Synthesis and Evaluation of DMPO-Type Spin Traps

The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-l-pyroHine N-oxide (4MDMPO) and 5,5-dimethyl- 4-phenyl-l-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-l-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol-water system. Several radicals, O₂, HO-, -CH₃, -CH₂OH, -CH(CH₃)OH, (CH₃)₃ CO and H radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O J with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O₂^-, 4PDMPO-O₂^- 4HMDMPO-O₂^- and DMPO-O₂^- were almost equal. In the trapping of the OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH.     

An Artifact in the ESR Spectrum Obtained by Spin Trapping with Dmpo

In order to overcome a common problem in spin trapping with high concentrations of 5.5-dimethyl-I-pyrroline-N-oxide (DMPO) where ESR spectra are obtained which include an unidentified set of lines composed of a triplet of doublets. commercial DMPO was analyzed for its impurities by high-performance liquid chromatography. mass spectrometry. and nuclear magnetic resonance spectroscopy. It has been determined that this undesirable ESR spectrum IS due to an impurity included in the spin trap. This compound has been assigned to the hydronylunine which is a DMPO-derivative having an epoxy ring located at the 2 and 3 positions.     

Dmpo Spin Trapping in the Presence of Fe Ion

Aminoxyl radical formation from DMPO in the presence of Fe ion was studied to clarify the ambiguous ESR signals obtained by spin trapping with DMPO. It was found that when DMPO was used in a Fenton system, a Fe-DMPO complex was formed immediately. This complex was subsequently attacked by oxidative species originating from H₂O₂ and thus oxidative degradation of DMPO was induced in the Fenton system. On the other hand, in the case of M, PO, the degradation was found to be very slow, indicating that the 3 position of DMPO was favorably attacked by the oxidative species. Some of the degradation products are probably aminoxyl radicals. This series of the degradation products are probably aminoxyl radicals. This series of reactions may compete with spin trapping and make it difficult to analyze ESR spectra obtained in the presence of Fe ion.     

电子自旋共振-自由基捕获技术在生物学中的应用

本文结合作者实验室的工作,简要回顾了电子自旋共振-自由基捕获技术在生物领域的应用,包括新型自由基捕获探针的分子设计与合成,以及该技术在细胞、植物体系中的应用实例,并结合该技术的研究现状初步讨论了它的未来发展前景。     

Photogeneration of superoxide by adriamycin and daunomycin. An electron spin resonance and spin trapping study

The hydroxyl and superoxide anion spin adducts of DMPO and 4-MePyBN, respectively, were obtained during photoirradiation of adriamycin and daunomycin solutions with visible light. Ethanol and dimethyl sulfoxide did not scavenge hydroxyl radicals in the photoirradiated drug solutions. Furthermore, the hydroxyl-DMPO spin adduct is not formed in the photolysis of air-free drug solutions, indicating that hydroxyl radicals are not directly produced in the photochemical reactions. Instead, the observed hydroxyl-DMPO is formed from the decay of the superoxide anion-DMPO spin adduct. The mechanism for generating the superoxide anion radical appears to be a direct electron transfer from the photoexcited adriamycin and daunomycin to dissolved oxygen.     

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.     

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 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.     

Oxygen Radical Formation in Well-Washed Rat Liver Microsomes: Spin Trapping Studies

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5, 5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O₂, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1, 2,4-triazole in the presence of H₂O₂ were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.     

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.     

Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.     

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     

Cancer chemopreventive oltipraz generates superoxide anion radical

The cancer chemopreventive actions of oltipraz, a member of a class of 1,2-dithiolethiones, have been primarily associated with the induction of phase 2 enzymes mediated by a 41bp enhancer element known as the anti-oxidant response element in the promoter regions of many phase 2 genes. It has been suggested that oxygen radical formation by oltipraz may be a critical mechanism by which it exerts chemoprevention. Therefore, in the present work, studies were performed to directly determine if oltipraz generates oxygen free radicals. Electron paramagnetic resonance (EPR) spin trapping demonstrated that oltipraz slowly reacts in the presence of oxygen to generate the superoxide anion radical. This formation of superoxide by oltipraz was concentration- and time-dependent. EPR oximetry studies showed that oxygen was also slowly consumed paralleling the process of superoxide formation. Thus, oltipraz induced superoxide formation occurs and could be involved in the mechanism by which it exerts chemoprotection.     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

α-2,6-Difluorophenyl-N-Tert-Butylnitrone: A Spin Trap for Distinguishing Different Types of Alkyl Radicals Based on Long-Range Fluorine Hyperfine Splitting

-2.6-Difluorophenyl-N-tert-butylnitrone (F₂PBN) was synthesized and evaluated. A number of alkyl adducts of F₂PBN were studied by ESR/ENDOR. An additional hyperfine splitting (a triplet of doublets of doublets) is reported. The existence of two (one large, one small) F-hfsc's from the ortho-fluorine atoms in the phenyl ring of most alkyl adducts was confirmed by ENDOR spectroscopy.     

Generation of Active Oxygen Species in Blood Platelets—Spin Trapping Analysis

Generation of active oxygen species by bovine blood platelets was examined by the electron spin resonance (ESR) spin trapping technique with 5,5-dimethyl-l-pyroline-l-oxide (DMPO). The hydroxyl spin-trapped adduct 5,5-dimethyl-2-hydroxy-l-pyrolidinyloxy (DMPO-OH) was formed in the presence of platelets, indicating the generation of hydroxyl radicals (· OH) by the platelets. Generation of · OH was observed even with platelets in the resting state, but was markedly enhanced when the platelets were activated with stimulants. Stronger stimulants such as the calcium ionophore ionomycin, induced greater radical gener-ation than the weaker stimulant ADP. When the platelets were stimulated by thrombin, generation of · OH was greatest after l.5 min, and depended on the dose of the stimulant. It was inhibited by inhibitors of platelet activation such as forskolin and phenolic antioxidants.     

Spin Trapping of Methyl Radicals by the Acyl Nitroso Compound PH-C (= O)NO Formed in the Photochemical Reaction Between Benzohydroxamic Acid, Dimethyl Sulfoxide and Hydrogen Peroxide. An Epr Study

By the use of EPR spectroscopy, it has been shown that acyl nitroso compounds can act as spin traps for short-lived radicals with the formation of acyl aminoxyl radicals. The reaction was studied for the system benzohydroxamicacid[Ph-C (= O)N(H)] - dimethyl sulfoxide - hydrogen peroxide. The acyl aminoxyl radicals appeared almost immediately when the reaction mixture was irradiated in situ in the EPR cavity with UV light. The trapping reaction involved two photochemical reactions, i.e. the oxidation of the hydroxamic acid to the acyl nitroso compound Ph-C (= O)NO, and the formation of methyl radicals from dimethyl sulfoxide. The EPR spectra are superpositions of the spectra of two species of acyl aminoxyl radicals, i.e. the radicals Ph-C (= O)N(O·)H formed by oxidation of the parent benzohydrox-amic acid, and the radical Ph-C (= O)N(O·)CH₃, formed by trapping of methyl radicals.