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1.
Radiat Phys Chem Oxf Engl 1993 ; 76(6): 982-987, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21960732

RESUMO

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to gamma-irradiation, and the resulting formation of single strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ((•)OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for (•)OH, we are able to estimate the rate coefficient for the reaction of (•)OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10(9) - 2×10(9) L mol(-1) s(-1) at 10(7) s(-1), and increases with increasing scavenging capacity.

2.
Int J Radiat Biol ; 81(1): 41-54, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15962762

RESUMO

PURPOSE: To measure the yield of DNA strand breaks and clustered lesions in plasmid DNA irradiated with protons, helium nuclei, and y-rays. MATERIALS AND METHODS: Plasmid DNA was irradiated with 1.03, 19.3 and 249 MeV protons (linear energy transfer = 25.5, 2.7, and 0.39 keV microm(-1) respectively), 26 MeV helium nuclei (25.5 keV microm) and gamma-rays (137Cs or 60Co) in phosphate buffer containing 2 mM or 200 mM glycerol. Single-and double-strand breaks (SSB and DSB) were measured by gel electrophoresis, and clustered lesions containing base lesions were quantified by converting them into irreparable DSB in transformed bacteria. RESULTS: For protons, SSB yield decreased with increasing LET (linear energy transfer). The yield of DSB and all clustered lesions seemed to reach a minimum around 3 keV microm(-1). There was a higher yield of SSB, DSB and total clustered lesions for protons compared to helium nuclei at 25.5 keV microm(-1). A difference in the yields between 137Cs and 60Co gamma-rays was also observed, especially for SSB. CONCLUSION: In this work we have demonstrated the complex LET dependence of clustered-lesion yields, governed by interplay of the radical recombination and change in track structure. As expected, there was also a significant difference in clustered lesion yields between various radiation fields, having the same or similar LET values, but differing in nanometric track structure.


Assuntos
Partículas alfa/efeitos adversos , Dano ao DNA , DNA/efeitos da radiação , Raios gama/efeitos adversos , Prótons/efeitos adversos , Lesões por Radiação/etiologia , DNA Bacteriano , Transferência Linear de Energia/efeitos da radiação , Modelos Biológicos , Plasmídeos/efeitos da radiação
3.
Int J Radiat Biol ; 80(9): 643-51, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15586884

RESUMO

PURPOSE: To define ionic conditions under which oligolysines condense DNA as assayed by radioprotection of a plasmid substrate. And to compare these conditions with those required by the well-characterized ligands spermidine and hexammine cobalt (III). This will enable a reversible compaction model for plasmid DNA to be devised that models more closely mammalian chromatin than those based on polyamines. MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA, sodium perchlorate and one of the five ligands trilysine, tetralysine, pentalysine, spermidine, or hexammine cobalt (III) were subjected to gamma-irradiation. The yields of the resulting single-strand breaks were quantified by gel electrophoresis. The effects of tetralysine and pentalysine were also examined by light scattering. RESULTS: The combination of low concentrations of the ligand and high concentrations of sodium perchlorate produced a relatively high yield of single-strand breaks. In contrast, the combination of high concentrations of the ligand and low concentrations of sodium perchlorate resulted in an approximately 25-fold lower single-strand break yield. The transition between these two break yields took place over very narrow concentration ranges of the ligand. A large change in light scattering occurred at the same concentration. The radioprotective ability of the ligands decreased in the order pentalysine > tetralysine > hexammine cobalt (III) > spermidine > trilysine. CONCLUSIONS: The effect of the oligolysines is qualitatively very similar to the previously reported radioprotection produced under similar conditions by the polyamines spermidine and spermine. It is caused by condensation of the DNA into a highly compacted form. As peptides, oligolysines are structurally more closely related than other ligands to naturally occurring DNA condensing agents such as histone proteins. Therefore, they may form the basis of a model system suitable for studying DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself).


Assuntos
Dano ao DNA , Oligopeptídeos/química , Plasmídeos/química , Plasmídeos/efeitos da radiação , Polilisina/química , Tolerância a Radiação/efeitos dos fármacos , Protetores contra Radiação/química , Relação Dose-Resposta à Radiação , Raios gama , Plasmídeos/análise , Doses de Radiação
4.
Nucleic Acids Res ; 31(21): 6258-63, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-14576314

RESUMO

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.


Assuntos
Aminoácidos/metabolismo , Dano ao DNA , Reparo do DNA , Guanina/metabolismo , Oxidantes/metabolismo , Plasmídeos/metabolismo , Aminoácidos/química , Dano ao DNA/efeitos da radiação , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/efeitos da radiação , Radicais Livres/química , Radicais Livres/metabolismo , Guanina/química , Cinética , Oxirredução , Plasmídeos/química , Plasmídeos/efeitos da radiação , Radiação Ionizante
5.
Int J Radiat Biol ; 78(8): 733-41, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12194757

RESUMO

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 gamma-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.


Assuntos
Dano ao DNA , DNA/química , Guanina/química , Antineoplásicos/uso terapêutico , Relação Dose-Resposta a Droga , Doxorrubicina/uso terapêutico , Escherichia coli/metabolismo , Radicais Livres , Modelos Estatísticos , Plasmídeos/metabolismo , Fatores de Tempo
6.
Biochemistry ; 41(27): 8683-8, 2002 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-12093286

RESUMO

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.


Assuntos
Dano ao DNA/efeitos da radiação , DNA/efeitos da radiação , Desoxirribonuclease IV (Fago T4-Induzido) , Proteínas de Escherichia coli , Plasmídeos/efeitos da radiação , Purinas/efeitos da radiação , Raios Ultravioleta , Sequência de Bases , Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA-Formamidopirimidina Glicosilase , Cinética , N-Glicosil Hidrolases/metabolismo , Oxirredução
7.
Int J Radiat Biol ; 78(5): 359-74, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12020427

RESUMO

PURPOSE: To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- 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 137Cs gamma-rays (662 keV). The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), 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, gamma-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 gamma-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*-. 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 gamma-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.


Assuntos
Proteínas de Escherichia coli , Plasmídeos/efeitos dos fármacos , Plasmídeos/efeitos da radiação , Selênio/farmacologia , Sítios de Ligação , Dano ao DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase , Relação Dose-Resposta à Radiação , Elétrons , Ferrocianetos/farmacologia , Raios gama , N-Glicosil Hidrolases/metabolismo , Oxirredução , Plasmídeos/química , Plasmídeos/metabolismo , Radiobiologia , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/efeitos da radiação , Ácido Selênico , Selênio/química , Selênio/efeitos da radiação , Compostos de Selênio/química , Compostos de Selênio/farmacologia , Compostos de Selênio/efeitos da radiação , Selenito de Sódio/química , Selenito de Sódio/farmacologia , Selenito de Sódio/efeitos da radiação
8.
Int J Radiat Biol ; 77(12): 1195-205, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11747544

RESUMO

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 one-electron 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 137Cs gamma-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 E7 = +1.39+/-0.01V.


Assuntos
DNA/efeitos da radiação , Raios gama , Guanina/química , Oxirredução , Plasmídeos/efeitos da radiação , Tiocianatos/química , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Concentração de Íons de Hidrogênio , Íons , Cinética , Modelos Químicos , Modelos Teóricos , Termodinâmica
9.
Int J Radiat Biol ; 77(11): 1095-108, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11683980

RESUMO

PURPOSE: It has been previously argued that the use of the one-electron oxidants (SCN)2(*-) and Br2(*-) 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 137Cs gamma-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.


Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA , Radicais Livres/química , Plasmídeos/química , Plasmídeos/efeitos da radiação , DNA/química , DNA/efeitos da radiação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/efeitos da radiação , DNA-Formamidopirimidina Glicosilase , Ferrocianetos , Sequestradores de Radicais Livres/química , Cinética , N-Glicosil Hidrolases/química , Oxirredução , Percloratos/química , Radiação Ionizante , Compostos de Sódio/química , Tálio/química , Tiocianatos/química , Tirosina
10.
Int J Radiat Biol ; 77(3): 281-93, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11258842

RESUMO

PURPOSE: It has been previously argued that gamma-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 137Cs gamma-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 (5 x 10(5), 2 x 10(5), 10(7) and 10(7) dm3 mol(-1) s(-1) for ferrocyanide, nitrite, promethazine and tryptophan respectively).


Assuntos
DNA Bacteriano/química , Radicais Livres/química , Guanina/química , Plasmídeos/genética , Brometos/química , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/efeitos da radiação , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/efeitos da radiação , DNA-Formamidopirimidina Glicosilase , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Sequestradores de Radicais Livres/química , Raios gama , Guanina/efeitos da radiação , Radical Hidroxila/química , Iodetos/química , N-Glicosil Hidrolases/metabolismo , Oxirredução/efeitos da radiação , Plasmídeos/efeitos da radiação , Tiocianatos/química
11.
Int J Radiat Biol ; 77(2): 155-64, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11236922

RESUMO

PURPOSE: To determine the increase in single- (SSB) and double-strand break (DSB) yields after post-high LET irradiation incubation of plasmid DNA with the endonuclease-III (endo-III) of Escherichia coli. MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with one of five radiation types: 137Cs gamma-rays (LET approximately 0.3keV microm(-1)), 244Cm alpha-particles (140-190 keV microm(-1)), 4He ions (97 keV microm(-1)), 56Fe ions (143 keV microm(-1)) or 197Au ions (1,440 keV microm(-1)). The irradiated samples were then incubated with endo-III. SSB and DSB yields were quantified by agarose gel electrophoresis. RESULTS: Endo-III incubation produced an increase in the SSB and DSB yields. The increases were in general lower after the high LET irradiation than after gamma-irradiation. This may reflect inhibition of the activity of endo-III by the nearby DNA damage expected from high LET radiation. It can be shown that even if the activity of endo remains unchanged, significantly lower increases in SSB and DSB yields would still be expected. CONCLUSION: The results provide evidence for clustered DNA damage after high LET irradiation.


Assuntos
Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Radioisótopos de Césio/metabolismo , Cúrio/metabolismo , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Radicais Livres , Raios gama , Ouro/metabolismo , Hélio/metabolismo , Íons , Ferro/metabolismo , Plasmídeos/efeitos dos fármacos , Plasmídeos/efeitos da radiação , Radioisótopos/metabolismo
12.
Int J Radiat Biol ; 76(11): 1475-83, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11098850

RESUMO

PURPOSE: To determine the increases in SSB and DSB yields after post gamma-irradiation incubation of plasmid DNA with the Escherichia coli base excision repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) and endonuclease III (endo III). MATERIALS AND METHODS: Aqueous solutions of plasmid DNA were irradiated with 137Cs gamma-rays in the presence of 10(-4) - 10(-1) mol dm(-3) formate. After irradiation, aliquots were treated with FPG and/or endo III. SSB and DSB yields were then determined using gel electrophoresis. RESULTS: Both SSB and DSB yields were found to increase after enzyme incubation, with the increase in the DSB yield being approximately equal to the square of the increase in the SSB yield. The correlation between the increases in the SSB and DSB yields was unaffected by the scavenger concentration during irradiation. CONCLUSION: Under the conditions used, the majority of DSB appear to be formed from two hydroxyl radical attacks.


Assuntos
Dano ao DNA , DNA/metabolismo , DNA/efeitos da radiação , Desoxirribonuclease (Dímero de Pirimidina) , Proteínas de Escherichia coli , DNA/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/efeitos da radiação , DNA-Formamidopirimidina Glicosilase , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Formiatos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Raios gama , Radical Hidroxila/metabolismo , N-Glicosil Hidrolases/metabolismo , Plasmídeos/efeitos dos fármacos , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação
13.
Int J Radiat Biol ; 76(10): 1305-14, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11057738

RESUMO

PURPOSE: It was previously shown that gamma-irradiation of aqueous solutions of plasmid DNA in the presence of millimolar concentrations of thiocyanate ions leads to the formation in very high yields of sites recognized by the base excision repair endonuclease formamido-pyrimidine-DNA N-glycosylase (FPG). The authors wished to characterize the mechanism responsible for the production of these FPG-sensitive sites. MATERIALS AND METHODS: An aqueous solution of plasmid DNA containing thiocyanate ions was irradiated with 137Cs gamma-rays. After irradiation, aliquots were treated with FPG. Break yields were determined using neutral agarose gel electrophoresis. RESULTS: The yield of FPG-sensitive sites decreased with decreasing enzyme activity, increasing thiocyanate concentration, increasing dose-rate, increasing ionic strength, increasing nitrite or iodide concentration, and decreasing oxygen concentration. CONCLUSION: The observations suggest that the monomeric thiocyanate radical SCN* is an intermediate in the reaction, and that the yields of FPG-sensitive sites are determined by competition between the disproportionation of the dimeric radical anion (SCN)*2- and the fate of a one-electron oxidized guanine species in DNA. The latter can react with oxygen to produce an FPG-sensitive site or can be reduced without producing an FPG-sensitive site. The results help to clarify the mechanisms responsible for DNA damage by the direct effect of ionizing radiation.


Assuntos
Dano ao DNA , DNA/efeitos da radiação , Tiocianatos/toxicidade , DNA-Formamidopirimidina Glicosilase , Relação Dose-Resposta à Radiação , Raios gama , Radical Hidroxila , N-Glicosil Hidrolases/metabolismo , Nitritos/farmacologia , Concentração Osmolar , Sódio/farmacologia , Superóxido Dismutase/farmacologia , Tiocianatos/metabolismo
14.
Radiat Res ; 151(6): 677-85, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10360787

RESUMO

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.


Assuntos
Cromossomos/efeitos da radiação , Animais , Bromodesoxiuridina/metabolismo , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Cricetinae , Dano ao DNA , Relação Dose-Resposta à Radiação , Humanos , Hibridização in Situ Fluorescente , Raios X
15.
Radiat Res ; 151(3): 334-42, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10073672

RESUMO

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.


Assuntos
Dano ao DNA , Plasmídeos/efeitos da radiação , DNA Ligases/metabolismo , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/efeitos da radiação , Escherichia coli/enzimologia , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/química , Radicais Livres/metabolismo , Radicais Livres/efeitos da radiação , Raios gama/efeitos adversos , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Radical Hidroxila/efeitos da radiação , Oxirredução , Plasmídeos/química , Plasmídeos/metabolismo
16.
Mutagenesis ; 12(6): 443-7, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9412998

RESUMO

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechst dye 33258 results in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB) yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis. Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is approximately 5-fold more efficient than SSB formation. Formation of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.


Assuntos
DNA Glicosilases , N-Glicosil Hidrolases/farmacologia , Oligopeptídeos/farmacologia , Vírus 40 dos Símios/efeitos dos fármacos , Vírus 40 dos Símios/metabolismo , Uracila/biossíntese , Uracila/efeitos da radiação , Bromodesoxiuridina/metabolismo , Quebra Cromossômica , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , DNA Viral/efeitos dos fármacos , DNA Viral/efeitos da radiação , Relação Dose-Resposta a Droga , Mutagênese/efeitos dos fármacos , Mutagênese/efeitos da radiação , Fotólise/efeitos dos fármacos , Vírus 40 dos Símios/genética , Raios Ultravioleta , Uracila-DNA Glicosidase
17.
Radiat Res ; 148(4): 325-9, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9339948

RESUMO

We have shown previously that the thiol N-(2'-mercaptoethyl)-1,3-diaminopropane (WR-1065) can attenuate the formation of strand breaks associated with ionizing radiation. The mechanism of this protection is predominantly the reduction of DNA radical species which otherwise would attenuate the chemical repair of DNA radical species which are strand break precursors. We had observed that the presence of a hydroxyl radical scavenger during irradiation resulted in a decrease in the ability of WR-1065 to attenuate the formation of strand breaks. Since ionic compounds are known to affect the binding of the dicationic WR-1065 with the polyanion DNA, the effect of the scavenger was initially attributed to its polar nature having a similar effect on the interaction of WR-1065 with DNA, and not as a consequence of its ability to scavenge hydroxyl radicals. After examining additional scavengers, we now conclude that an increased hydroxyl radical scavenging capacity does attenuate the repair of strand break precursors to some extent. The probable explanation for this observation is that an increased scavenging capacity results in a greater degree of radical clustering on the DNA, and that these clusters of multiple radicals are repaired more slowly than are single radical species.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA Bacteriano/efeitos da radiação , Sequestradores de Radicais Livres/farmacologia , Radical Hidroxila/metabolismo , Mercaptoetilaminas/farmacologia , Cinética , Plasmídeos/genética , Plasmídeos/efeitos da radiação
18.
Radiat Res ; 146(5): 510-3, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8896577

RESUMO

Using agarose gel electrophoresis, we have measured the yield of single-strand breaks (SSBs) induced by 137Cs gamma irradiation in a variety of plasmid DNA substrates ranging in size from 2.7 kb to 38 kb irradiated in aerobic aqueous solution in the presence of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO). Under these conditions DNA SSBs are caused mainly by the hydroxyl radical. Using the competition between DMSO and DNA for the hydroxyl radical, we have estimated the rate coefficient for the reaction of the hydroxyl radical with DNA. The results cannot be characterized by conventional steady-state competition kinetics. However, it is possible to describe the second-order rate constant for the reaction as a function of the scavenging capacity of the solution. The second-order rate constant increases with increasing scavenging capacity, rising from about 5 x 10(8) dm3 mol-1 s-1 at 10(5) s-1 to about 10(10) dm3 mol-1 s-1 at 10(10) s-1. This dependence of the second-order rate constant on the scavenging capacity appears to be more pronounced for larger plasmids.


Assuntos
DNA/metabolismo , Radical Hidroxila/metabolismo , Dimetil Sulfóxido/farmacologia
20.
Radiat Res ; 146(4): 436-43, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8927715

RESUMO

Using agarose gel electrophoresis, we have measured the yields of DNA single-strand breaks (SSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. Incubation after irradiation with the base damage repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III (endo III) results in an increase in the yield of SSBs. In the absence of dimethyl sulfoxide (DMSO) during irradiation, this increase is consistent with the yields of known substrates for FPG and endo III as determined by gas chromatography/mass spectrometry. After irradiation in the presence of 1 mol dm-3 DMSO, the increase in the yield of SSBs after enzyme incubation was further enhanced by a factor of about 5 to 7. The magnitude of this effect, the inability of acrylamide or oxygen to suppress it, and its attenuation by N,N,N',N'-tetramethylphenylenediamine (TMPD) or glycerol all suggest that the methylperoxyl radical (derived from DMSO) is involved as an intermediate. Reactions of the methylperoxyl radical (or some other species derived from it) do not result in strand break damage, but are responsible for DNA base damages which are recognized by FPG and endo III.


Assuntos
Dano ao DNA , DNA/efeitos da radiação , Soluções Tampão , DNA/química , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/efeitos da radiação , DNA-Formamidopirimidina Glicosilase , Desoxirribonuclease (Dímero de Pirimidina) , Dimetil Sulfóxido , Endodesoxirribonucleases/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Radicais Livres/efeitos da radiação , Raios gama , Técnicas In Vitro , N-Glicosil Hidrolases/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Radioquímica , Espécies Reativas de Oxigênio , Soluções , Água
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