RESUMO
Pretreatment of Escherichia coli cultures with the iron chelator 2,2-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H2O2 concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H2O2 concentrations (≥15 mM) induced mainly G:C→A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H2O2 concentrations in the absence of dipyridyl preferentially induced A:T→T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H2O2 alone (20X higher) was increased in 20 mM H2O2 and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H2O2 under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H2O2.
Assuntos
Quelantes/farmacologia , Dano ao DNA , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , /farmacologia , Citosina , Escherichia coli/genética , Metaloproteínas , Testes de MutagenicidadeRESUMO
Pretreatment of Escherichia coli cultures with the iron chelator 2,2'-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H(2)O(2) concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H(2)O(2) concentrations (>or=15 mM) induced mainly G:C-->A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H(2)O(2) concentrations in the absence of dipyridyl preferentially induced A:T-->T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H(2)O(2) alone (20X higher) was increased in 20 mM H(2)O(2) and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H(2)O(2) under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H(2)O(2).
Assuntos
2,2'-Dipiridil/farmacologia , Quelantes/farmacologia , Dano ao DNA , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Citosina , Escherichia coli/genética , Metaloproteínas , Testes de MutagenicidadeRESUMO
Iron ions mediate the formation of lethal DNA damage by hydrogen peroxide. However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected. Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions. Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators. The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain.
Assuntos
Cobre/metabolismo , Dano ao DNA/efeitos dos fármacos , Proteínas de Escherichia coli , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Peróxido de Hidrogênio/farmacologia , Deficiências de Ferro , 2,2'-Dipiridil/farmacologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Centrifugação com Gradiente de Concentração , Quelantes/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA-Formamidopirimidina Glicosilase , Escherichia coli/citologia , Escherichia coli/genética , Peróxido de Hidrogênio/antagonistas & inibidores , Ferro/metabolismo , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutagênicos/farmacologia , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Fenantrolinas/farmacologiaRESUMO
Despite 2,9-dimethyl 1,10-phenanthroline (NC) has been extensively used as a potential inhibitor of damage due to oxidative stress in biological systems, the incubation of E. coli cultures with the copper ion chelator NC prior to the challenge with hydrogen peroxide caused a lethal synergistic effect. The SOS response seems to be involved in the repair of the synergistic lesions through the recombination pathway. Furthermore, there is evidence for the UvrABC excinuclease participation in the repair of the synergistic lesions, and the base excision repair may also be required for bacterial survival to the synergistic effect mainly at high concentrations of H2O2, being the action of Fpg protein an important event. Incubation of lexA (Ind-) cultures with iron (II) ion chelator 2,2'-dipyridyl simultaneously with NC prevented the lethal synergistic effect. This result suggests an important role of the Fenton reaction on the phenomenon. NC treatment was able to increase the number of DNA strand breaks (DNAsb) induced by 10 mM of H2O2 in lexA (Ind-) strain and the simultaneous treatment with 2,2'-dipyridyl was able to block this effect.
