The role of Fpg protein in UVC-induced DNA lesions.

We previously demonstrated that reactive oxygen species (ROS) could be involved in ultraviolet-C (UVC)-induced DNA damage in Escherichia coli cells. In the present study, we evaluated the involvement of the GO system proteins in the repair of the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG, GO) lesion, which is ROS-induced oxidative damage. We first found that the mutant strain Δfur, which produces an accumulation of iron, and the cells treated with 2,2'-dipyridyl, a iron chelator, were both as resistant to UVC-induced lethality as the wild strain. The 8-oxoG could be mediated by singlet oxygen ((1)O(2)). The Fpg protein repaired this lesion when it was linked to C (cytosine), whereas the MutY protein repaired 8-oxoG when it was linked to A (adenine). The survival assay showed that the Fpg protein, but not the MutY protein, was important to UVC-induced lethality and interacted with the UvrA protein, a nucleotide excision repair (NER) protein involved in UVC repair. The GC-TA reversion assay in the mutant strains from the '8-oxoG-repair' GO system showed that UVC-induced mutagenesis in the fpg mutants, but not in the MutY strain. The transformation assay demonstrated that the Fpg protein is important in UVC repair. These results suggest that UVC could also cause indirect ROS-mediated DNA damage and the Fpg protein plays a predominant role in repairing this indirect damage.

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement.

We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.

Biological effects of stevioside on the survival of Escherichia coli strains and plasmid DNA.

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250-300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.

Reactive oxygen species mediate lethality induced by far-UV in Escherichia coli cells.

The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (1O2) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.

Antigenotoxic and antimutagenic potential of an annatto pigment (norbixin) against oxidative stress

Carotenoids are 40-carbon molecules with conjugated double bonds, making them particularly effective for quenching free radicals. They have always been believed to possess anticancer properties, which could be due to their antioxidant potential. Norbixin is an unusual dicarboxylic water-soluble carotenoid present as a component in the pericarp of the seeds of Bixa orellana L. (from the Bixaceae family), a tropical shrub commonly found in Brazil. The main carotenoids present in these seeds, bixin and norbixin, form a coloring material, known as annatto, which is mainly used in the food industry. As annatto is only used as a coloring material, most studies of annatto pigments have focused on the determination of annatto levels in food. However, little attention has been given to the biological properties of bixin and norbixin. We evaluated the effect of norbixin on the response of Escherichia coli cells to DNA damage induced by UV radiation, hydrogen peroxide (H2O2) and superoxide anions (O2*-)) and found that norbixin protects the cells against these agents. Norbixin enhanced survival at least 10 times. The SOS induction by UVC was inhibited 2.3 times more when cells were grown in the presence of norbixin. We also found that norbixin has antimutagenic properties, with a maximum inhibition of H2O2-induced mutagenic activity of 87%, based on the Salmonella mutagenicity test

Does UVB radiation induce SoxS gene expression in Escherichia coli cells?

The SoxRS regulon is induced when bacterial cells are exposed to redox-cycling agents such as menadione or paraquat. In this paper it is shown that a physical agent, such as ultraviolet radiation with a wavelength of 312 nm (UVB) can induce soxS gene expression. The results indicate that this induction involves the RpoS protein. Moreover, an unexpected increase of soxS gene expression independent of a functional soxR gene in UVB-irradiated cells has been verified. This increase could be explained by transcription of soxS gene in a rpoS-dependent pathway.

Adaptive response to H(2)O(2) protects against SnCl(2) damage: the OxyR system involvement.

The stannous ion, mainly the stannous chloride (SnCl(2)) salt form, is widely used as a reducing agent to label radiotracers with technetium-99m ((99m)Tc). These radiotracers can be employed as radiopharmaceuticals in nuclear medicine procedures. In this case, there is no doubt about absorption of this complex, because it is intravenously administered in humans, although biological effects of these agents have not been fully understood. In this work we used a bacterial system to study the cytotoxic potential of stannous chloride. It is known that SnCl(2) induces lesions that could be mediated by reactive oxygen species (ROS). We, thus, investigated the existence of cross-adaptive response between hydrogen peroxide (H(2)O(2)) and SnCl(2) and the role of the OxyR system known to promote cellular protection against oxidative damages. Here we describe the results obtained with prior treatment of different Escherichia coli strains with sub-lethal doses of H(2)O(2), followed by incubation with SnCl(2). Our data show that H(2)O(2) is capable of inducing cross-adaptive response against the lethality promoted by SnCl(2), suggesting the OxyR system participation through catalase, alkyl hydroperoxide reductase and superoxide dismutase enzymes

Effects of low iron conditions on the repair of DNA lesions induced by Cumene hydroperoxide in Escherichia coli cells.

In the present study, we evaluated the sensitivity of different Escherichia coli strains to Cumene hydroperoxide (CHP) treatment under distinct conditions of Fe2+ availability. Our results showed that the pretreatment with an iron chelator (dipyridyl) protects all the tested strains against CHP toxic effects, but it was not sufficient to abolish the CHP induced mutagenesis. On the other hand, simultaneous pretreatment with both dipyridyl and neocuproine (copper chelator) leads to a complete protection against CHP mutagenic effects. Our data suggest the participation of copper ion in the CHP mutagenesis induced in E. coli.

Participation of stress-inducible systems and enzymes involved in BER and NER in the protection of Escherichia coli against cumene hydroperoxide.

We studied the participation of the stress-inducible systems, as the OxyR, SoxRS and SOS regulons in the protection of Escherichia coli cells against lethal effects of cumene hydroperoxide (CHP). Moreover, we evaluated the participation of BER and NER in the repair of the DNA damage produced by CHP. Our results suggest that the hypersensitivity observed in the oxyR mutants to the lethal effect of CHP does not appear to be due to SOS inducing DNA lesions, but rather to cell membrane damage. On the other hand, DNA damage induced by CHP appears to be repaired by enzymes involved in BER and NER pathways. In this case, Fpg protein and UvrABC complex could be involved cooperatively in the elimination of a specific DNA lesion. Finally, we have detected the requirement for the uvrA gene function in SOS induction by CHP treatment.

H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background.

Pretreatment with 2.5 mM H2O2 protects E. coli cells against UV-C killing, a phenomenon independent of LexA cleavage. In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain [lexA (Ind-)]. Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response.

Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system?

Pretreatment with 2.5 mM H2O2 protects bacterial cells against cumene hydroperoxide killing. This response is independent of the OxyR system, but possibly involves the participation of Ahp protein, since ahp mutants are not protected. Treatment of bacterial cells with high H2O2 concentrations caused an alteration on the electrophoretic profile of the smaller subunit (22-kDa) of Ahp. This alteration does not require novel gene products and is not dependent on the OxyR protein. In this way, we propose that the modification of the 22-kDa subunit of Ahp by high H2O2 concentration may be responsible for the protection against the lethal effects of cumene hydroperoxide.

Hydrogen peroxide effects in Escherichia coli cells.

We analyzed DNA lesions produced by H2O2 under low iron conditions, the cross adaptive response and the synergistic lethal effect produced by iron chelator-o-phenanthroline, using different Escherichia coli mutants deficient in DNA repair mechanisms. At normal iron levels the lesions produced by H2O2 are repaired mainly by the exonuclease III protein. Under low iron conditions we observed that the Fpg and UvrA proteins as well as SOS and OxyR systems participate in the repair of these lesions. The lethal effect of H2O2 is strengthened by o-phenanthroline if both compounds are added simultaneously to the culture medium. This phenomenon was observed in the wild type cells and in the xthA mutant (hypersensitive to H2O2). E. coli cells treated with low concentrations of H2O2 (micromolar) acquire resistance to different DNA damaging agents. Our results indicate also that pretreatment with high (millimolar) H2O2 concentrations protects cells against killing, by UV and this phenomenon is independent of the SOS system, but dependent on RecA and UvrA proteins. H2O2 induces protection against lethal and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). H2O2 also protects the cells against killing by cumene hydroperoxide, possibly with the participation of Ahp protein.

Role of SOS and OxyR systems in the repair of Escherichia coli submitted to hydrogen peroxide under low iron conditions.

There are at least two mechanisms by which H2O2 induces DNA lesions in Escherichia coli: one in the presence of physiological iron levels and the other in low iron conditions. The survival as well as the induction of SOS response in different DNA repair mutant strains of E coli was evaluated after H2O2 treatment under low iron conditions (pretreatment with an iron chelator). Our results indicate that, in normal iron conditions RecA protein has a relevant role in recombination repair events, while in low iron conditions RecA protein is important as a positive regulator of the SOS response. On the other hand, the oxy delta R mutant is sensitive to the lethal effects of H2O2 only in low iron conditions and this sensitivity cannot be correlated with DNA strand breaks.

Hydrogen peroxide induces protection against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) effects in Escherichia coli.

Cross-adaptive response is defined as the capacity of cells to become resistant to a lethal agent when pretreated with a different lethal substance. In the present paper, the cross-adaptive response between hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in Escherichia coli repair mutants. Our results suggest that high doses of H2O2 induces protection against the lethal effects of MNNG in wild-type strain, ada, ogt, ada-ogt, aidB and alkA mutants. On the other hand, the MNNG induced mutagenesis is reduced by H2O2 pretreatment in wild-type and ogt mutant strains, but not in ada mutant. Furthermore, the protecting effect induced by H2O2 is time dependent: it decreases 15 min after the pretreatment and, after 30 min, is almost abolished. This reduction in the protecting effect is followed by an augmentation in the mutation frequency when MNNG is added 30 min after H2O2 pretreatment. This cross-adaptive response may be due to a modification of the MNNG alkylation pattern in the oxidized DNA.

Effects of 1,10-phenanthroline and hydrogen peroxide in Escherichia coli: lethal interaction.

It has been observed that when Escherichia coli cells are treated simultaneously with phenanthroline and H2O2, there is a lethal interaction. In order to analyze the mechanism of this lethal interaction, wild-type and xthA mutant cells of E. coli were treated with 2.5 mM H2O2 and 1 mM phenanthroline. This treatment was preceded by treatments with different metal chelators (dipyridyl for Fe2+, desferal for Fe3+ and neocuproine for Cu2+) or conducted simultaneously to other treatments with chelators and radical scavengers (thiourea, ethanol and sodium benzoate). The lethal interaction was observed in both the E. coli wild-type strain and xthA mutant strain, which is deficient in the exonuclease III repair enzyme. Nevertheless, the mutant strain was much more sensitive than the wild-type one. Dipyridyl pretreatment protected the cells against the lethal interaction, while desferal pretreament was unable to do so. This suggests that the lethal interaction requires Fe2+ and not Fe3+ ions. Ethanol and sodium benzoate were incapable of protecting bacterial cells against the lethal interaction. Even a 20-min pretreatment with benzoate did not confer protection. On the other hand, thiourea protected the cells completely. Based on our results, we propose that the lethal interaction may be caused not only by the reaction kinetics of phenanthroline and Fe, but also by the ability of phenanthroline to intercalate in DNA. After forming the mono and bis complexes, phenanthroline would serve as a shuttle and take the Fe2+ ions to the DNA. So, the Fenton reaction would take its course with the consequent generation of OH. radicals near DNA. This proximity to the DNA would protect the OH. radicals against the scavengers' action, thus optimizing the Fenton reaction.

Fpg and UvrA proteins participate in the repair of DNA lesions induced by hydrogen peroxide in low iron level in Escherichia coli.

The survival of different DNA repair mutant strains of Escherichia coli treated with H2O2 was evaluated in the presence or absence of an iron chelator (dipyridyl). Our results suggest that Fpg and UvrA proteins participate in vivo in the repair of DNA lesions produced by higher H2O2 concentrations in the presence of an iron chelator while UvrB and UvrC proteins seem to be ineffective in the repair of these lesions.

Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli.

The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture medium. If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times. It is known that the Fe(2+)-o-phenanthroline(I) and Fe(2+)-o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe(2+)-o-phenanthroline(III) complex is formed slowly. Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2. Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+.

Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli

The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture mediu. If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times. It is known that the Fe2+ -o-phenanthroline(I) and Fe2+ -o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe2+ -o-phenanthroline(III) complex is formed slowly, Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2. Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions.

In order to study the role of metallic ions in the H2O2 inactivation of Escherichia coli cells, H2O2-sensitive mutants were treated with metal ion chelators and then submitted to H2O2 treatment. o-Phenanthroline, dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H2O2 treatment was not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coli. On the other hand, prior treatment with iron chelators protected the cells against the H2O2 lethal effect, indicating that iron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNA degradation studies indicated that these chelators did not completely block the formation of DNA single-strand breaks by H2O2 treatment. Thiourea, a scavenger of OH, caused a reduction in both H2O2 sensitivity and DNA single-strand break production. The breaks observed after treatment with metal chelators and H2O2 were repaired 60 min after H2O2 elimination in xthA but not polA mutant cells. Therefore, we propose that there are at least two pathways for H2O2-induced DNA lesions: one produced by H2O2 through iron oxidation and OH production, in which lesions are repaired by the products of the xthA and polA genes, and the other produced by an iron-independent pathway in which DNA repair requires polA gene products but not those of the xthA gene.

Antioxidant capacity of cyclo-oxygenase and lipoxygenase inhibitors.

The present study analyzes the possible scavenger capacity of several anti-inflammatory drugs on growth of Escherichia coli K12, BW9 109, a strain hypersensitive to H2O2, in medium containing H2O2. Although all cyclo-oxygenase and/or lipoxygenase inhibitors protected the cells against H2O2, no correlation was found between their relative protective abilities and reported anti-inflammatory potencies.