Electron spin resonance studies of radicals obtained by the reaction of alpha-tocopherol and its model compound with superoxide ion.

alpha-Tocopherol (vitamin E) and its model compound, 6-hydroxy-2,2,5,7,8-pentamethylchroman, were found to be oxidized by O2- to yield free radicals which were detected at room temperature by ESR spectroscopy. The ESR spectra of these radicals showed seven main lines with additional hyperfine structure and have the same g-values at 2.0046. Assignments of the ESR spectra were done on the basis of the spectra of the free radicals of deuterated hydroxypentamethylchroman obtained from the same reaction with O2-. The radicals observed are chromanoxyls generated by the abstraction of hydrogen from the 6-hydroxy group of tocopherols.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

Stable free radicals in insect cuticles: electron spin resonance spectroscopy reveals differences between melanization and sclerotization

Insect cuticles (exuviae; cast skins) were examined for the first time by ESR spectroscopy for the presence of stable free radicals, as found in melanins. All cuticles, except those from a locust albino strain, irrespective of the presence of melanin, provided single-line signals of varied g-values and linewidths. The ESR signals of melanins, isolated or in cuticles, were characterized by g-values <2.004 and small linewidths in the range of 4-6G, while sclerotized cuticles, lacking melanin, showed g-values >2.004 and broad linewidths of 5-11 G. The melanin spectra were comparable to those reported for eumelanins with indol-based monomers. Minor signals ascribed to pheomelanins were found in several probes. The 'sclerotin' spectra were broader and displayed unresolved hyperfine structure in some cases. As for melanins, the location and environment of the radicals in cuticles giving rise to the two types of ESR spectra could not be assigned. Changes in the radical environment due to insecticide or solvent treatment can be detected by ESR spectroscopy.     

Evidence for the formation of strand-break precursors in hydroxy-attacked thymidine 5'-monophosphate by the spin trapping method

A method combining spin trapping, ESR, and HPLC was employed to obtain evidence for the formation of sugar radicals in OH-attacked TMP with special emphasis on the detection of strand-break precursors of DNA. OH radicals were produced by irradiating an N2O-saturated aqueous solution with X-rays. When an N2O-saturated aqueous solution containing TMP and a spin trapping reagent, MNP, was irradiated with X-rays, it was estimated on the basis of theoretical calculations using rate constants that 94% of the TMP radicals were induced by OH radicals. Since several spin adducts between TMP radicals and MNP, as well as the byproducts of the spin trapping reagent itself, were produced, reverse-phase HPLC was used to separate them. The presence of six spin adducts was confirmed by ESR examination. Further examination of these spin adducts by UV absorbance spectrophotometry showed the presence of a chromophore at 260 nm in three adducts. Since a gradual increase in the release of unaltered base from these adducts was observed when they were allowed to stand for 0-22 h at room temperature, they could be regarded as the spin adducts of sugar radicals and MNP. ESR spectra from the spin adducts were consistent with hydrogen abstraction radicals at the C1', C4', and C5' positions of the sugar moiety. These radicals appeared to be precursors of AP sites and strand breaks. In addition to these spin adducts, ESR spectra that were consistent with the spin adducts of base radicals (the C5 and C6 radicals) and MNP were observed.     

Esr and Spin-Trapping Study of Free Radicals in γ-Irradiated Solid Lysozyme

Free radicals produced by γ-irradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γ-irradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH₂-_-S^-, at disulfide bridges were produced in γ-irradiated solid lysozyme.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

过硫酸铵-N,N,N′,N′-四甲基乙二胺体系产生的氧自由基及其清除的研究

应用ESR和自旋捕集相结合的技术直接测定了过硫酸铵—N,N,N′,N′-四甲基乙二胺(AP-TEMED)体系产生的氧自由基,经计算机波谱模拟和计算波谱参数证实该体系产生的氧自由基是O_2~-和·OH。并用维生素C、茶多酚、超氧化物歧化酶等氧自由基清除剂,从聚丙烯酰胺凝胶法、化学发光法和脂质过氧化法不同角度研究了AP-TEMED体系在自由基研究方面的应用意义。     

Free radical generation by ultrasound in aqueous solutions of nucleic acid bases and nucleosides: an ESR and spin-trapping study

Direct evidence for the detection of intermediate radicals of nucleic acid constituents induced by ultrasound in argon-saturated aqueous solution is presented. The method of spin trapping with 3,5-dibromo-4-nitrosobenzene sulphonate, which is a water-soluble, non-volatile, aromatic nitroso spin trap, combined with ESR, was used for the detection of sonochemically induced radicals. Spin adducts were also generated by OH radicals produced by UV photolysis of aqueous solution containing H2O2. ESR spectra observed from these photolysis experiments were identical to those after sonolysis. The ESR spectra of the spin adducts suggest that the major spin-trapped radical of thymine and thymidine was the 5-yl radical, and that of cytosine, cytidine, uracil, and uridine was the 6-yl radical. To compare the radicals induced by sonolysis and photolysis, the decay of the ESR spectra of the thymine and thymidine spin adducts was investigated. The decay curves of thymine and thymidine after sonolysis indicated biphasic decay. However, after photolysis the spin adducts from both compounds showed very little decay. These results suggest that the observed spin adducts in the sonolysis of pyrimidine bases and nucleosides were formed by OH radical and H atom addition to the 5,6 double-bond.     

Spin label study of the structure and conformational properties of fibers from cotton grown from gamma-irradiated seeds]

According to ESR spectra of the radical - probe I it has been shown that there are two regions in cotton cellulose: ordered and disordered ones, pointing to inhibition and free rotation of the radical. 40 spin-labeled samples of cotton filaments and their mutants by the radicals II-IX chemically bound by the cellulose hydroxyl group were obtained. From ESR spectra strong inhibition of iminoxyl radical was established which was the evidenced of high rigidity of disordered regions of cellulose. Conformational changes of cotton cellulose grown from seeds exposed to different doses of gamma-irradiation.     

Computer analysis of electron paramagnetic resonance spectra of spin labels in the study of biological membranes

Application of computer analysis to ESR spectra of maleimide spin labels in erythrocyte ghosts and ESR spectra of "spin sacks"--erythrocyte ghosts and liposomes containing concentrated solution of non-penetrating spin label was described. The analysis of the ESR spectra of spin labels gives exhausting information about the parameters of spin hamiltonian, peculiarities of the movement of nitroxyl radicals and their distribution between the cell and medium.     

The fate of antioxidant radicals during lipid autooxidation. I. The tocopheroxyl radicals

Peroxidizing lipids were used to induce the formation of antioxidant radicals. It has been shown by electron spin resonance (ESR) spectroscopy and simulation of the first derivative ESR spectra that the radicals formed by this method are the already known tocopheroxyl radicals. dl-alpha-Tocopheroxyl radicals were formed in relatively high concentration but were rather rapidly destroyed as compared to the dl-delta-tocopheroxyl radicals, which were formed in rather low concentration and were destroyed rather slowly, dl-beta- and dl-gamma-tocopheroxyl radicals reacted in an intermediate way. Autooxidation induction times of the same lipids stabilized with the tocopherols show the well accepted series of antioxidant activities alpha less than beta congruent to gamma less than delta. Their relative antioxidant activity is nicely explained by the ESR experiment: the fast reacting dl-alpha-tocopherol is reacting more rapidly and traps the radicals more thoroughly and is therefore only available as an antioxidant for a short period of time as compared with the slowly reacting dl-delta-tocopherol. dl-beta- and dl-gamma-Tocopherols behave in an intermediate way.     

In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively.

On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.     

Analysis of free-radical products of near-UV irradiation at 77 K of frozen adenine and adenosine diphosphate solutions containing inorganic phosphate

Irradiation by near-UV light at 77 K of aqueous solutions of inorganic phosphate in weakly acidic conditions in the presence of the photosensitizers adenine and adenosine diphosphate results in the formation of free radicals of these compounds, photosensitized free radicals of phosphate itself, and H* and OH* radicals. The relative concentrations of free radical products were estimated by the analysis of total ESR signals registered in the region of g = 2.00 in the photosystems Ade + Pi and Adphi + Pi using the original computer program of ESR spectra simulation.     

The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.     

Formation of iminoxyl and nitroxide free radicals from nitrosonaphthols: an electron spin resonance study

The possible metabolic activation of nitrosonaphthols, suspected carcinogens, was investigated by electron spin resonance (ESR) spectroscopy. Free radicals were found to be the primary metabolites formed during both the reduction and oxidation of these compounds. Whereas the one-electron oxidation of nitrosonaphthols is enzymatic and catalyzed by the peroxidase prototype, horseradish peroxidase, their one-electron reduction by reducing cofactors such as NADH or NADPH was not enhanced by rat liver microsomal enzymes. The ESR spectra of the radicals found during the oxidation of nitrosonaphthols were analyzed and characterized as iminoxyl free radicals. The reduction pathway leads to nitroxide free radicals with unusually low nitrogen hyperfine constants.     

Reactions of superoxide ion with tocopherol and its model compounds: correlation between the physiological activities of tocopherols and the concentration of chromanoxyl-type radicals

Reactions of tocopherol model compounds with superoxide ion (02-) were investigated. 6-Hydroxy-2,2,5,7,8-pentamethylchroman (alpha-model), 6-hydroxy-2,2,5,7-tetramethylchroman and 6-hydroxy-2,2,5,8-tetramethylchroman (beta-model) were oxidized by O2- to yield chromanoxyl radicals which gave ESR spectra, but the radical species were not obtained from 6-hydroxy-2,2,7,8-tetramethylchroman (gamma-model) and 6-hydroxy-2,2-dimethylchroman, both of which do not have a methyl substituent at the C-5 position. ESR studies of the reactions of O2- with tocopherols or their model compounds indicate that the radical concentrations from tocopherol models correlate with the physiological activities of the tocopherols.     

Formation of .OH and .H in aqueous solutions by ultrasound using clinical equipment

Hydroxyl radicals and hydrogen atoms were produced in argon-saturated aqueous solutions exposed to ultrasound using clinical dental equipment. .OH and .H radicals were detected and identified by ESR and were spin trapped with 5,5-dimethyl-1-delta-pyrroline-N-oxide (DMPO) and alpha-4-pyridyl-1-oxide-N-tert-butylnitrone (POBN). The observed ESR spectra were compared with those obtained from sonolysis of argon-saturated water in an ultrasonic bath, from gamma radiolysis of air-saturated water, and from uv photolysis of aqueous hydrogen peroxide solutions.     

OH-induced free radicals in uridine studied by a method combining ESR, spin-trapping, and liquid chromatography

Free radicals produced by the reactions of OH radicals with uridine were investigated by a method combining ESR, spin-trapping, and liquid chromatography. A N2O-saturated aqueous solution of uridine, containing 2-methyl-2-nitrosopropane as a spin-trap, was X-irradiated and the resulting spin-adducts were separated by gel permeation chromatography and reverse-phase HPLC. ESR and uv-absorbance spectra obtained from the separated spin-adducts show that 5-yl and 6-yl radicals are produced by OH addition to the 5,6 double bond of the base moiety. It is also shown that radicals due to H abstraction from the sugar moiety at the C-4' and C-5' positions are produced.     

ESR spin trapping studies of free radicals generated from nitrofuran derivative analogues of nifurtimox by electrochemical and Trypanosoma cruzi reduction

Electron spin resonance (ESR) spectra of radicals obtained from two analogues of the antiprotozoal drug nifurtimox by electrolytic and Trypanosoma cruzi reduction were analyzed. The electrochemistry of these compounds was studied using cyclic voltammetry. STO 3-21G ab initio and INDO molecular orbital calculations were performed to obtain the optimized geometries and spin distribution, respectively. The antioxidant effect of glutathione on the nitroheterocycle radical was evaluated. DMPO spin trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. Nitro1 and Nitro2 nitrofuran analogues showed better antiparasitic activity than nifurtimox. Nitro2 produced oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of either the hydroxyl radical or the Nitro2 analogue radicals. These results are in agreement with the biological observation that Nitro2 showed anti-Chagas activity by an oxidative stress mechanism.