Reactions of the hydrated electron with pyrimidine nucleosides halogenated at the sugar moiety: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by the reactions of hydrated electrons with pyrimidine nucleosides halogenated at the sugar moiety (2'-chloro-2'-deoxyuridine and 2'-chlorothymidine) were studied by e.s.r. and spin-trapping. 2-Methyl-2-nitrosopropane was used as the spin-trap. The usual spin-trapping technique was extended to frozen and deoxygenated systems to avoid contamination of the trapped radicals with side-products by spin-trapping 2-methyl-2-nitrosopropane itself. When this method was applied to 2'-chloro-2'-deoxyuridine, a free radical at the C-2' position of the sugar moiety was spin-trapped together with a free radical at the C-5 position of the base moiety. This indicates that hydrated electrons both add to the base moiety and eliminate halogen anions from the halogenated sugar moiety. In the case of 2'-chlorothymidine, however, only a free radical attributed to H-addition at the C-6 position of the thymine base was observed. No radicals produced by the reaction of hydrated electrons with the halogenated sugar could be spin-trapped.     

Spin trapping of precursors of thymine damage in X-irradiated DNA

A spin-trapping method combined with ESR spectroscopy was utilized to obtain evidence for the presence of precursor radicals leading to damage in X-irradiated DNA. Two technical improvements were introduced to the conventional spin-trapping method to make possible its application to large molecules such as DNA: prior to X irradiation, sonolysis of aqueous DNA solution by 19.5-kHz ultrasound was made to get a highly concentrated DNA solution and to lower the viscosity of the solution; after precursor radicals in X-irradiated DNA were trapped by a spin-trapping reagent, the DNA was digested to oligonucleotides by DNase I to get an ESR spectrum with a well-resolved hyperfine structure. Thus, it was recognized that the ESR spectrum obtained after X irradiation of the aqueous solution containing DNA and the nitroso spin-trapping reagent 2-methyl-2-nitrosopropane consisted of at least three sets of signals in the DNA. Identification of free radicals was made by comparing the spectrum with that of thymidine, which was precisely examined by a spin-trapping method combining two kinds of spin traps (nitroso and nitrone compounds) with liquid chromatography. As a result, all the signals were identified as the spin adducts of radicals produced at the thymine base moiety of DNA. The 5-hydroxy-5,6-dihydrothymin-6-yl radical was identified as a precursor of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), the 6-hydroxy-5,6-dihydrothymin-5-yl radical as a precursor of 6-hydroxy-5,6-dihydrothymine, and the 5-methyleneuracil radical as a precursor of 5-(hydroxymethyl) uracil.     

γ射线辐照嘧啶核苷水溶液的电子自旋共振（ESR）研究

亚硝基特丁烷（ＭＮＲ）为捕集剂,利用自旋捕集技术,对γ射线辐照嘧啶苷水溶液进行了ＥＳＲ研究,确定了胸苷、尿苷和胞苷可产生的自肇捕集自由基的类别,并讨论了自由基形成的机制。     

Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.     

Hydrogen-deuterium exchange in gamma-irradiated polycrystalline DL-alanine: a spin-trapping and e.s.r. study

The hydrogen-deuterium exchange reactions in gamma-irradiated DL-alanine in the solid state were investigated by spin-trapping and electron spin resonance (e.s.r) using selectively deuterated DL-alanine. Subsequent to gamma-radiolysis at 30 degrees C, polycrystalline DL-alanine was dissolved in aqueous solutions of 2-methyl-2-nitrosopropane and the extent of H-D exchange of the deamination radicals was followed by e.s.r. After formation of the deamination radicals, four exchange reactions were found to occur between the radicals and the surrounding undamaged molecules. The first reaction, which occurs between the hydrogens of the C-2 carbon of the radicals and those of the methyl groups of the neighbouring molecules, can be followed at room temperature. The three other reactions could be conveniently monitored in gamma-irradiated polycrystalline alanine at 110 degrees C. The first of the other three reactions takes place between the methyl hydrogens of the radicals and the C-2 hydrogens of nearby molecules, while the remaining processes involve exchange between the hydrogen atoms of the amino group and those on the C-2 and C-3 carbon atoms of the deamination radical.     

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.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C] were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radical attack and the subsequent radical reactions in cytosine-related compounds were clarified.     

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.     

Electron transfer reactions of tris(toluene-3,4-dithiolato)molybdenum(VI) with organic compounds

The compound [Mo(tdt)₃] (tdt=toluene-3,4-dithiolate) reacts with aromatic hydrocarbons, N-heterocycles, riboflavin and molybdoenzyme substrates such as purines and aldehydes forming the reduced species [Mo(tdt)₃]⁻ and organic radicals. The reactions were followed with UV-Vis and ESR spectroscopy; organic radicals were trapped with 2-methyl-2-nitrosopropane and 5,5-dimethyl-1-pyrroline-N-oxide. The stoichiometry of the reaction with naphthalene and triphenylphosphine, determined by Job's method of continuous variations, was 1:1.The solvent was dichloromethane. For the reactions to occur the presence of a base was necessary, a reactant or added ethanol for the hydrocarbon reactions. The reactions involved electron transfer from the organic molecule to the molybdenum dithiolate.     

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.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline DL-alanine. A quantitative determination of radical yield

The quantitative aspects of determining free radicals in polycrystalline amino acids gamma-irradiated at room temperature and subsequently dissolved in spin-trap solutions were investigated. The deamination radical in DL-alanine was used for detailed studies and 2-methyl-2-nitrosopropane (MNP) was employed as the spin-trap. The spin-trapping efficiency (the number of radicals spin-trapped in solution divided by the number of radicals initially present in the gamma-irradiated solid) was found to be in the range 1 to 10 per cent for aqueous solutions depending on the experimental conditions. The effects of dose, particle size, pH, spin-trap concentration, age of spin-trap solution, MNP monomer to dimer ratio and the presence of organic solvents were investigated. Several reactions were found to decrease the spin-trapping efficiency; radical-radical recombination, the competition between the spin-adduct and the spin-trap for radicals and the reaction of radicals with the MNP dimer. The reaction of intact DL-alanine molecules with deamination radicals to produce H-abstraction radicals which are not spin-trapped does not significantly lower the spin-trapping efficiency. The results obtained with compounds such as glycine, glycylglycine, L-valine and L-proline suggest that the low spin-trapping efficiency found for DL-alanine may be representative of polycrystalline amino acids.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Photochemistry of pyrimidine bases as studied by e.s.r. and spin-trapping

The direct photoexcitation of pyrimidine bases in D2O solutions yields free radicals which could be conveniently identified by spin-trapping with 2-methyl-2-nitrosopropane. Most of the radicals formed were attributed to D-addition to one end of the 5,6 double bond. However, orotic acid and iso-orotic acid yielded N(3) centred free radicals, formed by homolytic cleavage of the N-H bond. No indication could be found for a free radical involvement in the photocleavage of cyclobutane-type pyrimidine dimers.     

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.     

Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat.

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.     

OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3'-deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2'-deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2'-deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety.     

Oxidation of orcinol by ceruloplasmin and mixed-ligand copper complexes

Unable to oxidize orcinol (3,5-dihydroxytoluene) under conventional conditions, ceruloplasmin (Cp) catalyzes its oxidation when superoxide radicals are injected into the solution with the aid of a high-voltage generator. The O2-. to oxidized orcinol ratio in solution is close to 2:1. The concentration of hydrogen peroxide, which is the product of the Cp-catalyzed dismutase reaction, is about half that of O2-. No slower than by the native enzyme, orcinol in the presence of O2-. is oxidized by Cp depleted of all its type 2 coppers and partly of type 1 Cu2+. Copper complexes with oxalate and pyrophosphate are able to oxidize orcinol under aerobic conditions, one molecule of oxygen being consumed per each oxidized molecule of orcinol. Both the oxidation of orcinol by Cp and by copper complexes are inhibited by cyanide. Orcinol oxidation seems to be caused by singlet oxygen produced in the Cp-catalyzed dismutase reaction.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.