Spin Trapping Of Superoxide Research Articles

Reprint of: Spin Trapping of Superoxide and Hydroxyl Radical: Practical Aspects

Reprint of: Spin Trapping of Superoxide and Hydroxyl Radical: Practical Aspects

On 'Spin trapping of superoxide and hydroxyl radical: Practical aspects' by Eli Finkelstein, Gerald M. Rosen and Elmer J. Rauckman

This commentary describes a highly cited paper by Eli Finkelstein, Gerald M. Rosen, and Elmer J. Raukman that appeared in Archives of Biochemistry and Biophysics published in 1980. They reviewed many reports being regularly appearing in the literature describing spin trapping and hydroxyl radicals from various sources and contributed to the development and progress that has been made in oxidative stress research.

High-Throughput Screening of NOX Inhibitors.

Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.

Embedding cyclic nitrone in mesoporous silica particles for EPR spin trapping of superoxide and other radicals

The generation of superoxide radical anion in biological systems is one of the major initiating events in the redox biology of NADPH oxidases and mitochondrial redox signalling. However, the pallette of chemical tools for superoxide detection is very limited, hampering progress in understanding the chemical biology of superoxide. Although EPR spin trapping is regarded as the most rigorous technique for superoxide detection, rapid reduction of the EPR-active superoxide spin adducts to EPR-silent hydroxylamines, or to hydroxyl radical adducts by bioreductants, significantly limits the applicability of this technique in biological systems. To overcome these limitations, in this work, we report the synthesis and characterization of a new mesoporous silica functionalized with a phosphorylated cyclic spin trap (DIPPMPO nitrone). The DIPPMPO-grafted silica is a versatile spin-trap agent enabling the identification of a wide range of carbon or oxygen-centered transient radicals in organic and in aqueous media. Moreover, superoxide was efficiently trapped under in vitro conditions in both cell-free and cellular systems. The generated superoxide adduct exhibited an exceptional half-life of 3.5 h and a resistance toward bioreductant agents such as glutathione for several hours.

Changes in mitochondrial functioning with electromagnetic radiation of ultra high frequency as revealed by electron paramagnetic resonance methods

Purpose: To study the effects of electromagnetic radiation (EMR) of ultra high frequency (UHF) in the doses equivalent to the maximal permitted energy load for the staffs of the radar stations on the biochemical processes that occur in the cell organelles.Materials and methods: Liver, cardiac and aorta tissues from the male rats exposed to non-thermal UHF EMR in pulsed and continuous modes were studied during 28 days after the irradiation by the electron paramagnetic resonance (EPR) methods including a spin trapping of superoxide radicals.Results: The qualitative and quantitative disturbances in electron transport chain (ETC) of mitochondria are registered. A formation of the iron-nitrosyl complexes of nitric oxide (NO) radicals with the iron-sulphide (FeS) proteins, the decreased activity of FeS-protein N2 of NADH-ubiquinone oxidoreductase complex and flavo-ubisemiquinone growth combined with the increased rates of superoxide production are obtained.Conclusions: (i) Abnormalities in the mitochondrial ETC of liver and aorta cells are more pronounced for animals radiated in a pulsed mode; (ii) the alterations in the functioning of the mitochondrial ETC cause increase of superoxide radicals generation rate in all samples, formation of cellular hypoxia, and intensification of the oxide-initiated metabolic changes; and (iii) electron paramagnetic resonance methods could be used to track the qualitative and quantitative changes in the mitochondrial ETC caused by the UHF EMR.

Electron Paramagnetic Resonance Study of Tumor Affected Bone Marrow

To study the mechanism of interaction of tumor cells with bone marrow cells continuous wave electron paramagnetic resonance (EPR) experiments at 9GHz including a spin trapping of superoxide were carried out. The common features of the EPR spectra in healthy and tumor affected tissues of donors and rats as well as their difference are presented and discussed. It is proposed that labile iron pool plays a significant role in mechanisms of tumor invasion. We hope that the observed EPR features could be used to study the mechanisms of invasion and progression of tumor in different organs.

Impact of SOD-Mimetic Manganoporphyrins on Spin Trapping of Superoxide and Related Artifacts

The superoxide dismutase (SOD)-mimetic effectiveness of [meso-tetrakis (R)porphyrinato]manganese with R = 1,3-di-N-ethylimidazolium-2-yl (Mn-TDEIP), 1,3-di-N-methylimidazolium-2-yl (Mn-TDMIP), 1,3-di-N-propylimidazolium-2-yl (Mn-TDPIP), N-ethyl-2-pyridyl (Mn-T2EPyP), 4-sulphonatophenyl (Mn-TSP), 1-methyl-4-pyridyl (Mn-T4PyP), 4-carboxyphenyl (Mn-TBAP), and β-octabromomeso-tetrakis(4-carboxyphenyl porphyrinato)manganese (MnBr8TBAP) was compared with Cu, Zn SOD. Superoxide generated by reaction of xanthine oxidase with hypoxanthine was trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), forming BMPO-OOH, which was monitored by electron paramagnetic resonance. Manganoporphyrins with redox potentials ranging from -0.190 to 0.346 V relative to the standard hydrogen electrode were selected for this study. With 0.1 µM manganoporphyrins and 20 mM BMPO, the effectiveness of the manganoporphyrins in inhibiting formation of BMPO-OOH increases in the order Mn-TSP < Mn-TBAP < MnBr8TBAP < Mn-T4PyP < Mn-T2EPyP < Mn-TDEIP ~ Mn-TDMIP ~ Mn-TDPIP ~ Cu, Zn SOD. However, at higher concentrations of manganoporphyrin and BMPO, a BMPO-OH signal was observed. The formation of BMPO-OH was not inhibited by catalase or dimethylsulfoxide, which demonstrated that it was not produced from hydroxyl radical. The artifactual formation of BMPO-OH is attributed to oxidation of the water adduct of BMPO by the manganoporphyrins or decomposition of BMPO-OOH. Although spin trapping is an effective method for evaluating SOD-mimetic efficacy, caution must be exercised to ensure that artifact signals are not interpreted improperly.

Production of extracellular superoxide by human lymphoblast cell lines: Comparison of electron spin resonance techniques and cytochrome C reduction assay

Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O2−), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O2− production in cultured human lymphoblast cell lines by three different methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein–Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected two to five times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)–SOD and extracellular Cu,Zn–SOD inhibited O2− detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O2− generation in human lymphoblasts. Our study describes a new technique for O2− measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O2− in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.

Inefficient spin trapping of superoxide in the presence of nitric-oxide: Implications for studies on nitric-oxide synthase uncoupling

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate l-arginine or the cofactor (6 R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H 2O 2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide (DEPMPO) as a spin trap. As expected, the Ca 2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H 2O 2 in the absence of added l-arginine and BH4. Addition of BH4 (10 μM) did not significantly affect the rate of H 2O 2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H 2O 2. Although l-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H 2O 2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50–100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.

Spin trapping of superoxide by diester-nitrones

The nitrone N-[(1-oxidopyridin-1-ium-4-yl)-methylidene]-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPyON) was synthesized and used as a spin trapping agent. The kinetic aspects of the superoxide detection by this new spin trap and by two other diester-nitrones, i.e. 2,2-diethoxycarbonyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPO) and N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN), were examined by determining the rate constants for the trapping reaction and for the spin adduct decay at pH 7.2. Comparing the results obtained to those given by analogous monoester-nitrones showed that both the spin trapping and the adduct decay reactions were faster in the presence of a second ester group in the cyclic nitrone series, while the superoxide trapping capacities of linear diester-nitrones were found to be dramatically weak. It follows from this study that DEPO and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) are superior when it comes to superoxide detection. Below 0.005 mol dm(-3), DEPO is to date the only nitrone capable of clearly detecting superoxide, while EMPO should be preferred at higher spin trap concentration.

Superoxide Dismutase versus Ferricytochrome C: Determining Rate Constants for the Spin Trapping of Superoxide by Cyclic Nitrones

Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.

Spin trapping of superoxide, alkyl- and lipid-derived radicals with derivatives of the spin trap EPPN

The N- t-butyl-α-phenylnitrone derivative N-2-(2-ethoxycarbonyl-propyl)-α-phenylnitrone (EPPN) has recently been reported to form a superoxide spin adduct ( t 1/2=5.25 min at pH 7.0), which is considerably more stable than the respective N- t-butyl-α-phenylnitrone or 5,5-dimethylpyrroline N-oxide adducts ( t 1/2 ∼10 and 45 s, respectively). In continuation of our previous studies on structure optimization of 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide derivatives, a series of six different EPPN derivatives was synthesized and characterized by 1 H NMR, 13 C NMR and IR spectroscopy. The ethoxy group of EPPN was replaced by a propoxy, iso-propoxy, n-butoxy, sec-butoxy, and tert-butoxy moiety, as well as the phenyl by a pyridyl ring. Electron spin resonance spectra and stabilities of the superoxide adducts of the propoxy derivatives were found to be similar to those of the respective EPPN adduct, whereas the electron spin resonance spectra of the superoxide adducts of N-2-(2-ethoxycarbonyl-propyl)-α-(4-pyridyl) nitrone and the butoxy derivatives were accompanied by decomposition products. In contrast to the 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide series, no significant improvement of the superoxide adduct stability could be obtained when the ethoxy group was replaced by other substituents. Carbon centered radical adducts derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide were more stable than those of 5,5-dimethylpyrroline N-oxide, whereas among the alkoxyl radicals only the methoxyl radical adduct could be detected.

Spin trapping of superoxide in the presence of beta-cyclodextrins.

The trapping of superoxide anion with DMPO and DEPMPO has been carried out in the presence of a methylated beta-cyclodextrin, Me-beta-CD; inclusion of the spin adducts in the cavity of Me-beta-CD resulted in a seven-fold increase of their half-life (t1/2 = 96 min for DEPMPO-superoxide spin adduct) and in their protection towards glutathione peroxidase (Gpx) and also ascrobate anion in the case of DEPMPO.

EPR spin trapping of superoxide from nitric oxide synthase

15 N)- EMPO has significantly improved the sen- sitivity of superoxide detection by electron para- magnetic resonance (EPR). Unlike 5,5-dimethyl-1- pyrroline N-oxide (DMPO), a commonly used superoxide spin trap, both DEPMPO and EMPO form superoxide adducts that are persistent, which do not decay to the corresponding hydroxyl adduct. Using these new spin traps, the EPR-detection of superoxide is now straightfor- ward and more sensitive. These new superoxide traps in combination with the loop gap resonator technology have enabled us to demonstrate unequivocally the formation of superoxide from the endothelial and neuronal isoforms of nitric oxide synthase. Superoxide is formed at the oxy- genase domain of the enzyme by a calcium/ calmodulin dependent mechanism. The effect of tetrahydrobiopterin cofactor and NOS inhibitors on the release of superoxide is discussed.

Spin trapping of superoxide in aqueous solutions of fresh and aged cigarette smoke

Superoxide generation in aqueous solutions of cigarette smoke was determined as a function of the age of smoke using spin trapping. As a spin trap, 5,5-dimethylpyrroline- N-oxide (DMPO) was used. The superoxide adduct of DMPO was detected in a solution of fresh main-stream smoke for over 1 h. The superoxide-generating potential of smoke was rapidly lost as the smoke was kept in a plastic syringe. The smoke that was aged for 3 min did not generate superoxide. Additional evidence of superoxide generation in aqueous solutions of cigarette smoke was obtained by the chemiluminescence method.

The spin trapping of superoxide and hydroxyl free radicals with DMPO (5,5-dimethylpyrroline-N-oxide): more about iron.

The reaction of superoxide with the spin trap DMPO (5,5-dimethylpyrroline-N-oxide) is widely used to study superoxide production as well as issues associated with the superoxide-related formation of hydroxyl radical. However, the interpretation of observed intensities of DMPO/.OOH and DMPO/.OH signals in electron paramagnetic resonance spin trapping experiments is not without its difficulties. In this paper, I report experiments that demonstrate: 1. That the flux of superoxide formation in a DMPO spin trapping experiment can alter the apparent importance of weak DMPO/.OH signals; 2. That iron can influence the DMPO/.OOH spin trapping results; 3. That there is very little spontaneous breakdown of DMPO/.OOH to form DMPO/.OH.

Spin Trapping of Superoxide Released by Opsonized Asbestos from Human Promyelocytic Leukemia Cell Line, HL60

By ESR using 5,5-dimethyl-1-pyrroline-1-oxide as a spin trap, superoxide (O 2 −) production was proved upon stimulation of dimethyl sulfoxide-differentiated HL60 by crocidolite opsonized with fresh or refrigerated serum, as well as by phorbol myristate acetate (PMA) . Crocidolite, unopsonized or opsonized with frozen-thawed or heat-inactivated serum, did not induce O 2 − release. Addition of iron chelators or superoxide dismutase inhibited O 2 − release completely. Neither undifferentiated nor PMA-differentiated HL60 released O 2 − upon stimulation with opsonized crocidolite.

Spin Trapping of Superoxide from Glass Adherent Polymorphonuclear Leukocytes Induced by N-Formylmethionyl-Leucyl-Phenylalanine

Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.

Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.

Insight Into the Nature and Site of Oxygen-Centered Free Radical Generation by Endothelial Cell Monolayers Using a Novel Spin Trapping Technique

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA-stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy-radical generation.