Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and an excess of dimethyl sulfoxide, we previously reported that in the absence of an exogenous iron catalyst, human neutrophils will not generate hydroxyl radical, manifested as the catalse-inhibitable methyl radical spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy (DMPO-CH3) (Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986) J. Biol. Chem. 261, 4426-4431). However, superoxide destroys the preformed hydroxyl radical spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH), and DMPO-CH3. The present study was undertaken to better resolve the limits of sensitivity of the spin-trapping method. Photolytically generated DMPO-CH3 and DMPO-OH slowly decomposed in the presence of a low flux (1 microM/min) of enzymatically (xanthine/xanthine oxidase)-generated superoxide, but more rapid decomposition of these adducts occurred with higher superoxide flux (5 microM/min). Inclusion of cysteine markedly increased the rate of DMPO-OH and DMPO-CH3 decomposition, masking the effect of superoxide alone. The addition of varying concentrations of superoxide dismutase did not lead to increased formation of DMPO-OH or DMPO-CH3, as should have occurred if these adducts were being destroyed by superoxide. As a positive control, we employed an iron-supplemented system with phorbol 12-myristate 13-acetate-stimulated neutrophils or xanthine/xanthine oxidase to generate DMPO-CH3. Addition of superoxide dismutase increased the magnitude of DMPO-CH3, primarily by increasing the rate of hydrogen peroxide formation, and to a lesser extent by prolonging the half-life of DMPO-CH3. Although spin-trapped adducts can be destroyed by a high concentration of superoxide, or by lower concentrations of superoxide in the presence of thiol-containing compounds, our results demonstrate that such decomposition does not interfere with the ability of the spin-trapping method to detect hydroxyl radical generated by human neutrophils. These data do not support the capacity of neutrophils to generate hydroxyl radical in the absence of an exogenous Haber-Weiss catalyst.     

Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical

The generation of oxygen reduction products by Neisseria gonorrhoeae FA1090 upon exposure to streptonigrin (SNG) and paraquat (PQ2+) and their toxicity was examined. N. gonorrhoeae exhibited maximal cyanide-insensitive respiration, which was employed as an indicator of superoxide (O2-) formation, in the presence of 0.064 mM streptonigrin and 90 mM PQ2+, respectively. Using the concentrations of SNG and PQ2+ described above, complete lethality (greater than 10(8) cells/ml) was observed among cells exposed to SNG, whereas PQ2+ reduced viability by only 3 logs. In an attempt to determine the oxygen radical species generated by gonococci when exposed to SNG, dimethyl sulfoxide, Fe3+, KCN, and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect .OH manifested as the methyl adduct (DMPO-CH3). The production of the latter species was not inhibited by catalase, suggesting intracellular .OH generation. When PQ2+ was substituted for SNG, only low levels of DMPO-CH3 were observed, the production of which ceased within 8 min. SNG and PQ2+, added to a O2(-)- generating system in the presence of Fe3+, promoted increased .OH generation. The iron chelator diethyl-enetriaminepentaacetic acid enhanced the generation of spin-trapped .OH and O2- in the presence of PQ2+. The addition of catalase to this system, however, eliminated the DMPO-CH3 signal, showing that the .OH in this system was extracellular. PQ2+-mediated generation of extracellular .OH in the presence of Fe3+-diethylenetriaminepentaacetic acid EDTA did not enhance the killing of gonococci by PQ2+. These data show that the lethality of SNG relative to PQ2+ is due to the inherent ability of SNG to catalyze the formation of critical levels of intracellular .OH, detectable through the use of spin trapping techniques.     

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Neutrophil degranulation inhibits potential hydroxyl-radical formation. Relative impact of myeloperoxidase and lactoferrin release on hydroxyl-radical production by iron-supplemented neutrophils assessed by spin-trapping techniques.

Hydroxyl radical (.OH) formation by neutrophils in vitro requires exogenous iron. Two recent studies [Britigan, Rosen, Thompson, Chai & Cohen (1986) J. Biol. Chem. 261, 17026-17032; Winterbourn (1987) J. Clin. Invest. 78, 545-550] both reported that neutrophil degranulation could potentially inhibit the formation of .OH, but differed in their conclusions as to the responsible factor, myeloperoxidase (MPO) or lactoferrin (LF). By using a previously developed spin-trapping system which allows specific on-line detection of superoxide anion (O2-) and .OH production, the impact of MPO and LF release on neutrophil .OH production was compared. When iron-diethylenetriaminepenta-acetic acid-supplemented neutrophils were stimulated with phorbol myristate acetate or opsonized zymosan, .OH formation occurred, but terminated prematurely in spite of continued O2- generation. Inhibition of MPO by azide increased the magnitude, but not the duration, of .OH formation. No azide effect was noted when MPO-deficient neutrophils were used. Anti-LF antibody increased both the magnitude and duration of .OH generation. Pretreatment of neutrophils with cytochalasin B to prevent phagosome formation did not alter the relative impact of azide or anti-LF on neutrophil .OH production. An effect of azide or anti-LF on spin-trapped-adduct stability was eliminated as a confounding factor. These data indicate that neutrophils possess two mechanisms for limiting .OH production. Implications for neutrophil-derived oxidant damage are discussed.     

Cautionary note for DMPO spin trapping in the presence of iron ion

2-Hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH), which is known to be produced by spin trapping of hydroxyl radicals (.OH) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and has been a good monitor for detecting .OH in biological systems, has been examined by EPR for its production scheme in the presence of iron ion. In an aqueous DMPO solution containing ferric ion (Fe3+), DMPO-OH was produced and addition of methanol, a good scavenger for .OH, to this solution led to an aminoxyl radical, DMPO-OCH3, instead of DMPO-CH2OH which is produced by DMPO spin trapping of .CH2OH arising from H-abstraction by .OH. Also EPR measurements at 77K indicated the formation of a chelate between DMPO and Fe3+. Based on these, it has been elucidated that DMPO-OH as well as DMPO-OCH3 is formed by the nucleophilic attack of water and methanol to the chelating DMPO, respectively.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

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

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.     

The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.

Uroporphyrin I, haematoporphyrin and haematoporphyrin derivative had no effect on O2-. generation during oxidation of hypoxanthine by xanthine oxidase and on the formation of hydroxyl radicals (OH.) in the hypoxanthine/xanthine oxidase/Fe3+-EDTA/deoxyribose system. On the other hand, these porphyrins strongly inhibited O2-. formation in a horseradish peroxidase/H2O2/NADPH mixture, whereas they augmented OH. generation in this system after addition of Fe3+-EDTA. Experimental evidence suggests that these observations should be ascribed to the formation of a porphyrin anion radical in the horseradish peroxidase/NADPH system. The formation of this anion radical was confirmed by e.s.r. spectroscopy. This radical is apparently unable to reduce cytochrome c, but it can replace O2-. in the OH.-generating Haber-Weiss reaction.     

Spin trapping evidence for the lack of significant hydroxyl radical production during the respiration burst of human phagocytes using a spin adduct resistant to superoxide-mediated destruction

Failure to detect hydroxyl radical (.OH)-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide in electron spin resonance (ESR) spin trapping experiments has been offered as evidence for the lack of the endogenous capacity of stimulated human phagocytes (neutrophils, monocytes, and monocyte-derived macrophages (MDM] to generate .OH. Recent reports that 5,5-dimethyl-1-pyrroline N-oxide spin adducts are unstable in the presence of superoxide-generating systems such as stimulated neutrophils has raised concerns regarding the sensitivity of spin trapping techniques for assessment of phagocyte free radical formation. Consequently, we have employed a new approach that uses the spin trap N-t-butyl-alpha-phenyl-nitrone (PBN) and dimethyl sulfoxide. In the presence of dimethyl sulfoxide and PBN, the formation of .OH via three different mechanisms in air-saturated aqueous solutions all yielded a single nitroxide species whose ESR peak amplitude remained stable in the presence of superoxide (.O2-). This nitroxide, which we have assigned as PBN/.OCH3, appears to be an oxygen-centered radical derived from the spin trapping of the reaction product of O2 and methyl radical. When neutrophils, monocytes, or MDM were stimulated with phorbol 12-myristate 13-acetate or opsonized zymosan in the presence of exogenous iron, catalase-inhibitable PBN/.OCH3 was the sole nitroxide detected. In the absence of exogenous iron, no nitroxide was observed, providing evidence for the lack of the endogenous capacity of neutrophils, monocytes, and MDM to generate .OH.     

Hydroxyl radical production by stimulated neutrophils reappraised

Release of active oxygen species during the human neutrophil respiratory burst is thought to be mandatory for effective defense against bacterial infections and may play an important role in damage to host tissues. Part of the critical bacterial and host tissue damage has been attributed to hydroxyl radicals produced from superoxide and hydrogen peroxide. Because of the short life time of the very reactive hydroxyl radical, direct study of hydroxyl radical production is not possible; therefore, indirect detection methods such as electron spin resonance (ESR) coupled with appropriate spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been used. Superoxide production during the oxidative burst has been unambiguously demonstrated. Recent reports claim that hydroxyl radicals are not made during neutrophil stimulation and offer as an explanation the presence of granular components that interfere with hydroxyl radical production. When using the spin-trap agent DMPO, absence of the relatively long-lived adducts DMPO-OH and DMPO-CH3 has been assumed to be prima facie evidence for lack of hydroxyl radical participation. We show that high superoxide flux produced during stimulation of human neutrophils rapidly destroys both DMPO-OH and DMPO-CH3. In accord with previous implications, our results provide an alternative explanation for the absence of .OH adduct in spin-trapping studies and corroborate results obtained using other methods that implicate hydroxyl radical production during neutrophil stimulation.     

Cytochrome b translocation to human neutrophil plasma membranes and superoxide release. Differential effects of N-formylmethionylleucylphenylalanine, phorbol myristate acetate, and A23187

The role of specific granules and cytochrome b in superoxide (O(2)) release was studied by comparing the effects of three different stimuli on normal human neutrophils, neutrophils congenitally deficient in specific granules, and granule-free normal neutrophil cytoplasts. Phorbol myristate acetate (PMA) stimulated normal neutrophils to release more O(2) than did N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), which stimulated greater release than the calcium ionophore A23187. Neutrophils lacking specific granules produced variable amounts of O(2) in response to all stimuli. Stimulation with PMA, fMet-Leu-Phe, and A23187 produced maximal rates of O(2) release that were 32, 55, and 21% of that by normal cells. Likewise, granule-free neutrophil cytoplasts released 24, 20, and 0% of the O(2) released by intact cells. These data suggest that the stimuli require different mechanisms for activation. Three subcellular fractions (azurophil granule rich, specific granule rich, and plasma membrane rich) were separated by Percoll gradients from normal resting and stimulated neutrophils. In resting neutrophils, the cytochrome b content in the plasma membrane was 31% of the total, with the rest in the specific granule-rich fraction. Ten minutes after stimulation, PMA, fMet-Leu-Phe, and A23187 induced translocation of 27, 8, and 49%, respectively, of the cytochrome b from the specific granule-rich fraction to the plasma membrane. Although our data support a role for specific granule factors in A23187-induced O(2) release, there is no correlation between the amount of cytochrome b incorporated into the plasma membrane and the extent of O(2) production activated by the different stimuli.     

Iron and xanthine oxidase catalyze formation of an oxidant species distinguishable from OH.: comparison with the Haber-Weiss reaction

O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, or without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron-dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme.     

Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals

The effects of thiols and thiocarbamides on hydroxyl radical (.OH) formation by the hypoxanthine(HYP)-xanthine oxidase(XOD)-Fe3+ .EDTA system were investigated in the range of 0.5-5 mM by colorimetrically measuring salicylate hydroxylation. Thiocarbamides powerfully inhibited the hydroxylation while thiols showed a paradoxical effect, enhancing it at low concentrations, but inhibiting it at high ones. Thiols in the presence of Fe3+ .EDTA generated superoxide anions (O2-.) and .OH during the oxidation, but thiocarbamides did not. A study of the effect of ergothioneine, a thiocarbamide present in mammals, on the .OH spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by EPR spectrometry showed that it effectively decreased the .OH spin adduct without causing the appearance of other signals. Reaction mechanisms are proposed for the O2-. evolution and .OH formation by the thiols themselves in the presence of Fe3+ .EDTA and .OH with thiols and thiocarbamides.     

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

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

Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes

Phenol and 1-naphthol, products of benzene and naphthalene biotransformation, are metabolized during O2- generation by xanthine oxidase/hypoxanthine and phorbol myristate acetate (PMA)-stimulated human neutrophils. The addition of 1-naphthol to xanthine oxidase/hypoxanthine incubations resulted in the formation of 1,4-naphthoquinone (1,4-NQ) whereas phenol addition yielded only small quantities of hydroquinone, catechol and a unidentified reducible product but not 1,4-benzoquinone. This formation of 1,4-NQ was dependent upon hypoxanthine, xanthine oxidase, and 1-naphthol and was inhibited by the addition of superoxide dismutase (SOD) demonstrating that the conversion was O2-mediated. During O2- generation by PMA-stimulated neutrophils, the addition of phenol interfered with luminol-dependent chemiluminescence and resulted in covalent binding of phenol to protein. Protein binding was 80% inhibited by the addition of azide or catalase to the incubations indicating that bioactivation was peroxidase-mediated. In contrast, the addition of 1-naphthol to PMA-stimulated neutrophils interfered with superoxide-dependent cytochrome c reduction as well as luminol-dependent chemiluminescence and also resulted in protein binding. Protein binding was only partially inhibited by azide or catalase. The addition of SOD in combination with catalase resulted in a significantly greater inhibition of binding when compared to that of catalase alone. The results of these experiments indicate that phenol and 1-naphthol are converted to reactive metabolites during superoxide generating conditions but by different mechanisms. The formation of reactive metabolites from phenol was almost exclusively peroxidase-mediated whereas the bioactivation of 1-naphthol could occur by two different mechanisms, a peroxidase-dependent and a direct superoxide-dependent mechanism.     

Detection of phagocyte-derived free radicals with spin trapping techniques: effect of temperature and cellular metabolism

Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20 degrees C with either stimuli, neutrophil superoxide production was manifested predominantly as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37 degrees C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese.

When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2.- is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid-reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.--dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+-EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.