Immunoexpression of tumour necrosis factor-α, interleukin-1α and interleukin-10 on odontogenic cysts and tumours.

AIM: To analyse the immunoreactivity of IL-1α, TNF-α and IL-10 in odontogenic cysts and tumours and to investigate possible associations with established biological behaviours of these different lesions. METHODOLOGY: Immunohistochemical expression of anti-IL-1α, anti-TNF-α and anti-IL-10 antibodies was assessed on epithelium and mesenchyme of 20 radicular cysts (RCs), 20 residual cysts (RECs), 20 dentigerous cysts (DCs), 18 solid ameloblastomas (SAs), 20 keratocystic odontogenic tumours (KCOTs) and 15 dental follicles (DFs). Comparative analysis of data was performed using the nonparametric Wilcoxon signed-rank test and Kruskal-Wallis's test. RESULTS: Significantly greater expression of IL-1α in the epithelium was noted in RC, KCOT and SA (P = 0.01), whilst IL-10 and TNF-α was in the epithelium of RC, DC and KCOT (P < 0.01). In the mesenchyme, significantly greater immunopositivity was observed for IL-1α, IL-10 and TNF-α in KCOT, DC and RC (P < 0.01). In epithelial and mesenchymal tissues, there were a significant number of cases of RC and DC with IL-1α < IL-10 ratio (P < 0.01), whilst SA and KCOT showed IL-1α > IL-10 (P < 0.01). There was a significantly greater percentage of DF, DC and KCOT with TNF-α > IL10 ratio (P < 0.01). CONCLUSION: These results suggest involvement of the proteins in the pathogenesis of odontogenic cysts and tumours, with emphasis on the highest immunoreactivity of osteolysis stimulating factors in tumours with aggressive biological behaviour, such as SA and KCOT.

Nuclear and mitochondrial genome instability induced by senna (Cassia angustifolia Vahl.) aqueous extract in Saccharomyces cerevisiae strains.

Cassia angustifolia Vahl. (senna) is commonly used in self-medication and is frequently used to treat intestine constipation. A previous study involving bacteria and plasmid DNA suggested the possible toxicity of the aqueous extract of senna (SAE). The aim of this study was to extend the knowledge concerning SAE genotoxicity mechanisms because of its widespread use and its risks to human health. We investigated the impact of SAE on nuclear DNA and on the stability of mitochondrial DNA in Saccharomyces cerevisiae (wt, ogg1, msh6, and ogg1msh6) strains, monitoring the formation of petite mutants. Our results demonstrated that SAE specifically increased Can(R) mutagenesis only in the msh6 mutant, supporting the view that SAE can induce misincorporation errors in DNA. We observed a significant increase in the frequency of petite colonies in all studied strains. Our data indicate that SAE has genotoxic activity towards both mitochondrial and nuclear DNA.

Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.

Nitrogen mustard- and half-mustard-induced damage in Escherichia coli requires different DNA repair pathways.

Bifunctional alkylating agents are used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. Nitrogen mustards are commonly used chemotherapeutic agents that can bind mono- or bifunctionally to guanines in DNA. Mustard HN1 is considered a monofunctional analog of bifunctional mustard HN2 (mechlorethamine). Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) or base excision repair (BER) were submitted to increasing concentrations of HN2 or HN1, and the results revealed that damage induced by each chemical demands different DNA repair pathways. Damage induced by HN2 demands the activity of NER with a minor requirement of the BER pathway, while HN1 damage repair depends on BER action, without any requirement of NER function. Taken together, our data suggest that HN1 and HN2 seem to induce different types of damage, since their repair depends on distinct pathways in E. coli.

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.

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 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.

Damage induced by stannous chloride in plasmid DNA.

Stannous chloride (SnCl(2)) is widely used in daily human life, for example, to conserve soft drinks, in food manufacturing and biocidal preparations. In nuclear medicine, stannous chloride is used as a reducing agent of Technetium-99m, a radionuclide used to label different cells and molecules. In spite of this, stannous chloride is able to generate reactive oxygen species (ROS) which can damage DNA. In this work, plasmid DNA (pUC 9.1) was incubated with SnCl(2) under different conditions and the results analyzed through DNA migration in agarose gel electrophoresis. Our data reinforce the powerful damaging effect induced by stannous ion and suggest that this salt can play a direct role in inducing DNA lesions.

Characterization of Staphylococcus epidermidis mutants sensitive to ultraviolet radiation.

Five UV-sensitive mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphylococcus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr- Umu-; 1 mutant that appears analogous to Uvr-; 1 mutant that resembles LexA- and 1 mutant that exhibits a RecA- phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.

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.

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.

Inhibition and induction of SOS responses in Escherichia coli by cobaltous chloride.

Mutagenesis induced by several genotoxic agents has been reported to be inhibited by cobaltous chloride. In order to study the effects of this metal in some SOS functions we evaluated mutagenesis, lysogenic induction and phage reactivation in Escherichia coli cells treated with CoCl2. We detected that cobaltous chloride, when present in the plating medium, was able to block mutagenesis and lysogenic induction promoted by UV irradiation. We also found that CoCl2 blocked protein synthesis, so we propose that this effect can be responsible for the antimutagenic and antilysogenic effects of this metal. On the other hand, if the cells were treated for a short period of time with CoCl2, in the absence of Mg, we observed that cobaltous chloride per se was able to promote lysogenic induction as well as to enhance the phage reactivation induced by UV irradiation. We conclude that depending on experimental conditions, cobaltous chloride may act either as an inhibitor or as an inducer of the SOS functions.

Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli.

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.

Genotoxic and mutagenic effects of guarana (Paullinia cupana) in prokaryotic organisms.

Aqueous extracts of Paullinia cupana (guarana), a species that belongs to the Sapindaceae family, were analyzed for the presence of genotoxic activities in bacterial cells. The extracts of guarana were genotoxic as assessed by lysogenic induction in Escherichia coli and they were also able to induce mutagenesis in Salmonella typhimurium. Addition of S9 microsomal fraction, catalase, superoxide dismutase or thiourea counteracted the genotoxic activity of guarana, suggesting that oxygen reactive species play an essential role in the genotoxicity of aqueous guarana extracts. The genotoxic activity in the extracts was related to the presence of a molecular complex formed by caffeine and a flavonoid (catechin or epicatechin) in the presence of potassium.

Synergistic lethal effect between hydrogen peroxide and neocuproine (2,9-dimethyl 1,10-phenanthroline) in Escherichia coli.

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.

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.

Mutagenic and genotoxic effects of aqueous extracts of Achyrocline satureoides in prokaryotic organisms.

Aqueous extracts of Achyrocline satureoides (Marcela and/or Macela) were tested for the presence of genotoxic activity in microorganisms. This species belongs to the family Compositae and is used on a large scale by the population of South Brazil. The extracts showed genotoxic activity in the presence of S9 mix in the Ames test TA100, TA98 and TA102 strains, 'SOS' spot chromotest and Microscreen phage-induction assay. The positive results were related to the presence of quercetin and caffeic acid in the aqueous extracts.

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.

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.

Mutational potentiality of stannous chloride: an important reducing agent in the Tc-99m-radiopharmaceuticals.

Stannous chloride (SnCl2) is frequently used in nuclear medicine as a reducing agent to label many radiopharmaceutical products with technetium-99m (99mTc). The aim of the present paper was to study the role of DNA repair genes in the repair of SnCl2-induced damage, using mutant strains of Escherichia coli lacking one or more DNA repair genes. Our results suggest that the product of the xthA gene, exonuclease III, is required for the repair of lesions induced by SnCl2. We further investigated the mutagenic properties of SnCl2 to a molecular level by using the supF tRNA gene as target in a forward mutational system. We have found that the survival of E. coli cells was strongly reduced with increasing concentrations of SnCl2. Moreover, when the shuttle vector pAC189 carrying the supF gene was treated with SnCl2, and then transfected to E. coli, we observed that its transformation efficiency dropped when compared to the non-treated control, with a parallel increase in mutation frequency after the damaged plasmids have replicated in bacterial cells. The mutation spectrum induced by SnCl2 reveals a high frequency of base substitutions, involving guanines. Sequence analysis of 41 independent supF mutant plasmids revealed that 39 mutants contained base substitutions, with 21 G:C to T:A and 17 G:C to C:G transversions. G to T transversions presumably resulted from 8-oxoG. However, the G to C one may be due to a yet unidentified lesion.