthine-oxidase pathway, and the increase of antioxidative enzymes significantly contributes to mediating the protection by the hormone against ischemia-reperfusion gastric injury.     

In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used alpha-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration.     

Free radical involvement in the oxidative phenomena induced by tert-butyl hydroperoxide in erythrocytes

Free radical involvement in the oxidative events induced by tert-butyl hydroperoxide in erythrocytes has been demonstrated by the use of the electron spin resonance technique of spin trapping with the spin trap 5.5-dimethyl-1-pyrroline-N-oxide (DMPO). The reactions of tert-butyl hydroperoxide with haemoglobins and intact cell systems were studied. Oxyhaemoglobin-containing system showed exclusive production of the t-butyloxy radical spin adduct of DMPO (DMPO-OBut), indicating t-butyloxy radical production. Methaemoglobin-containing systems showed the production of an oxidised derivative of DMPO, 5,5-dimethyl-2-ketopyrrolidino-1-oxyl (DMPOX)-previously associated with the generation of highly oxidised haem-iron. Carbon monoxyhaemoglobin-containing systems show the production of both DMPO-OBut and DMPOX but markedly slower than in either of the other haemoglobin systems. Generally, free radical production in haemoglobin systems was faster than in intact cell systems, indicating a membrane transport rate-limiting step for the tert-butyl hydroperoxide-mediated effects. Data from the use of free radical scavengers to inhibit DMPO-OBut production was consistent with the known reactivities of the scavengers toward t-butyloxy radicals. These and previously reported results (Trotta, R. J., Sullivan, S. G. and Stern, A. (1981) Biochim. Biophys. Acta 679, 230-237 and (1982) Biochem. J. 204, 405-415) implicate important roles for t-butyloxy radicals and haem intermediates in tert-butyl hydroperoxide-induced lipid peroxidation and haemoglobin oxidation in erythrocytes, respectively.     

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.     

Spin trapping evidence for alcohol-associated oxidative stress

The spin trapping method combined with EPR spectroscopy has been employed by a number of laboratories to study alcohol-induced oxidative stress. Ethanol is converted to a free radical metabolite, the 1-hydroxyethyl radical in chemical reactions that generate hydroxyl radicals, in incubations of rat liver microsomes and in vivo. Furthermore, both acute and chronic ethanol administration leads to formation of radicals in the liver that are presumed to be products of lipid peroxidation. In general, overall radical production is enhanced by treatments known to be involved in alcoholic liver injury, such as chronic alcohol administration along with high levels of dietary fat.     

病毒感染致病与自由基的损伤

以流感病毒A/FM1/1/47(H₁N₁)鼠适应株,鼻腔内接种感染小鼠为模型,探讨了病毒感染过程中,自由基的产生以及在致病过程中的作用.结果表明,感染病毒的小鼠肺组织中氧自由基水平和黄嘌呤氧化酶活性显著升高,并与肺组织损伤和死亡率之间呈正相关.提示,氧自由基参与了病毒感染小鼠的致病过程,是造成组织损伤的重要因素.     

Iron and free radical oxidations in cell membranes.

Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive.     

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.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro

Free radical reactions involved in the metabolism of carbon tetrachloride by rat liver have been considered to be a cause of at least part of the injury resulting from exposure to this halocarbon. In an earlier study employing electron spin resonance and spin-trapping techniques, we demonstrated that trichloromethyl (13.CCl3) radicals are readily observed in rat liver microsomes metabolizing 13CCl4, and that the same radical could be shown to form in vivo in the liver of intact rats given a single dose of 13CCl4. This report describes the production of lipid dienyl (L.) and oxygen-centered lipid radicals (LO. or LOO., or both) in in vitro systems metabolizing 13CCl4, and also the formation of lipid dienyl radicals (L.) in liver of intact animals exposed to CCl4. The radicals appear to be produced in a sequence of reactions governed among other things by the oxygen tension in the system. The lipid radicals (L.) which form in intact liver of CCl4-treated rats are apparently the result of an attack on lipids of the endoplasmic reticulum by 13.CCl3 radicals formed by reductive cleavage to CCl4 and are the initial intermediates in the process of lipid peroxidation. These investigations demonstrate that while the events occurring in liver microsomes in vitro appear to parallel those which take place in intact liver in vivo, the conditions in vivo make the spin-trapping studies of radicals in intact animals much more selective than it is in vitro for a given spin trap, and requires the use of more than one type of spin-trapping agent to detect different radical species in vivo.