Desulfonation of a colitis inducer 2,4,6-trinitrobenzene sulfonic acid produces sulfite radical.

2,4,6-Trinitrobenzene sulfonic acid (TNBS) has been used in vivo to induce colitis. With the nitroreductase of intestinal cells, TNBS underwent redox cycling to produce TNBS-nitro and superoxide radical anions which are thought to be involved in initial oxidative reactions that lead to colonic injury. In this study, we demonstrated that the TNBS desulfonative reaction with tissue amino acids produces sulfite which is subsequently oxidized to sulfite radical. Sulfite radical was measured using a spin trapping methodology. Sulfite radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were detected in a mixture of TNBS and lysine, xanthine oxidase, red blood cells, colonic mucosal or submucosal muscle tissues. TNBS alone did not produce sulfite radical, indicating that its formation required the presence of amino acids. Because sulfite radical is the precursor of highly reactive sulfiteperoxyl and sulfate radicals, our data imply that these sulfite-derived free radicals may also contribute to oxidative reactions leading to colonic injury in TNBS-induced colitis.

Generation of nitro and superoxide radical anions from 2,4,6-trinitrobenzenesulfonic acid by rat gastrointestinal cells.

Reactive oxygen and nitrogen species have been implicated in the inflammation of the gastrointestinal tract. The objective of this study was to investigate mechanisms of free radical formation from the colitis inducer 2,4,6-trinitrobenzene sulfonic acid (TNBS). We showed that TNBS was rapidly metabolized to TNBS nitro radical anion via metabolic reduction by flavinmononucleotide/NADPH, xanthine/xanthine oxidase as well as the rat small intestine and colon. TNBS nitro radical anion was directly detected with electron paramagnetic resonance (EPR) spectroscopy. EPR spectra of TNBS nitro radical anion showed hyperfine coupling constants from the proximal nitrogen, two hydrogens and the two distal nitrogens with respective magnitudes of a(N)(4) = 9.7 G; a(H)(3,5) = 3.2 G (2); and a(N)(2,6) = 0.25 G. EPR spin trapping using 5.5-dimethyl-1-pyrroline N-oxide in aerobic incubations of isolated enterocytes (or colonocytes, or red blood cells) and TNBS, in the presence or absence of NADPH, produced radical adducts characteristic of superoxide and hydroxyl radicals. Our EPR data showing generation of TNBS nitro and superoxide radical anions demonstrate that one-electron reduction of TNBS may be an initial step in the cascade of the in vivo inflammatory events in TNBS-induced colitis.

Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite.

The objective of this study was to investigate the ability of immune cells of the small intestine to produce highly reactive free radicals from the food additive sulfites. These free radicals were characterized with a spin-trapping technique using the spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the presence of glucose, purified lymphocytes from intestinal Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stimulated with phorbol 12-myristate 13-acetate (PMA) to produce superoxide and hydroxyl DEPMPO radical adducts. The formation of these adducts was inhibited by superoxide dismutase or diphenyleneiodonium chloride, indicating that these cells produced superoxide radical during reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. With the treatment of sodium sulfite, PMA-stimulated PP lymphocytes produced a DEPMPO-sulfite radical adduct and an unknown radical adduct. When DEPMPO was replaced with DMPO, DMPO-sulfite and hydroxyl radical adducts were detected. The latter adduct resulted from DMPO oxidation by sulfate radical, which was capable of oxidizing formate or ethanol. Oxygen consumption rates were further increased after the addition of sulfite to PMA-stimulated lymphocytes, suggesting the presence of sulfiteperoxyl radical. Taken together, oxidants generated by stimulated lymphocytes oxidized sulfite to sulfite radical, which subsequently formed sulfiteperoxyl and sulfate radicals. The latter two radicals are highly reactive, contributing to increased oxidative stress, which may lead to sulfite toxicity, altered functions in intestinal lymphocytes, or both.

The catalytic activity of iron in synovial fluid as monitored by the ascorbate free radical.

Human synovial fluid, from a patient with synovitis disease, was examined by electron spin resonance spectroscopy for evidence of free radicals. The ascorbate free radical was observed and its intensity was affected by iron chelating agents, demonstrating that the iron in the synovial fluid is indeed available for oxidative catalysis.

Free radical formation from organic hydroperoxides in isolated human polymorphonuclear neutrophils.

We have demonstrated with electron paramagnetic resonance (EPR) that organic hydroperoxides are decomposed to free radicals by both human polymorphonuclear leukocytes (PMNs) and purified myeloperoxidase. When tert-butyl hydroperoxide was incubated with either PMNs or purified myeloperoxidase, peroxyl, alkoxyl, and alkyl radicals were trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the case of ethyl hydroperoxide, DMPO radical adducts of peroxyl and alkyl (identified as alpha-hydroxyethyl when trapped by tert-nitrosobutane) radicals were detected. Radical adduct formation was inhibited when azide was added to the incubation mixture. Myeloperoxidase-deficient PMNs produced DMPO radical adduct intensities at only about 20-30% of that of normal PMNs. Our studies suggest that myeloperoxidase in PMNs is primarily responsible for the decomposition of organic hydroperoxides to free radicals. The finding of the free radical formation derived from organic hydroperoxides by PMNs may be related to the cytotoxicity of this class of compounds.

Nitrosylation of blood hemoglobin and renal nonheme proteins in autoimmune MRL-lpr/lpr mice.

MRL-lpr/lpr mice spontaneously develop manifestations of autoimmunity including arthritis, vasculitis, and glomerulonephritis. The paramagnetic molecule nitric oxide has been implicated as an effector molecule in initiation and propagation of these inflammatory conditions. In this study, we utilized electron paramagnetic resonance spectroscopy to directly detect nitrosylated protein complexes as products of nitric oxide in whole blood and in kidneys of MRL-lpr/lpr mice. Electron paramagnetic resonance spectra of blood samples from MRL-lpr/lpr mice showed nitrosyl hemoglobin species. Amounts of blood nitrosyl hemoglobin in MRL-lpr/lpr mice were significantly increased as compared to age-matched control mice. Electron paramagnetic resonance spectra of MRL-lpr/lpr kidney tissue exhibited a signal characteristic of a dinitrosyl-iron-dithiolate complex at g approximately 2.04. Formation of nitrosylated nonheme protein in diseased kidneys is associated with development of glomerulonephritis in the autoimmune mice. The presence of nitrosylated nonheme protein indicates the formation of nitric oxide within the kidneys of the diseased mice signifying in situ renal nitric oxide formation.

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism.

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.

EPR studies of nitric oxide interactions of alkoxyl and peroxyl radicals in in vitro and ex vivo model systems.

A model compound of lipid peroxidation, tert-butyl hydroperoxide (tBOOH), was used in vitro to investigate (i) the generation of tBOOH-derived free radicals by hematin or rat enterocytes and (ii) the modulation of cell-generated free radical production by a nitric oxide (NO) donor, or when these cells were primed to produce NO. In hematin-catalyzed decomposition of tBOOH, NO from nitrosoglutathione, or S-nitroso-N-acetylpenicillamine suppressed the generation of peroxyl radicals (measured by direct electron paramagnetic resonance) and tert-butylalkoxyl, methoxyl, and methyl radicals (measured by electron paramagnetic resonance spin trapping). Similarly, co-incubation of S-nitroso-N-acetylpenicillamine or nitrosoglutathione with tBOOH caused significant decreases in tBOOH-derived free radical generation catalyzed by enterocytes. Epithelial cells are the known source of the inducible form of NO synthase in the intestine of rats challenged with lipopolysaccharide (LPS). Enterocytes isolated from LPS-treated rats produced decreased levels of tBOOH-derived radicals. These decreases in free radical production were further decreased when these cells were treated with LPS in vitro. These findings demonstrated that exogenously added or endogenously produced NO could modulate the extent of tBOOH-derived free radical generation in enterocytes. These decreases in free radical production could, at least in part, describe the protective role of NO from hydroperoxide-induced injury.

Nitrosyl complex formation during endotoxin-induced injury in the rat small intestine.

The objective of this study was to demonstrate nitric oxide (NO) production and determine its role in the rat small intestine following endotoxin treatment. By using electron paramagnetic resonance (EPR) spectroscopy, we were able to detect high concentrations of nitrosylated proteins in the small intestines of rats administered 1 mg/kg lipopolysaccharide (LPS) and sacrificed 6 h later. EPR spectra of non-heme and heme nitrosyl complexes were detected in the epithelium layer and intestinal wall. Only EPR spectra characteristic of nitrosyl hemoprotein complexes were detected in the luminal contents of these rats. LPS administration elevated the concentrations of intestinal lipid peroxidation biomarkers, thiobarbituric acid-reactive substances, and conjugated dienes. These changes were attenuated by NO synthase inhibitor treatment. We conclude that oxidants associated with NO formation were at least in part involved in the oxidation of tissue lipids. This process may be one of the mechanisms of intestinal injury induced by LPS.

Endotoxin-induced oxidative stress in the rat small intestine: role of nitric oxide.

Reactive oxygen species have been implicated in the gastrointestinal pathogenesis of septic and endotoxic shock. The objective of this study was to investigate the role of inducible nitric oxide synthase during endotoxin-induced formation of oxidants by cells of the small intestine. After intravenous Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) injection, nitric oxide production was measured as nitrosyl complex formation in the ileum using electron paramagnetic resonance spectroscopy. Oxidative stress biomarkers were determined as duodenal mucosal-reduced thiols, the ileal lipid peroxidation and luminal free radical production using spin trapping methodology. Demonstration of nitrosyl complex formation commenced at 3 h and diminished 24 h post-LPS. Mucosal thiol levels were decreased at 3, 6, 12, and 18 h post-LPS treatment. At these time point, the ileal lipid peroxidation also increased as did luminal formation of hydroxyl radical adduct. Nitric oxide synthase inhibitors reversed the elevation of hydroxyl radical formation and reversed the decrease in mucosal-reduced thiol levels in the LPS-treated rats. Our data indicate that nitric oxide or its oxidant product(s), such as peroxynitrite, contribute to oxidative injury in the small intestine of rats treated with endotoxin.

Priming of Peyer's patch lymphoid cells by hemorrhagic shock and resuscitation to produce superoxide radical.

Hemorrhagic shock (HS) can cause whole body ischemia including the gastrointestinal tract. We investigated whether cells from small intestine Peyer's patches (PP) were capable of producing superoxide radical when animals underwent HS or HS followed by resuscitation (HS/RS). HS was initiated by removing 60% of the blood volume of surgically prepared guinea pigs. PP lymphoid cells were purified and stimulated with phorbol 12-myristate 13-acetate in the presence of spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Electron paramagnetic resonance spectra of PP lymphoid cells from sham-treated control, HS, and HS/RS animals produced DEPMPO radical adducts characterized as the adducts of superoxide (DEPMPO/*OOH) and hydroxyl (DEPMPO/*OH) radicals. The formation of both radical adducts was totally inhibited by superoxide dismutase or a nicotinamide adenine dinucleotide phosphate (reduced form) oxidase inhibitor, diphenyleneiodonium chloride. HS/RS increased radical adduct formation, expressed as a percentage control, by 160% and 225% for DEPMPO/*OOH, and DEPMPO/*OH, respectively. When animals were allowed to recover for 24 h post-HS/RS treatment, PP cells decreased the superoxide generation to the same level as controls. Thus, RS following HS may prime PP lymphoid cells for increased nicotinamide adenine dinucleotide phosphate (reduced form) oxidase-dependent superoxide generation, and this process may have cytotoxic and/or immunomodulatory effects on the host.

Phenyl N-tert-butyl nitrone forms nitric oxide as a result of its FE(III)-catalyzed hydrolysis or hydroxyl radical adduct formation.

Phenyl N-tert-butyl nitrone (PBN) is commonly employed in spin-trapping studies. We report here evidence that PBN in aqueous solutions is decomposed by two pathways leading to the generation of nitric oxide (.NO). The first pathway is by hydrolysis of PBN, which is strongly catalyzed by ferric iron. The second pathway is via PBN-hydroxyl radical adduct formation. .NO was trapped in the presence of cysteine and ferrous iron to form a [(cys)2Fe(NO)2]-3 complex, which was measured by use of electron paramagnetic resonance (EPR) spectroscopy. A concomitant metabolite, benzaldehyde, was detected from both reaction mixtures. We propose that PBN is hydrolyzed by Fe3+ or attacked by hydroxyl radical, leading eventually to a common transient species, tert-butyl hydronitroxide [t-BuN(O.)H], which is further oxidized to a .NO source, t-BuNO. Our data imply that PBN may decompose to .NO when used in biological models with oxidative stress conditions.

Electron paramagnetic resonance investigations of nitrosyl complex formation during endotoxin tolerance.

The prior administration of low dose endotoxin induces a state of hyporesponsiveness or tolerance to the lethal effects of endotoxin. It is generally accepted that macrophages are main cellular components in the development of tolerance, hence, nitric oxide (.NO) as one of the macrophage mediators may play a role in host defense mechanisms during tolerance. In this study, we utilized EPR spectroscopy to directly detect nitrosyl complexes as products of .NO in whole blood, livers and intestines of lipopolysaccharide (LPS)-tolerant rats. Male Sprague-Dawley rats were injected with a "low dose" LPS (0.5 mg/kg) 12-168 h prior to a "high dose" LPS (3 mg/kg), then sacrificed 6 h later. EPR signals of nitrosyl hemoprotein complexes were detected in specimens after high dose LPS. The post-LPS EPR signals of nitrosyl complexes from all samples were attenuated by a prior injection of low dose LPS. The signals of dinitrosyl-iron-dithiolate became apparent in samples from tolerant rats as signals of nitrosyl hemoprotein decreased. The maximal tolerance in terms of diminished .NO production was observed when low dose LPS was given 48-96 h prior to high dose LPS. Hemoglobin concentrations in the intestine used as biomarkers of hemorrhagic damage, were concomitantly attenuated in the jejunum of tolerant rats. These results together with our previous studies indicate that suppression of .NO production may contribute to the amelioration of hepatic and intestinal injury during endotoxin tolerance.

Tumor necrosis factor-alpha and nitric oxide production in endotoxin-primed rats administered carbon tetrachloride.

Tumor necrosis factor-alpha (TNF alpha) is elevated in the sera of rats administered non-lethal doses of carbon tetrachloride (CCl4) followed by endotoxin. Elevated TNF alpha levels are correlated with the increased release of hepatic enzymes indicating hepatic damage. Under these conditions, nitric oxide (NO) was also produced in the liver as evidenced by the formation of nitrosyl complexes which were measured by electron paramagnetic resonance (EPR) spectroscopy. Decreased nitrosyl complex formation occurred in livers following treatment with either an inhibitor or macrophage activation (gadolinium trichloride; GdCl3), an inhibitor of cytokine responses (dexamethasone) or a NO synthase inhibitor (NG-monomethyl-L-arginine; 1-NMA), GdCl3 or dexamethasone treatment decreased, while 1-NMA treatment increased, TNF alpha serum level. Taken together, these data suggest that TNF alpha and NO are induced following CCl4 and LPS exposure and may be important regulators in the hepatotoxicity of this liver injury model.

Nitric oxide inhibited peroxyl and alkoxyl radical formation with concomitant protection against oxidant injury in intestinal epithelial cells.

A model compound of lipid peroxidation, tert-butyl hydroperoxide (tBOOH), was used in vitro to investigate (i) the generation of tBOOH-derived peroxyl and alkoxyl radicals by rat intestinal epithelial cells or enterocytes and (ii) the role of nitric oxide (NO) on cell-generated free radical formation and cellular cytotoxicity. Peroxyl, alkoxyl, and methyl radicals were detected and characterized by direct and spin-trapping electron paramagnetic resonance spectroscopy in incubations containing tBOOH and hematin, enterocytes, or intestinal epithelial cell line-6 cells. The direct interactions of tBOOH-derived radicals and NO from nitrosoglutathione (GSNO), nitrosoacetyl penicillamine (SNAP), or 1-¿b3-aminopropy-4-(3-aminopropylammonio)¿ butylamino-diazeniumdiolate (SpNONOate) were demonstrated as their levels were depleted in these incubations. SNAP, not GSNO or SpNONOate, was capable of trapping methyl radical produced during hematin-catalyzed decomposition of tBOOH. Cellular cytotoxicity expressed by percentage of dead cells and lactate dehydrogenase was increased with tBOOH treatment. Addition of GSNO, SNAP, or SpNONOate suppressed tBOOH-induced elevation of cell cytotoxicity. The NO donor precursor glutathione, acetylpenicillamine, or spermine did not have any effects on tBOOH-derived radical generation or cell cytotoxicity. These findings demonstrated free radical-free radical reactions between NO- and tBOOH-derived alkoxyl and peroxyl radicals generated by enterocytes. These reactions, at least in part, describe the protective role of NO from hydroperoxide-induced injury in intestinal epithelial cells.

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations.

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.

Nitric oxide and liver injury in alcohol-fed rats after lipopolysaccharide administration.

Earlier studies showed that alcohol-fed animals were more susceptible than controls to injurious effects of endotoxin. Increased superoxide radical production by hepatocyte organelles, Kupffer cells, and neutrophils from alcohol-fed animals has been well documented. In this study, electron paramagnetic resonance spectroscopy was used to detect nitrosyl protein complexes indicating nitric oxide (.NO) production. We showed that the concentrations of nitrosyl complexes in whole blood and in liver tissues of alcohol-fed rats treated with lipopolysaccharide (alc + LPS), increased 3-fold, compared with those from rats on control diet treated with LPS (con+LPS). Electron paramagnetic resonance spectra of whole blood and liver tissues from the alc + LPS-treated group exhibited features characteristic of hemoglobin nitrosyl complexes. Plasma levels of the hepatic ASTs and ALTs from the alc + LPS-treated group were increased 2- to 3-fold, compared with those from the con+LPS-treated group. Inhibition of .NO production of aminoguanidine treatment attenuated plasma hepatic enzyme levels in the alc + LPS-treated group. Thus, under the conditions of elevated inflammatory oxidative states caused by chronic alcohol feeding, endotoxin treatment enhanced liver injury as a result of the actions of .NO, and/or the cytotoxic species derived from .NO.

Alkyl free radicals from the beta-scission of fatty acid alkoxyl radicals as detected by spin trapping in a lipoxygenase system.

2-Methyl-2-nitrosopropane (tNB)-radical adducts from incubation mixtures of fatty acids and soybean lipoxygenase in borate buffer (pH 9.0) were measured by electron paramagnetic resonance (EPR). In addition to the previously reported six-line signal of secondary carbon-centered radicals (RCHR'), a weak signal submerged in the baseline was detected after the peroxidation phase was finished. We propose that this radical is a decomposition product formed via beta-scission of fatty acid alkoxyl radicals. EPR spectra of tNB-radical adducts formed in mixtures of either linoleic acid, arachidonic acid, or 15-hydroperoxyeicosatetraenoic acid with lipoxygenase exhibited hyperfine structure characteristic of tNB/.CH2CH2-R with hyperfine coupling constants: aN = 17.1 G; aH beta = 11.2 G (2H); and aH gamma = 0.6 G (2H). In the case of linolenic acid, this radical tNB/.CH=CH-R' with hyperfine coupling constants: aN = 17.1 G; aH beta = 10.9 G (2H); aH gamma = 1.1 G; and aH delta = 0.5 G. In accord with the decomposition scheme of hydroperoxides derived from unsaturated fatty acids, the radical adducts tNB/.CH2CH2-R and tNB/.CH2-CH=CH-R' were assigned as the pentyl and 2-pentenyl radicals, respectively.

When are metal ion-dependent hydroxyl and alkoxyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide artifacts?

The formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.OH adduct of the spin trap DMPO has been reported to occur through nucleophilic addition of water in the presence of aqueous ferric chloride (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080). Due to the serious implications of these findings with respect to many spin trapping studies, the suitability of DMPO as a hydroxyl radical spin trap was studied in typical Fenton systems. Using 17O-enriched water, we show conclusively that nucleophilic addition of water occurs at the nitrone carbon (or C-2 position) of DMPO in the presence of either Fe or Cu ions. Furthermore, our results demonstrate that this nucleophilic reaction is a major pathway to the DMPO/.OH adduct, even during the reaction of Fe(II) or Cu(I) with hydrogen peroxide. Primary alkoxyl adducts of DMPO also form in aqueous solution through nucleophilic addition in the presence of both Fe(III) and Cu(II). Attempts to obtain secondary and tertiary alkoxyl adducts by this mechanism were unsuccessful, possibly due to steric effects. When the reaction is carried out in various buffers, however, or in the presence of metal ion chelators, nucleophilic addition to DMPO from Fe(III) is effectively suppressed. Chelators also suppress the reaction with Cu(II). Hence, under most common experimental conditions in biochemical free radical research, nucleophilic addition to DMPO should not be of major concern.

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.