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1.
Free Radic Res ; 54(7): 517-524, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32781874

RESUMO

The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing conditions (air, N20/O2 (4:1), N2O, vacuum) were employed to elucidate the contribution of several alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all systems, Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine, formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental data strongly suggest that the addition of molecular oxygen to the neutral guanine radical G(-H)• plays a major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may compete with recombination of G(-H)• with superoxide and/or alkyl peroxyl radicals. The production of Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.


Assuntos
Dano ao DNA , DNA/química , Radicais Livres/química , Radical Hidroxila/química , Imidazóis/química , 8-Hidroxi-2'-Desoxiguanosina/química , Animais , DNA/efeitos da radiação , Diaminas/química , Guanosina/química , Masculino , Salmão
2.
Radiat Res ; 192(3): 324-330, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31298612

RESUMO

The production of 2-deoxyribonolactones (C1'-oxidation product), C4'-oxidized abasic sites and C5'-carbonyl terminated strand scission products was investigated in complexes of double-stranded DNA with protamine, poly-L-lysine and spermine exposed to X-ray radiation. The lesions were quantified by high-performance liquid chromatography through the release of the corresponding low-molecular-weight products 5-methylenefuran-2(5H)-one, N-(2'-hydroxy-ethyl)-5-methylene-D3-pyrrolin-2-one and furfural, respectively. All binders were found to increase the relative yield of C1' oxidation up to 40% of the total 2-deoxyribose damage through the indirect effect versus approximately 18% typically found in homogeneous solutions by the same technique. On the contrary, the yield of C5'-oxidation was found to be suppressed almost completely, while in homogeneous solutions it constituted approximately 14% of the total. The observed change in end product distribution is attributed to free valence transfer to and from the complexing agent, although the mechanisms associated with this process remain unclear.


Assuntos
Dano ao DNA/efeitos da radiação , Peptídeos/metabolismo , Poliaminas/metabolismo , Açúcares Ácidos/metabolismo , Oxirredução/efeitos da radiação
3.
Free Radic Res ; 50(7): 756-66, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27043476

RESUMO

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO4(•-) were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,' C4,' and C5' positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2'-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2'-deoxy-ß-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4' oxidation. The contribution of C1' oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1'-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1' was found.


Assuntos
Ânions/química , Dano ao DNA , Guanina/química , Imidazóis/química , Sulfatos/química , Hidrogênio/química , Oxirredução
4.
Radiat Res ; 181(2): 131-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24410455

RESUMO

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4'-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ(3)-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1', C4' and C5'-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1'-oxidation; 43% FBR). The production of nucleoside-5'-aldehydes (C5'-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4'-radicals into malondialdehyde and 3'-phosphoglycolates, our results identify the C4'-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dano ao DNA , DNA/química , DNA/genética , Desoxirribose/metabolismo , Raios gama/efeitos adversos , DNA/metabolismo , Oxirredução/efeitos da radiação
5.
Radiat Res ; 174(5): 645-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20954863

RESUMO

This study reports the effects of denaturation and deoxygenation on radiation-induced formation of 2-deoxyribonolactone (2-dL) and 5'-aldehyde (5'-Ald) lesions in highly polymerized DNA. The radiation-chemical yields of 2-dL were determined through quantification of its dephosphorylation product 5-methylenefuranone (5MF). The formation of 5'-Ald was monitored qualitatively through the release of furfural (Fur) under the same conditions. The yields of 2-dL were found to be 7.3 ± 0.3 nmol J(-1), or about 18% of the yield of free base release measured in the same samples. Denaturation increased the efficiency of 2-dL formation approximately twofold while deoxygenation resulted in a fourfold decrease. The release of Fur is about twofold lower than that of 5MF in aerated native DNA samples and is further reduced by denaturation of the DNA. Unlike 5MF, the formation of Fur requires the presence of molecular oxygen, which is consistent with peroxyl radical-mediated oxidation of C5' radicals into 5'-Ald. In contrast, the existence of an oxygen-independent pathway of 2-dL formation suggests that C1' sugar radicals can also be oxidized by radiation-produced oxidizing intermediates such as electron-loss centers on guanines.


Assuntos
Carbono/metabolismo , Adutos de DNA/genética , Adutos de DNA/metabolismo , Dano ao DNA , Animais , Bovinos , Adutos de DNA/química , Furaldeído/metabolismo , Desnaturação de Ácido Nucleico/efeitos da radiação , Oxirredução/efeitos da radiação , Açúcares Ácidos/metabolismo
6.
J Phys Chem B ; 113(23): 8183-91, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19492855

RESUMO

The question of how NA base sequence influences the yield of DNA strand breaks produced by the direct effect of ionizing radiation was investigated in a series of oligodeoxynucleotides of the form (d(CG)(n))(2) and (d(GC)(n))(2). The yields of free base release from X-irradiated DNA films containing 2.5 waters/nucleotide were measured by HPLC as a function of oligomer length. For (d(CG)(n))(2), the ratio of the Gua yield to Cyt yield, R, was relatively constant at 2.4-2.5 for n = 2-4 and it decreased to 1.2 as n increased from 5 to 10. When Gua was moved to the 5' end, for example going from d(CG)(5) to d(GC)(5), R dropped from 1.9 +/- 0.1 to 1.1 +/- 0.1. These effects are poorly described if the chemistry at the oligomer ends is assumed to be independent of the remainder of the oligomer. A mathematical model incorporating charge transfer through the base stack was derived to explain these effects. In addition, EPR was used to measure the yield of trapped-deoxyribose radicals at 4 K following X-irradiation at 4 K. The yield of free base release was substantially greater, by 50-100 nmol/J, than the yield of trapped-deoxyribose radicals. Therefore, a large fraction of free base release stems from a nonradical intermediate. For this intermediate, a deoxyribose carbocation formed by two one-electron oxidations is proposed. This reaction pathway requires that the hole (electron loss site) transfers through the base stack and, upon encountering a deoxyribose hole, oxidizes that site to form a deoxyribose carbocation. This reaction mechanism provides a consistent way of explaining both the absence of trapped radical intermediates and the unusual dependence of free base release on oligomer length.


Assuntos
Dano ao DNA , Oligodesoxirribonucleotídeos/química , Radiação Ionizante , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância de Spin Eletrônica
7.
Radiat Res ; 171(3): 342-8, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19267561

RESUMO

2-Deoxyribonolactone lesions were identified as major products of radiation damage to DNA mediated by o,o'-diphenyleneiodonium cations in a hydroxyl radical-scavenging environment. The highest selectivity toward deoxyribonolactone formation (up to 86% of all sugar-phosphate damages) and the overall reaction efficiency (up to 40% of all radiation-generated intermediates converted into products) was displayed by derivatives with positively charged (2-aminoethylthio)acetylamino and (2-aminoethylamino)acetylamino side chains. The reaction can be useful for random single-step incorporation of deoxyribonolactone lesions into single- and double-stranded oligonucleotides and highly polymerized DNA directly in commonly used buffers (PBS, phosphate, Tris-HCl, etc.) at room temperature. In combination with HPLC separation, this technique can serve as a source of short (<6 mer) sequences containing deoxyribonolactone lesions at known positions.


Assuntos
Dano ao DNA , DNA/genética , DNA/metabolismo , Oniocompostos/química , Oniocompostos/farmacologia , Efeitos da Radiação , Açúcares Ácidos/metabolismo , Animais , Sequência de Bases , Tampões (Química) , Bovinos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta à Radiação , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Oniocompostos/metabolismo , Oxirredução , Especificidade por Substrato
8.
Radiat Res ; 168(3): 367-81, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17705640

RESUMO

Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.


Assuntos
Pareamento de Bases/efeitos da radiação , Dano ao DNA/efeitos da radiação , Desoxirribose/química , Desoxirribose/efeitos da radiação , Modelos Químicos , Oligonucleotídeos/química , Oligonucleotídeos/efeitos da radiação , Simulação por Computador , Relação Dose-Resposta à Radiação , Radicais Livres/efeitos da radiação , Modelos Genéticos , Doses de Radiação , Raios X
9.
Radiat Res ; 166(1 Pt 1): 9-18, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16808625

RESUMO

Radioprotection of DNA from direct-type radiation damage by histones has been studied in model systems using complexes of positively charged polypeptides (PCPs) with DNA. PCPs bind to DNA via ionic interactions mimicking the mode of DNA-histone binding. Direct radiation damage to DNA in films of DNA-PCP complexes was quantified as unaltered base release, which correlates closely with DNA strand breaks. All types of PCPs tested protected DNA from radiation, with the maximum radioprotection being approximately 2.5-fold compared with non-complexed DNA. Conformational changes of the DNA induced by PCPs or repair of free radical damage on the DNA sugar moiety by PCPs are considered the most feasible mechanisms of radioprotection of DNA. The degree of radioprotection of DNA by polylysine (PL) increased dramatically on going from pure DNA to a molar ratio of PL monomer:DNA nucleotide approximately 1:2, while a further increase in the PL:DNA ratio did not offer more radioprotection. This concentration dependence is in agreement with the model of PCP binding to DNA that assumes preferential binding of positively charged side groups to DNA phosphates in the minor groove, so that the maximum occupancy of all minor-groove PCP binding sites is at a molar ratio of PCP:DNA = 1:2.


Assuntos
Dano ao DNA , DNA/química , DNA/efeitos da radiação , Modelos Químicos , Peptídeos/química , Peptídeos/efeitos da radiação , Protetores contra Radiação/química , Simulação por Computador , Relação Dose-Resposta à Radiação , Ligação Proteica/efeitos da radiação , Doses de Radiação , Eletricidade Estática
11.
Radiat Res ; 163(1): 85-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15606311

RESUMO

Release of 5-methylene-2-furanone (5-MF), a characteristic marker of DNA deoxyribose oxidative damage at the C1' position, was observed in significant quantities from X-irradiated DNA. This observation, which held for DNA irradiated either in aqueous solution or as a film, requires postirradiation treatment at 90 degrees C in the presence of polyamines and divalent metal cations at biological pH. The 5-MF product was quantified by using reverse-phase HPLC. The radiation chemical yield of 5-MF comprised more than 30% of the yield of total unaltered base release. Polylysine, spermine and Be(II) showed the strongest catalytic effect on 5-MF release, while Zn(II), Cu(II), Ni(II), putrescine and Mg(II) were substantially less efficient. We have hypothesized that the 5-MF release from irradiated DNA occurs through catalytic decomposition of the 2'-deoxyribonolactone (dL) precursor through two consecutive beta- and delta-phosphate elimination reactions. A stepwise character of the process was indicated by the S-shaped time course of 5-MF accumulation. If dL proves to be the precursor to 5-MF formation, it would then follow that dL is a very important lesion generated in DNA by ionizing radiation.


Assuntos
Dano ao DNA/efeitos da radiação , DNA/química , DNA/efeitos da radiação , Furanos/síntese química , Metais/química , Poliaminas/química , Catálise , Cátions Bivalentes/química , Cátions Bivalentes/efeitos da radiação , Relação Dose-Resposta à Radiação , Metais/efeitos da radiação , Poliaminas/efeitos da radiação , Polieletrólitos , Doses de Radiação , Soluções , Temperatura , Água/química , Raios X
12.
J Phys Chem B ; 108(7): 2432-7, 2004 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-17375182

RESUMO

In this study we report analytical solutions for both time-dependent and steady-state problems of unbiased charge transfer through a regular DNA sequence via a hopping mechanism. The phenomenon is treated as a diffusion of charge in a one-dimensional array of equally spaced and energetically equivalent temporary trapping sites. The solutions take into account the rates of charge hopping (k), side reactions (k(r)), and charge transfer to a terminal charge acceptor (k(t)). A detailed analysis of the time-dependent problem is performed for the diffusion-controlled regime, i.e., under the assumption that k(t) >> k, which is also equivalent to the fast relaxation limit of charge trapping. The analysis shows that the kinetics of charge hopping through DNA is always multiexponential, but under certain circumstances it can be asymptotically approximated by a single-exponential term. In that case, the efficiency of charge transfer can be characterized by a single rate constant k(CT) = 1.23kN(-2) + k(r), where N is the DNA length expressed in terms of the number of equidistant trapping sites and k(r) is the rate of competing chemical processes. The absolute yield of charge transfer under steady-state conditions in general is obtained as Y(infinity) = omega [alpha sinh(alphaN) + omega cosh(alphaN)](-1), where alpha = (2k(r)/k)(1/2) and omega = 2k(t)/k. For the diffusion-controlled regime and small N, in particular, it turns into the known "algebraic" dependence Y(infinity) = [1 + (k(r)/k)N(2)](-1). At large N the solution is asymptotically exponential with the parameter alpha mimicking the tunneling parameter beta in agreement with earlier predictions. Similar equations and distance dependencies have also been obtained for the damage ratios at the intermediate and terminal trapping sites in DNA. The nonlinear least-squares fit of one of these equations to experimental yields of guanine oxidation available from the literature returns kinetic parameters that are in reasonable agreement with those obtained by Bixon et al. [Proc. Natl. Acad. Sci. U.S.A.1999, 96, 11713-11716] by numerical simulations, suggesting that these two approaches are physically equivalent.

13.
Radiat Res ; 160(3): 334-9, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12926992

RESUMO

Direct ionization of crystalline d(CGCGCGCG) and d(CGCGCGCGCG) oligomers produces 3'- and 5'-phosphate-terminated fragments as the main strand breakage products detectable by ion-exchange chromatography. The nature of the base has no effect on the probability of strand breakage at the given site. The yields of 3'-phosphates are systematically lower than the yields of the 5'-phosphates originating from the same cleavage site, pointing to the possible presence of unidentified products with sugar remnants attached to the 3'-end. These results show that direct ionization is efficient at producing single-strand breaks in DNA and its action is relatively indiscriminate with respect to base sequence.


Assuntos
DNA/efeitos da radiação , Oligodesoxirribonucleotídeos/efeitos da radiação , Raios X , Sítios de Ligação , Cromatografia por Troca Iônica , Cristalização , Dano ao DNA , Íons
14.
Radiat Res ; 159(5): 663-9, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12710878

RESUMO

This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3'- and 5'-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 +/- 0.03 micro mol/J and 0.07 +/- 0.02 micro mol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.


Assuntos
Dano ao DNA , DNA/química , DNA/efeitos da radiação , Sequência de Bases , Cristalização , Relação Dose-Resposta a Droga , Raios X
15.
Radiat Res ; 159(4): 543-9, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12643799

RESUMO

o,o'-Diphenyleneiodonium cations (DPI) convert relatively harmless radiation-produced electrons into efficient DNA cleaving agents. The cleavage products are unaltered DNA bases, 5-methylenefuranone (5-MF), and a complete set of 3' and 5'-phosphorylated DNA fragments. The production of alkali-labile sites is a minor factor in the process. Based on the production of 5-MF, it is concluded that DNA cleavage by DPI cations involves (but may not be limited to) the C1' chemistry. The loss of 3-aminoDPI (ADPI) cations bound to highly polymerized calf thymus DNA appears to be due to a short-chain reaction with an apparent length of up to 2.1 ADPI cations consumed for each radiation-produced electron. The suggested chain reaction mechanism includes the one-electron oxidation of DNA radicals (including the C1' sugar radical) by ADPI cations bound to the same duplex. The yields of DNA loss in complexes formed by ADPI with short synthetic duplexes indicate that there is more than a 60% probability of DNA damage after one-electron reduction of ADPI.


Assuntos
Dano ao DNA , DNA/efeitos da radiação , Elétrons , Inibidores Enzimáticos/efeitos da radiação , Raios gama , Oniocompostos/efeitos da radiação , Animais , Cátions , Bovinos , Radioisótopos de Cobalto , DNA/metabolismo , Inibidores Enzimáticos/metabolismo , Furanos/análise , Cinética , Modelos Químicos , Oniocompostos/metabolismo , Piperidinas/farmacologia
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