Guanyl Radicals Research Articles

Redox factors in the antioxidant activity of nitroxides toward DNA guanyl and 2-deoxyribose-peroxyl radicals

A series of eight nitroxide compounds (four substituted piperidines, three pyrrolidines and one oxo-piperidine) are found to undergo electron transfer to 2’-deoxyribose-peroxyl and the guanyl radical. One-electron oxidation potentials of the nitroxides to oxoammonium cations (oxoammonium reduction potential), E 0’, have been measured against a common redox indicator, chlorpromazine, and found to span the range 751 ± 15 mV to 973 ± 15 mV. Fast chemical reduction of the 2’-deoxyribose-peroxyl radical to the hydroperoxide, generated by • OH radical attack on 2-deoxyribose, dR, in oxygenated aqueous solution, is a redox-dependent reaction, with rate constants of 0.8–3.5 x 107 M−1 s−1.The guanyl radicals, produced upon one-electron oxidation of 2’-deoxyguanosine monophosphate, dG, by the selenite radical, SeO3 • -, react with the nitroxides in a redox-independent reaction with diffusion rate constants of 1–2 x 108 M−1 s−1. These findings represent a possible antioxidant role for nitroxides in the fast chemical repair of DNA radicals, which is supported by an in vitro strand break study using a plasmid.

Modeling the Influence of Histone Proteins on the Sensitivity of DNA to Ionizing Radiation

The DNA-binding proteins that are present in chromatin significantly affect the sensitivity of cells to ionizing radiation and to the radiation chemistry of DNA damage. The interaction between protein and DNA modifies the radiation chemistry of the latter. To model these processes, we have examined the effects of ionizing radiation on the minichromosome form of SV40 (which contains histone proteins arranged in nucleosomes) and also on plasmid DNA in the presence of lysozyme. Although high concentrations of lysozyme can bring about an extensive radioprotection by condensation of the plasmid, at lower levels it still produces significant radioprotective effects under conditions where this associative phase separation does not take place. The presence of histones or of lysozyme decreases the yield of modified guanines produced by ionizing radiation. Comparison with previous observations made with oligopeptides suggests that the mechanism responsible is electron donation to guanyl radicals in the DNA by tryptophan and tyrosine residues in the proteins. However, there was no evidence for DNA-protein crosslink formation.

Structure reactivity relationship in the reaction of DNA guanyl radicals with hydroxybenzoates

In DNA, guanine bases are the sites from which electrons are most easily removed. As a result of hole migration to this stable location on guanine, guanyl radicals are major intermediates in DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself and not through the intermediacy of water radicals). We have modeled this process by employing gamma irradiation in the presence of thiocyanate ions, a method which also produces single electron oxidized guanyl radicals in plasmid DNA in aqueous solution. The stable products formed in DNA from these radicals are detected as strand breaks after incubation with the FPG protein. When a phenolic compound is present in the solution during gamma irradiation, the formation of guanyl radical species is decreased by electron donation from the phenol to the guanyl radical. We have quantified the rate of this reaction for four different phenolic compounds bearing carboxylate substituents as proton acceptors. A comparison of the rates of these reactions with the redox strengths of the phenolic compounds reveals that salicylate reacts ca. 10-fold faster than its structural analogs. This observation is consistent with a reaction mechanism involving a proton coupled electron transfer, because intra-molecular transfer of a proton from the phenolic hydroxyl group to the carboxylate group is possible only in salicylate, and is favored by the strong 6-membered ring intra-molecular hydrogen bond in this compound.

Protection Against Radiation-Induced DNA Damage by Amino Acids: A DFT Study

Direct and indirect radiation-induced DNA damage is associated with the formation of radical cations (G(+)) and radical anions (G(-)) of guanine, respectively. Deprotonation of G(+) and dehydrogenation of G(-) generate guanine neutral radical [G(-H)] and guanine anion [G(-H)(-)], respectively. These products are of worrisome concern, as they are involved in reactions that are related to certain lethal diseases. It has been observed that guanyl radicals can be repaired by amino acids having strong reducing properties that are believed to be the residues of DNA-bound proteins such as histones. As a result, repair of G(-H) and G(-H)(-) by the amino acids cysteine and tyrosine has been studied here in detail by density functional theory in both the gas phase and aqueous medium using the polarized continuum and Onsager solvation models of self-consistent reaction field theory. Solvation in aqueous medium using three explicit water molecules was also studied. Four equivalent tautomers of each the above radical and anion that will be formed through proton and hydrogen loss from all of the nitrogen centers of guanine radical cation and guanine radical anion, respectively, were considered in the present study. It was found that in both the gas phase and aqueous medium, normal guanine can be retrieved from its radical-damaged form by a hydrogen-atom-transfer (HT) mechanism. Normal guanine can also be retrieved from its anionic damaged form in both the gas phase and aqueous medium through a two-electron-coupled proton-transfer (TECPT) mechanism or a one-step hydrogen-atom- and electron-transfer (OSHET) mechanism. The present results are discussed in light of the experimental findings.

Reactivity of DNA Guanyl Radicals with Phenolate Anions

Guanine bases are the most easily oxidized sites in DNA. Electron-deficient guanine species are major intermediates produced in DNA by the direct effect of ionizing radiation (ionization of the DNA itself) because of preferential hole migration within DNA to guanine bases. By using thiocyanate ions to modify the indirect effect (ionization of the solvent), we are able to produce these single-electron-oxidized guanine radical species in dilute aqueous solutions of plasmid DNA where the direct effect is negligible. The guanyl radical species produce stable modified guanine products. They can be detected in the plasmid by converting them to strand breaks after incubation with a DNA repair enzyme. If a phenol is present during irradiation, the yield of modified guanines is decreased. The mechanism is reduction of the guanine radical species by the phenol. It is possible to derive a rate constant for the reaction of the phenol with the guanyl radical. The pH dependence shows that phenolate anions are more reactive than their conjugate acids, although the difference for guanyl radicals is smaller than with other single-electron-oxidizing agents. At physiological pH values, the reduction of a guanyl radical entails the transfer of a proton in addition to the electron. The relatively small dependence of the rate constant on the driving force implies that the electron cannot be transferred before the proton. These results emphasize the potential importance of acidic tyrosine residues and the intimate involvement of protons in DNA repair.

Involvement of proton transfer in the reductive repair of DNA guanyl radicals by aniline derivatives

The most easily oxidized sites in DNA are the guanine bases, and major intermediates produced by the direct effect of ionizing radiation (ionization of the DNA itself) are electron deficient guanine species. By means of a radiation chemical method (gamma-irradiation of aqueous thiocyanate), we are able to produce these guanyl radicals in dilute aqueous solutions of plasmid DNA where the direct effect would otherwise be negligible. Stable modified guanine products are formed from these radicals. They can be detected in the plasmid conversion to strand breaks after a post-irradiation incubation with a DNA base excision endonuclease enzyme. If aniline compounds are also present, the yield of modified guanines is strongly attenuated. The mechanism responsible for this effect is electron donation from the aniline compound to the guanyl radical, and it is possible to derive rate constants for this reaction. Aniline compounds bearing electron withdrawing groups (e.g., 4-CF3) were found to be less reactive than those bearing electron donating groups (e.g., 4-CH3). At physiological pH values, the reduction of a guanyl radical involves the transfer of a proton as well as of an electron. The mild dependence of the rate constant on the driving force suggests that the electron is not transferred before the proton. Although the source of the proton is unclear, our observations emphasize the importance of an accompanying proton transfer in the reductive repair of oxidative damage to guanine bases which are located in a biologically active double stranded plasmid DNA substrate.

Repair of oxidative guanine damage in plasmid DNA by indoles involves proton transfer between complementary bases.

We have used the single electron oxidizing agent (SCN)(2)(*)(-) (generated by gamma-irradiation of aqueous thiocyanate) to produce guanyl radicals in plasmid DNA. The stable product(s) formed from these radicals can be detected after conversion with a base excision repair endonuclease to single strand breaks. The yield of enzyme-induced breaks is decreased by the presence during irradiation of indole compounds. Rate constants for the reduction of DNA guanyl radicals by these indoles can be calculated from the concentration dependence of the attenuation in the yield of enzyme sensitive sites. Indoles bearing electron-donating groups (methoxy or methyl) appear to react at the diffusion-controlled rate, but those bearing electron-withdrawing groups (cyano or nitro) are significantly less reactive. At physiological pH values, the reduction of a DNA guanyl radical involves the transfer of a proton as well as an electron. Comparison of the kinetic results with literature thermodynamic data suggests that the source of this proton is the complementary base-paired cytosine.

Repair of guanyl radicals in plasmid DNA by electron transfer is coupled to proton transfer.

By using gamma-irradiation in the presence of thiocyanate ions, we have generated guanyl radicals in plasmid DNA. These can be detected by using an Escherichia coli base excision repair endonuclease to convert their stable end products to strand breaks. The yield of enzyme-sensitive sites is strongly attenuated by the presence of micromolar concentrations of one of a series of singly substituted phenols, and it is possible to derive bimolecular rate constants for the reduction of DNA guanyl radicals by these phenols. More strongly reducing phenols were found to react more rapidly. This electron-transfer reaction also involves a proton transfer. By comparing the expected energetics of the reaction with the observed rate constants, the electron transfer is found to be mechanistically coupled with the proton transfer.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions

Purpose : Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation. The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss. This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation. The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them. Materials and methods : Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with γ-irradiation. These conditions result in the very efficient production of guanyl radicals in the plasmid. We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined. Results : The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed. The additional presence of nitrite decreased this effect of doxorubicin. Conclusion : Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical. Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime. Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime. The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals. It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

One-electron oxidation of plasmid DNA by selenium(V) species

Purpose : To employ the γ-radiation-generated selenium(V) one-electron-oxidizing agent SeO 3 •- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with 137 Cs γ-rays (662 keV). The solutions also contained up to 4 ×10 -2 mol dm -3 sodium selenate (Na 2 SeO 4) and/or up to 10 -1 mol dm -3 sodium biselenite (NaHSeO 3) , as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. Results : In general, γ-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after γ-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after γ-irradiation in the presence of 10 -2 mol dm -3 sodium selenate and 10 -1 mol dm -3 glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO 3 •-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. Conclusion : By comparing the yields of breaks produced after γ-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.

Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation

Purpose : It has been previously argued that the use of the one-electron oxidants (SCN) 2 •- and Br 2 •- with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl II OH +) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. Materials and methods : Aqueous solutions of plasmid DNA containing 10 -3 mol dm -3 thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10 -7 -10 -4 mol dm -3) were irradiated with 137 Cs γ-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. Results : High yields of FPG-sensitive sites produced by the oxidants (SCN)2 •- and Tl II OH + were strongly attenuated by the presence of the reducing agents. Conclusions : From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10 4 -10 7 dm 3 mol -1 s -1. Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.

Redox reactivity of guanyl radicals in plasmid DNA

Purpose : It has been previously argued that γ-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E. coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG), and there that derive from an intermediate guanyl radical species. The wish was to characterize the reactivity of this intermediate with reducing agents. Materials and methods : Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10 -3 to 10 -1 mol dm -3) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10 -7 to 10 -3 mol dm -3) were subjected to 137 Cs γ-irradiation (662 keV). After irradiation, the plasmid was incubated with FPG. Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions. Results : The very high yields of FPG-sensitive sites in the presence of SCN - or Br - decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least. Conclusions : From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5x10 5, 2x10 5, 10 7 and 10 7 dm 3 mol -1 s -1 for ferrocyanide, nitrite, promethazine and tryptophan respectively).

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength

Purpose : Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN) 2 £ -. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN) 2 £ - is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of oneelectron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. Materials and methods : Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137 Cs n -irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. Results : The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). Conclusions : It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN) 2 £ - and DNA-G £ +. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G £ + /DNA of E 7 = +1.39 - 0.01V.

Monophotonic ionization of poly[A,G] and DNA in alkaline aqueous solution by 248 nm laser light: Formation of guanyl radical

Photoionization of 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), polyadenylic-guanylic acid (poly[A,G]) and single-stranded DNA (ssDNA) in alkaline aqueous solution by 248 nm laser pulses was investigated. Unlike the reactions in neutral solution, the ionization at pH > 9 was found to be monophotonic. The transition from mono- to biphotonic ionization with increasing pH was shown to be associated with deprotonation of guanine and the corresponding decrease of ionization potential. In the case of Poly[A,G] and ssDNA, photoionization occurs at deprotonated guanine moieties, thus giving guanyl radicals. The quantum yields of formation of hydrated electrons ( Φ e−) were determined at room temperature with KI solutions used as a reference.

The reactivity of adenyl and guanyl radicals towards oxygen

A strong oxidant [Tl(II)] oxidizes adenine (A) and guanine (G) to adenyl and guanyl radical cations, A + ̇ and G + ̇ , respectively. In neutral solutions these cation radicals lose protons and become adenyl, ·A(-H), and guanyl, ·G(-H), radicals. From the pulse radiolysis experiments and the measurements of oxygen uptake, it was concluded that, in contrast with carbon centered radicals, the adenyl and guanyl radicals do not react rapidly with oxygen. Their reaction rate constants are estimated to be of the order of 10 2M -1s -1 or less. The low reactivity of ·A(-H) and ·G(-H) with oxygen is suggested to be a consequence of the unpaired electron being mostly on the oxygen and nitrogen rather than on carbon. In contrast, OH-adducts generated in the reaction of ·OH radical with guanine (·G-OH) and adenine (·A-OH) react with oxygen rapidly.