OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Radical anions from one-electron-reduced adrenochrome. Detection and identification by electron spin resonance spectroscopy

Free radicals from the one-electron reduction of adrenochrome have been studied in aqueous solutions. These radicals have been detected and identified by electron spin resonance spectroscopy, using spin stabilization methods (complexation with diamagnetic metal ions) to enhance radical concentrations. It is shown that the radicals have a characteristic ESR spectrum enabling their identification in complex systems. The spin density distribution in the radicals has been studied as a function of complexing metal ions and solvent composition. In the presence of oxidants (e.g., oxygen) the spectrum of the radical is replaced by that derived from the one-electron exidation of adrenochrome.     

The enzymatic one-electron reduction of porphyrins to their anion free radicals

The anaerobic enzymatic one-electron reduction of uroporphyrin I (in the absence of light) by the ferredoxin/ferredoxin:NADP+ oxidoreductase system was investigated using NADPH as the source of reducing equivalents. The porphyrin anion free radical metabolite formed by one-electron reduction of the parent molecule was detected with ESR spectroscopy. The ESR spectrum exhibited a singlet (g = 2.0021) with a 5.4-G peak-to-peak linewidth. The reduction process was also investigated under aerobic conditions. The reduction of molecular oxygen to superoxide anion radical by the porphyrin anion radical was demonstrated by using the ESR technique of spin trapping. The ESR spectra of the spin-trapped oxygen-derived radicals were superoxide dismutase-sensitive and catalase-insensitive, supporting the assignment of the trapped radical to the superoxide anion radical. These aerobic experiments demonstrate electron transfer from the porphyrin anion radical to molecular oxygen. The anaerobic reduction of Photofrin II by hepatic microsomes and the ferredoxin/ferredoxin:NADP+ oxidoreductase system to a porphyrin anion radical was also investigated. Free radical formation by ferredoxin: NADP+ oxidoreductase is totally dependent upon ferredoxin. The ESR spectrum of this porphyrin free radical also exhibited a singlet (g = 2.0026) with a 15-G peak-to-peak linewidth.     

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.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Characterization of the structure and reactions of free radicals from serotonin and related indoles

The ESR spectra of the free radicals formed by the autoxidation of serotonin, 5-hydroxyindole, and 5-hydroxytryptophan in 1 N NaOH are presented. The analysis of the hyperfine splitting constants in H2O and D2O characterize these free radicals as semiquinone-imines, the one-electron oxidation product of the corresponding indole. At alkaline pH, autoxidation of these compounds ultimately leads to solid precipitate and unresolved ESR spectra characteristic of polymeric material. The reduction of cytochrome c at pH 7.4 by a wide variety of indoles correlates with the amplitude of the ESR signal in 1 N NaOH, as do other processes thought to be related to 5-hydroxyindole free radical formation. Relative to the rate of cytochrome c reduction, neither serotonin nor the serotonin free radical appears to react with oxygen to form superoxide. In the presence of NAD(P)H, the serotonin radical most probably oxidizes NAD(P)H to form the NAD(P). radical. The NAD(P). radical then reacts with oxygen to form superoxide, which ultimately reduces cytochrome c.     

Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

The detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody sensitivity and specificity greatly expands the utility of the spin trapping technique, which is no longer dependent on the quantum mechanical electron spin resonance (ESR). The specificity of the reactions of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Now the development of an immunoassay for the nitrone adducts of protein radicals brings the power of immunological techniques to bear on free radical biology. Polyclonal antibodies have now been developed that bind to protein adducts of the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In initial studies, anti-DMPO was used to detect DMPO protein adducts produced on myoglobin and hemoglobin resulting from self-peroxidation by H2O2. These investigations demonstrated that myoglobin forms the predominant detectable protein radical in rat heart supernatant, and hemoglobin radicals form inside red blood cells. In time, all of the immunological techniques based on antibody-nitrone binding should become available for free radical detection in a wide variety of biological systems.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

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 of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.     

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.     

Reaction of vanadium(V) with thiols generates vanadium (IV) and thiyl radicals

The in vivo toxicity of vanadium(V) has been found to correlate with the depletion of cellular glutathione and related non-protein thiols. With a view to understanding the mechanism for this observation, we have investigated the oxidation of glutathione, cysteine N-acetylcysteine and penicillamine by vanadium(V), using electron spin resonance (ESR) and ESR spin trapping methodology. The spin trap used was 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). It is found that the oxidation of these thiols by vanadium(V) generates the corresponding thiyl radicals and vanadium- (IV) complexes. The results suggest that free radical reactions play a significant role in the depletion of cellular thiols by vanadium(V) and hence in vanadium(V) toxicity.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/^·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

DMPO-Alkyl radical spin trapping: an in situ radiolysis steady-state ESR study

Short-lived free radicals formed in the reaction of 11 substrates and radiolytically produced hydroxyl radicals were trapped successfully with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in dilute aqueous solution. The in situ radiolysis steady-state ESR spectra of the spin adducts were analyzed to determine accurate ESR parameters for these spin adducts in a uniform environment. Parent alkyl radicals include methyl, ethyl, 1-propyl and 2-propyl (1-methylethyl). Hydroxyalkyl parent radicals were hydroxymethyl, hydroxyethyl, 2-hydroxy-2-propyl (1-methyl-1-hydroxyethyl), 1-hydroxypropyl and 2-hydroxy-2-methylpropyl. Carboxyl radical (carbon dioxide anion, formate radical) and sulfite anion radical were the sigma radicals studied. The DMPO spin adduct of 1-propyl was identified for the first time. For most spin adducts, g factors were also determined for the first time. In DMPO spin adducts of hydroxyalkyl radicals, nitrogen and C(2)-proton hyperfine coupling constants are smaller than those of alkyl radical adducts; the hydroxyalkyl spin adducts possess larger g values than their unsubstituted counterparts. These changes are ascribed to the spread of pi conjugation to include the hydroxyl group. Strong evidence of spin addend-aminoxyl group interaction can be seen in the asymmetrical line shapes in the hydroxyethyl and the hydroxypropyl spin adducts.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Effects of pH and exocyclic substitution on flavosemiquinone reactivity with redox proteins and inorganic oxidants

The effect of pH on the reaction of free flavosemiquinone analogs generated by laser-flash photolysis with oxidized Chromatium vinosum high-potential iron-sulfur protein, other iron-containing redox proteins, and nonbiological one-electron oxidants has been investigated. The results demonstrate that the second-order rate constant for the oxidation of lumiflavin flavosemiquinone increases dramatically with increasing pH for the redox proteins and some of the other oxidants. The pH-rate constant profiles for the redox proteins closely follow the ionization of the proton at the N-5 position of the neutral lumiflavin flavosemiquinone, suggesting a higher intrinsic reactivity for the anionic lumiflavin flavosemiquinone. This increased reactivity apparently results from changes in the redox potential and in the electron spin density distribution between the two protonic forms of the semiquinone. Similar pH dependencies are observed for a number of other flavin structural analogs, yielding estimates of the N-5 pK values for these analogs. The data are consistent with the involvement of both the N-5-dimethylbenzene ring portion and the C-4a position of the flavin macrocycle in flavosemiquinone oxidation by one-electron oxidants.