Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Abstract

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.     

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H₂O₂, hydroxyl radical, or peroxynitrite (ONOO⁻). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondria-generated ROS?

In this study we report the synthesis and biological application of a novel cyclic nitrone spin trap containing a phosphonium cation. This new spin trap ([4-(2-methyl-1-oxy-3, 4-dihydro-2H-pyrrole-2-carbonyloxy)-butyl]-triphenyl-phosphonium bromide, MitoBMPOBr) is a derivative of the cyclic nitrone, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO). MitoBMPOBr forms radical adducts upon trapping of superoxide and hydroxyl radicals that exhibit highly distinct and characteristic EPR spectra. The stability of these adducts is comparable to those of BMPO. Because of the presence of a positively-charged phosphonium moiety, MitoBMPOBr may be suitable for trapping reactive oxygen species (ROS) in the mitochondria.     

Evaluation of spin trapping agents and trapping conditions for detection of cell-generated reactive oxygen species

Electron paramagnetic resonance with spin trapping is a useful technique to detect reactive oxygen species, such as superoxide radical anion (O2*-), a key species in many biological processes. We evaluated the abilities of four spin traps in trapping cell-generated O2*-: 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), 2-diethoxyphosphoryl-2-phenethyl-3,4-dihydro-2H-pyrrole N-oxide (DEPPEPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Optimal experimental conditions for obtaining maximal signal intensity of O2*- adduct in a cellular system were first studied. The maximal intensities of BMPO, DEPMPO, and DMPO adducts were similar while DEPPEPO did not trap cell-generated O2*- induced by 1,6-benzo[a]pyrene quinone in a human mammary epithelial cell line (MCF-10A). BMPO and DEPMPO adducts were more stable, considering the stability of their maximal signal, than DMPO adduct in the tested cellular systems. In addition, we observed that O2*- spin adducts were reduced to their corresponding hydroxyl adducts in the cellular system. The selection of optimal spin trap in trapping cell-generated O2*- is discussed.     

Spin Trapping Using 2,2-Dimethyl-2H-Imidazole-1-Oxides

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na₂SO₃ and NaHSO₃. From the data obtained, a successful application of these new spin traps in biological systems can be expected.     

Detection and characterization of cyclic hydroxylamine adducts by mass spectrometry

Two cyclic hydroxylamines (cHA) bearing pyrrolidine (CPH) and piperidine moieties (TMTH) were evaluated to trap hydroxyl, peptide and phospholipid free radicals using mass spectrometry for their detection. The cHA ionized as [M+H](+) ions, showing higher relative abundances when compared to the DMPO, probably due to higher ionization efficiency. In the presence of hydroxyl radicals, both cHA generated new ions that could be attributed to loss of (*)H and (*)CH(3), most likely deriving from decomposition reactions of the nitroxide spin adduct. Addition of cHA to Leucine-enkephalin and palmitoyl-lineloyl-glycerophosphatidylcholine free radicals promoted the formation of cHA biomolecule adducts, which were confirmed by MS/MS data. Results suggest that the cHA are not suitable for hydroxyl radical trapping but can be used for trapping biomolecule radicals, having the advantage, over the most used cyclic nitrones, of being water soluble. The biomolecule adducts identified by MS are ESR silent, evidencing the importance of MS detection.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals

A novel cyclic nitrone spin trap, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) as a pure white solid has been synthesized for the first time. BMPO offers several advantages over the existing spin traps in the detection and characterization of thiyl radicals, hydroxyl radicals, and superoxide anions in biological systems. The corresponding BMPO adducts exhibit distinct and characteristic electron spin resonance (ESR) spectral patterns. Unlike the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-derived superoxide adduct, the BMPO superoxide adduct does not non-enzymatically decompose to the BMPO hydroxyl adduct. This feature is clearly perceived as a definite advantage of BMPO in its biological applications. In addition, the ESR spectrum of the BMPO glutathionyl adduct (BMPO/*SG) does not fully overlap with the spectrum of its hydroxyl adduct. This spectral feature is again distinctly different from that of DMPO because the ESR spectral lines of DMPO glutathionyl and hydroxyl radical adducts largely overlap. Finally, the ESR spectra of BMPO-derived adducts exhibit a much higher signal-to-noise ratio in biological systems. These favorable chemical and spectroscopic features make BMPO ideal for the detection of superoxide anions, hydroxyl and thiyl radicals in biochemical oxidation and reduction.     

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2. as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. No significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.     

Spin trapping endogenous radicals in MC-1010 cells: Evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO₂) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO₂ oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A_N=15.9 G; A_H=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO₂ treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe²⁺ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO₂ oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A_H4=1.8 G.Abbreviation EPR Electron Paramagnetic Resonance     

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree

Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.     

CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems

The cis/trans diastereoisomeric composition of hydroxyl radical adducts to chiral cyclic nitrones can be used to approach mechanisms of free radical formation in biological systems. Such determination is greatly simplified when both diastereoisomers have ESR spectra with at least two non-overlapping lines. To achieve this prerequisite, a series of DEPMPO-derived spin traps bearing one unsubstituted or alkyl-substituted 2-oxo-1,3,2-dioxaphosphorinane ring were synthesized and their structures were confirmed by X-ray diffraction, (1)H, (13)C and (31)P NMR. These CyDEPMPOs nitrones showed variable lipophilicities and LD(50) values on murine fibroblasts compatible with a safe use in biological spin trapping. All CyDEPMPOs formed persistent spin adducts with a series of free radicals, including superoxide and hydroxyl (i.e., CyDEPMPOs-OH) and the in vitro half-life times of these two latter were at least as extended as those of parent DEPMPO. Using four methods of CyDEPMPOs-OH formation, the cis-CyDEPMPOs-OH percentage was found significantly varied with substitution on the P-containing ring and, more interestingly, with the generating system.     

Use of high-performance liquid chromatography to detect hydroxyl and superoxide radicals generated from mitomycin C

Distinguishing between short-lived reactive oxygen species like hydroxyl and superoxide radicals is difficult; the most successful approaches employ electron spin resonance (ESR) spin-trapping techniques. Using the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO) to selectively trap various radicals in the presence and absence of ethanol, an HPLC system which is capable of separating the hydroxyl- and superoxide-generated DMPO adduct species has been developed. The radical-generated DMPO adducts were measured with an electrochemical detector attached to the HPLC system and confirmed by spin-trapping techniques. The HPLC separation was carried out on an ODS reverse-phase column with a pH 5.1 buffered 8.5% acetonitrile mobile phase. The advantage of the HPLC system described is that it permits the separation and detection of hydroxyl and superoxide radicals without requiring ESR instrumentation. The antineoplastic bioreductive alkylating agent mitomycin C, when activated by NADPH-cytochrome c reductase, was shown to generate both hydroxyl and superoxide radicals.     

Radical Trapping and Inhibition of Iron-Dependent CNS Damage by Cyclic Nitrone Spin Traps

Abstract: Oxidative damage in the CNS is proposed to play a role in many acute and chronic neurodegenerative disorders. Accordingly, the nitrone spin trap α-phenyl- N - tert -butylnitrone (PBN), which reacts covalently with free radicals, has shown efficacy in a variety of animal models of CNS injury. We have synthesized a number of cyclic variants of PBN and examined their activity as radical traps and protectants against oxidative damage in CNS tissue. By using electron spin resonance spectroscopy, the cyclic nitrones MDL 101,002 and MDL 102,832 were shown to trap radicals in a manner similar to that of PBN. All cyclic nitrones tested prevented hydroxyl radical-dependent degradation of 2-deoxyribose and peroxyl radical-dependent oxidation of synaptosomes more potently than PBN. The radical scavenging properties of the cyclic nitrones contributed to a three- to 25-fold increase in potency relative to PBN against oxidative damage and cytotoxicity in cerebellar granule cell cultures. Similar to the phenolic antioxidant MDL 74,722, the nitrones minimized seizures and delayed the time to death in mice following central injection of ferrous iron. Although iron-induced lipid peroxidation was inhibited by MDL 74,722, the nitrones had no effect on this biochemical end point, indicating that iron-induced mortality does not result solely from lipid peroxidation and suggesting additional neuroprotective properties for the nitrones. These results indicate that cyclic nitrones are more potent radical traps and inhibitors of lipid peroxidation in vitro than PBN, and their ability to delay significantly iron-induced mortality in vivo suggests they may be useful in the treatment of acute and chronic neurodegeneration. Furthermore, the stability of the spin trap adducts of the cyclic nitrones provides a new tool for the study of oxidative tissue injury.     

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.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Spin Trapping of Free Radicals Formed During Peroxidation of Sarcolemmal Membranes (SLM)

The generation of free radicals in a superoxide (O₂-)driven Fe⁺³ catalysed reactions with isolated myocytic sarcolemma using electron spin resonance was investigated. Incubation of highly purified canine myocytic sarcolemma in the presence of the spin trap, 2-methyl-2-nitrosopropane (MNP). followed by the addition of dihydroxyfurmarate (DHF) and Fe⁺³-ADP resulted in the generation and detection of radical adducts of this spin trap. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl radical adduct following exposure to DHF/Fe⁺³-ADP. With sarcolemma and the alkyl nitroso compound, the only radical product trapped was the methyl radical formed by β-scission of alkoxyl radical. The participation of hydroperoxide-derived radicals in this system verified that the decomposition of unsaturated hydroperoxy fatty acid does proceed via a free radical mechanism.     

Investigating the free radical trapping ability of NXY-059, S-PBN and PBN

The spin trapping ability of the nitrones 2,4-disulphophenyl-N-tert-butyl nitrone (NXY-059), 2-sulphophenyl-N-tert-butyl nitrone (S-PBN) and alpha-phenyl-N-tert-butyl nitrone (PBN) for both hydroxyl and methanol radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy. The radicals of interest were generated in situ in the spectrometer under constant flow conditions in the presence of each nitrone. The spin adducts formed were detected by EPR spectroscopy. This approach allowed for quantitative comparison of the EPR spectra of the spin adducts of each nitrone. The results obtained showed that NXY-059 trapped a greater number of hydroxyl and methanol radicals than the other two nitrones, under the conditions studied.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.