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.     

Monte carlo simulations of site-specific radical attack to DNA bases

An atomistic biophysical model permitting the calculation of initial attacks to a 38-bp representation of B-DNA base moieties by water radicals is presented. This model is based on a previous radiation damage model developed by Aydogan et al. (Radiat. Res. 157, 38-44, 2002). Absolute efficiencies for radical attack to the 38-bp DNA molecule are calculated to be 41, 0.8 and 15% for hydroxyl radical ((.)OH), hydrogen radical (H(.)), and hydrated electron (e(aq))(,) respectively. Among the nucleobases, guanine is found to have the highest percentage (.)OH attack probability at 36%. Adenine, cytosine and thymine moieties have initial attack probabilities of 24, 18 and 22%, respectively. A systematic study is performed to investigate (.)OH attack probabilities at each specified attack site in four molecular models including free bases, single nucleotides, single base pairs, and the central eight base pairs of the 38-bp DNA molecule. Cytosine is the free base moiety for which the closest agreement is observed between the model prediction and the experimental data. The initial (.)OH attack probabilities for cytosine as the free base are calculated to be 72 and 28%, while experimental data are reported at 87 and 13% for the C5 and C6 positions on the base, respectively. In this study, we incorporated atomic charges to scale the site-specific (.)OH reaction rates at the individual atomic positions on the pyrimidine and purine bases. Future updates to the RIDNA model will include the use of electron densities to scale the reaction rates. With respect to reactions of the aqueous electron with DNA, a comparison of the initial distribution of electron attack sites calculated in this study and experimental results suggests an extremely rapid and extensive redistribution of the e(-)(aq) after their initial reactions with DNA.     

Polysaccharide degradation by Fenton reaction--or peroxidase-generated hydroxyl radicals in isolated plant cell walls

The formation of hydroxyl radicals (OH*) by peroxidase was confirmed by EPR spectroscopy using ethanol/alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin-trapping system specific of OH*. The effect of OH*, generated either non-enzymatically with the Fenton reaction (H(2)O(2) + Fe(2+)) or with horseradish peroxidase in the presence of O(2) and NADH, on cell walls isolated from maize (Zea mays) coleoptiles or soybean (Glycine max) hypocotyls was investigated. OH* produced by these reactions attack polysaccharides in the wall, demonstrated by the release of a heterogeneous mixture of polymeric breakdown products into the incubation medium. The peroxidase-catalyzed degradation of cell-wall polysaccharides can be inhibited by KCN and superoxide radical (O(2)*) or OH* scavengers. These data support the hypothesis that OH*, produced by cell-wall peroxidases in vivo, act as wall-loosening agents in plant extension growth.     

Hot spot kinetics of the sonolysis of aqueous acetate solutions

Water and acetate solutions were irradiated under argon by 300 kHz ultrasonic waves. Oxygen was found to be generated besides the products H2 and H2O2, already known. In the presence of acetate the O2 yield decreased rapidly while that of H2O2 decreased more slowly. Succinic acid was found as a product of the attack of OH radicals on acetate. Appreciable amounts of glyoxylic and glycolic acid and smaller amounts of formaldehyde and carbon dioxide were also detected. They resulted from the reaction of sonolytically generated oxygen with CH2CO2- radicals, produced upon attack of OH on acetate. Methane was a minor product of sonolysis. At acetate concentrations above 0.4 mol dm-3 CO2 and CO became the predominant products of sonolysis. This is explained by a second kind of action of ultrasound on dissolved acetate, i.e. by a thermal decomposition. This decomposition is possibly facilitated by radical attack on acetate. The results are discussed in terms of a 'structured hot spot' model, in which three regions for the occurrence of chemical reactions are postulated: a hot gaseous nucleus, an interfacial region with radial gradient in temperature and local radical density; and the bulk solution at ambient temperature.     

A pulse radiolysis study of some free radical reactions with erythrocyte membranes.

The reactions of the free radicals eaq- minus, OH and Br2- minus with haemoglobin-free erythrocyte ghost membranes have been studied by producing the radicals by pulse radiolysis and monitoring their reactions by optical spectroscopy. Hydrated electrons react rapidly with the membrane, but no attack at disulphide links was observed. Hydroxyl radical attack produced transient species absorbing weakly in the ultraviolet, which may arise from carbohydrate residues, such as N-acetyl neuraminic acid and N-acetyl glucosamine, on the membrane surface. No evidence was obtained for OH attack at ring-containing amino acid residues of the protein component. The Br2- minus radical, a more selective electrophile than OH, reacted only slowly with erythrocyte ghosts. Solubilization of the membranes with dodecylsulphate or digestion with alkali exposed protein containing tyrosine and tryptophan residues which reacted with Br2- minus. These results support other evidence for the absence of reactive protein at the membrane surface.     

Radical-Induced Damage to Proteins: E.S.R. Spin-Trapping Studies

The reactions of hydroxyl radicals generated from Fe¹¹/H₂O₂ and Cu¹¹/H₂O₂ redox couples with a variety of proteins (BSA, histones, cytochrome c, lysozyme and protamine) have been investigated by e.s.r. spin trapping. The signals obtained, which are generally anisotropic in nature, characterize the formation of partially-immobilized spin-adducts resulting from attack of the HO- radicals on the protein and subsequent reaction of the protein-derived radicals with the spin trap. Similar spin adducts are observed on incubation of two haem-proteins (haemoglobin and myoglobin) with H₂O₂ in the absence of added metal ions implying a reaction at the haem centre followed by internal electron transfer reactions.

Two strategies have been employed to obtain information about the site(s) of radical damage in these proteins. The first involves the use of a variety of spin traps and in particular DMPO: with this particular trap the broad spectra from largely immobilized radicals show characteristic a(β-H) values which enable carbon-, oxygen- and sulphur-centred radicals to be distinguished. The second involves the use of enzymatic cleavage of first-formed adducts to release smaller nitroxides, with isotropic spectra, which allow the recognition of β-proton splittings and hence information about the sites of radical damage to be obtained. These results, which allows backbone and side-chain attack to be distinguished, are in agreement with random attack of the HO^. radical on the protein and are in accord with studies carried out on model peptides. In contrast the use of less reactive attacking radicals [N₃·, ·CH(CH₃)OH] and oxidising agents (Ce⁴⁺) provides evidence for selective attack on these proteins at particular residues.     

Site-specific and bulk-phase generation of hydroxyl radicals in the presence of cupric ions and thiol compounds.

Addition of a thiol compound to a solution containing Cu2+ and H2O2 resulted in the generation of hydroxyl radicals (OH.). These radicals were able to oxidize salicylic acid and tryptamine in a reaction that was strongly inhibited by the OH.-scavenger mannitol. Covalent coupling of the thiol compound to tryptamine did not significantly influence the degradation of the indole moiety subsequent to addition of H2O2 and Cu2+. The inhibiting effect of mannitol, however, was strongly reduced, indicating that the scavenger could not interfere with site-specific reactions of OH..     

Oxidation of hydroxylamine to nitrite as an assay for the combined presence of superoxide anions and hydroxyl radicals.

The pulse-radiolytic oxidation of hydroxylamine by either hydroxyl radicals (OH), superoxide anions (O₂^?), or a combination of both radicals was investigated. It was found that only OH radicals efficiently attack the substrate, while O₂^? is necessary for the subsequent formation of nitrite. Determination of the latter reaction thus allows the detection of the combined presence of both oxygen radical species.     

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.     

The hydroxylation of the salicylate anion by a Fenton reaction and T-radiolysis: a consideration of the respective mechanisms

The yield of 2,3- and 2,5-dihydroxybenzoates (dHB's) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.     

The Effects of Hydroxyl Radical Attack on Dopa,Dopamine, 6-Hydroxydopa,and 6-Hydroxydopamine

High pressure liquid chromatography with electrochemical detection (HPLC-ED) was employed in conjugation with a sensitive and specific salicylate hydroxylation assay to evaluate the immediate effects of hydroxyl radical (·OH) attack on four catechol intermediates of eumelanin, dopamine (3,4-dihydroxyphenylethylamine), its precursor dopa (3,4-dihydroxyphenylalanine), and their respective neurotoxic trihydroxyphenyl derivatives, 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine,6-OHDA) and 6-hydroxydopa(2,4,5-trihydroxyphenylalanine, TOPA). Semiquinone and quinone species were identified as the initial products of the oxidation of these four catechol substrates. The enhanced oxidations of the catechols when exposed to ·OH attack was accompanied by marked decreases in the level of each semiquinone species. Quinone levels were elevated in reactions involving ·OH attack on dopamine and 6-OHDA, but absent in reactions involving radical attack on dopa or TOPA, suggesting that dopaquinone (DOQ) and TOPA p-quinone (TOPA p-Q) are oxidized more rapidly by‘OH than are the quinones of dopamine and 6-OHDA. The formation of 6-OHDA p-quinone (6-OHDA p-Q) in incubations involving DA and ·OH suggest that the ·OH-mediated hydroxylation of DA may be a mechanism for generating this potentially cytotoxic trihydroxyphenyl. The results of this study demonstrate for the first time that semiquinone and quinone intermediates of eumelanin are the initial products derived from the ·OH-mediated oxidations of dopa, DA, TOPA, and 6-OHDA. These observations suggest that if ·OH is generated beyond the capabilities of cytoprotective mechanisms, the radical can rapidly oxidize catechol precursors, augment melanogenesis, and generate additional cytotoxic quinoid intermediates of eumelanin.     

NADH-dependent microsomal interaction with ferric complexes and production of reactive oxygen intermediates

The interaction of NADPH with ferric complexes to catalyze microsomal generation of reactive oxygen intermediates has been well studied. Experiments were carried out to characterize the ability of NADH to interact with various ferric chelates to promote microsomal lipid peroxidation and generation of .OH-like species. In the presence of NADH and iron, microsomes produced .OH as assessed by the oxidation of a variety of .OH scavenging agents. Rates of NADH-dependent .OH production were 50 to 80% those of the NADPH-catalyzed reaction. The oxidation of dimethyl sulfoxide or t-butyl alcohol was inhibited by catalase and competitive .OH scavengers but not by superoxide dismutase or carbon monoxide. NADH-dependent .OH production was effectively catalyzed by ferric-EDTA and ferric-diethylenetriaminepentaacetic acid (DTPA), whereas ferric-ATP and ferric-citrate were poor catalysts. All these ferric chelates were reduced by microsomes in the presence of NADH (and NADPH). H2O2 was produced in the presence of NADH in a reaction stimulated by the addition of ferric-EDTA, consistent with the increase in .OH production. The latter appeared to be limited by the rate of H2O2 generation rather than the rate of reduction of the ferric chelate. NADH-dependent lipid peroxidation was much lower than the NADPH-catalyzed reaction and showed an opposite response to catalysis by ferric complexes compared to .OH generation as production of thiobarbituric acid-reactive material was increased with ferric-ATP and -citrate, but not with ferric-EDTA or- DTPA, and was not affected by catalase, SOD, or .OH scavengers. These results indicate that NADH can support microsomal reduction of ferric chelates, with the subsequent production of .OH-like species and peroxidation of lipids. The pattern of response of the NADH-dependent reactions with respect to catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with NADPH. Many of the metabolic actions of ethanol have been ascribed to production of NADH as a consequence of oxidation by alcohol dehydrogenase. Since the cytosol normally maintains a highly oxidized NAD+/NADH redox ratio, it is interesting to speculate that increased availability of NADH from the oxidation of ethanol may support microsomal reduction of iron complexes, with the subsequent generation of reactive oxygen intermediates.     

Production of hydroxyl free radical in the xanthine oxidase system with addition of 1-methyl-3-nitro-1-nitrosoguanidine

We have examined the mechanism of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced gastric cancer with respect to the production of hydroxyl free radical (OH). Nucleophilic attack by H2O2 on the nitroso group of MNNG produces 1-methyl-3-nitroguanidine (MNG) and the intermediate peroxynitric acid (ONOOH), which splits into hydroxyl free radical (OH) and nitrogen dioxide leading to the formation of nitric and nitrate ions in water. Xanthine oxidase (XO) induces the production of O2.- or H2O2 from molecular oxygen, depending on the overall level of enzyme reduction. In this study, we examined OH production by the reaction of MNNG with H2O2 derived from the XO-HX system containing XO and the purine substrate hypoxanthine by ESR using the spin trapping reagent 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). OH was produced in the XO-HX-DMPO system with addition of MNNG (the MNNG-XO-HX-DMPO system) under aerobic conditions, but was not in the XO-HX-DMPO system, and production of OH was inhibited by catalase but not by superoxide dismutase, suggesting that OH was produced by the reaction of MNNG with H2O2 derived from the XO-HX system. The production of OH was significantly increased with increase in the reducing activity of XO, though that of O2.- was not, also suggesting the O2(.-)-independent .OH production. The productions of nitrite ion and MNG in the MNNG-XO-HX system were determined by the colorimetric method and HPLC, respectively. Based on these findings, we conclude that .OH was produced by homolytic split of the intermediate ONOOH formed by nucleophilic attack of H2O2 derived from the XO-HX system on MNNG.     

Site-specific OH attack to the sugar moiety of DNA: a comparison of experimental data and computational simulation.

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na(+) counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5')+2H(5'), H(4'), H(3'), 1H(2')+2H(2'), H(1')] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738-9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4') is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5')+2H(5') > H(4') > H(3') > 1H(2')+2H(2') > H(1').     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Spontaneous nucleophilic addition of hydroxide ions to the meso-position of high-valent antimony-oxo porphyrin complexes

Bimolecular reactions of the antimony(V) porphyrin complexes SbO(tpp)OH, 1 and SbO(oep)OH, 2 with tetra-n-butylammonium hydroxide were investigated at 298 K in acetonitrile solution (tpp, dianion of 5,10,15,20-tetraphenylporphyrin and oep, dianion of 2,3,7,8,12,13,17,18-octaethylporphyrin). Spontaneous nucleophilic addition of hydroxide ions to the non-oxidized porphyrin macrocycle leads to novel hydroxyphlorin derivatives, which contain a saturated meso-carbon bridge. While this process is a reversible equilibrium reaction for the TPP derivative, the OEP complex undergoes subsequent demetallation and oxidative ring cleavage in the presence of dioxygen. Possible implications for the competitive inhibition of heme-oxygenase by high-valent metalloporphyrin therapeutics are discussed.     

Phosphorylation of AMP by inorganic phosphorylating agents

AMP was phosphorylated by inorganic phosphorylating agents: cyclo-triphosphate and diphosphonate, in aqueous solution (70-80 degrees C, pH 6-12). The molecular structures of phosphorylated products were established by use of phosphorus-31 NMR and high-performance liquid chromatography (HPLC). The OH groups on AMP were phosphorylated by both phosphorylating agents to form 2'- or 3'-phosphate but an OH group on dAMP was not phosphorylated. Phosphorylation of OH group proceeds in two steps: formation of hydrogen bond between OH group and phosphorylating agent; subsequent nucleophilic attack of OH group on a phosphorus atom. Phosphate group on AMP was phosphorylated by diphosphonate but not by cyclo-triphosphate. The difference in the reactivities is explained in terms of charge repulsion between AMP and agents.     

Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.     

Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.     

Improved hybrid optimization algorithm for 3D protein structure prediction

Diuron, a chlorine-substituted dimethyl herbicide, is widely used in agriculture. Though the degradation of diuron in water has been studied much with experiments, little is known about the detailed degradation mechanism from the molecular level. In this work, the degradation mechanisms for OH-induced reactions of diuron in water phase are investigated at the MPWB1K/6–311+G(3df,2p)//MPWB1K/6–31+G(d,p) level with polarizable continuum model (PCM) calculation. Three reaction types including H-atom abstraction, addition, and substitution are identified. For H-atom abstraction reactions, the calculation results show that the reaction abstracting H atom from the methyl group has the lowest energy barrier; the potential barrier of ortho- H (H1’) abstraction is higher than the meta- H abstraction, and the reason is possibly that part of the potential energy is to overcome the side chain torsion for the H1’ abstraction reaction. For addition pathways, the ortho- site (C (2) atom) is the most favorable site that OH may first attack; the potential barriers for OH additions to the ortho- sites (pathways R7 and R8) and the chloro-substituted para- site (R10) are lower than other sites, indicating the ortho- and para- sites are more favorable to be attacked, matching well with the -NHCO- group as an ortho-para directing group.