Alvaro C. Leitão

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 Gordonia sp. strain F.5.25.8 capable of dibenzothiophene desulfurization and carbazole utilization

A dibenzothiophene (DBT)-degrading bacterial strain able to utilize carbazole as the only source of nitrogen was identified as Gordonia sp. F.5.25.8 due to its 16S rRNA gene sequence and phenotypic characteristics. Gas chromatography (GC) and GC–mass spectroscopy analyses showed that strain F.5.25.8 transformed DBT into 2-hydroxybiphenyl (2-HBP). This strain was also able to grow using various organic sulfur or nitrogen compounds as the sole sulfur or nitrogen sources. Resting-cell studies indicated that desulfurization occurs either in cell-associated or in cell-free extracts of F.5.25.8. The biological responses of F.5.25.8 to a series of mutagens and environmental agents were also characterized. The results revealed that this strain is highly tolerant to DNA damage and also refractory to induced mutagenesis. Strain F.5.25.8 was also characterized genetically. Results showed that genes involved in desulfurization (dsz) are located in the chromosome, and PCR amplification was observed with primers dszA and dszB designed based on Rhodococcus genes. However, no amplification product was observed with the primer based on dszC.

Several pathways of hydrogen peroxide action that damage the E. coli genome

Hydrogen peroxide is an important reactive oxygen species (ROS) that arises either during the aerobic respiration process or as a by-product of water radiolysis after exposure to ionizing radiation. The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress condition that can result in damage to cell components such as proteins, lipids and principally to DNA, leading to mutagenesis and cell death. Escherichia coli cells are able to deal with these adverse events via DNA repair mechanisms, which enable them to recover their genome integrity. These include base excision repair (BER), nucleotide excision repair (NER) and recombinational repair. Other important defense mechanisms present in Escherichia coli are OxyR and SosRS anti-oxidant inducible pathways, which are elicited by cells to avoid the introduction of oxidative lesions by hydrogen peroxide. This review summarizes the phenomena of lethal synergism between UV irradiation (254 nm) and H2O2, the cross-adaptive response between different classes of genotoxic agents and hydrogen peroxide, and the role of copper ions in the lethal response to H2O2 under low-iron conditions.

The anabolic androgenic steroid nandrolone decanoate disrupts redox homeostasis in liver, heart and kidney of male Wistar rats.

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g−1 body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state.

Genotoxic effects of stannous chloride (SnCl2) in K562 cell line.

The toxic effects of SnCl2 in K562 cells were analyzed in this study. This cell line is resistant to reactive oxygen species (ROS) making it suitable to evaluate the impact of SnCl2 in culture either through ROS or by direct toxicity using Trypan blue dye exclusion, comet and flow cytometry assays. An important loss of viability induced by SnCl2 in a dose-response manner was observed in cells treated in Tris-buffered saline (TBS). This necrotic cell death was further confirmed by flow cytometry. On the other hand, there was no loss of viability when cells were treated in rich medium (RPMI). DNA damage was visualized in SnCl2-treated K562 cells in both tested conditions. The data indicate that SnCl2 induces DNA damage and reduces K562 viability. Both actions seem to be correlated with ROS formation and direct linkage to DNA.

Non-coherent visible and infrared radiation increase survival to UV (254 nm) in Escherichia coli K12.

Interactions between visible or infrared (IR) and ultraviolet (UV, 254 nm) radiation have been studied in E. coli. Pre-illumination with non-coherent monochromatic 446, 466, 570 and 685 nm radiation, as well as with polychromatic red and IR radiation at room temperature, leads to increased cell survival after a subsequent irradiation with UV light. In the thermic range of the spectrum (red and IR), IR but not red light pre-treatment is able to increase cell survival to a subsequent lethal heat (51 degrees C) challenge, suggesting that increased UV survival may be due to IR-induced heat-shock response. On the other hand, visible-light-induced resistance may be due to a different mechanism, possibly involved with unknown bacterial light receptors.

Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L.) Using In Vitro Assays

Papain, a phytotherapeutic agent, has been used in the treatment of eschars and as a debriding chemical agent to remove damaged or necrotic tissue of pressure ulcers and gangrene. Its benefits in these treatments are deemed effective, since more than 5000 patients, at the public university hospital at Rio de Janeiro, Brazil, have undergone papain treatment and presented satisfactory results. Despite its extensive use, there is little information about toxic and mutagenic properties of papain. This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains. Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest and Plasmid-DNA treatment, and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties. Papain exhibited negative results for all tests. This agent presented an activity protecting cells against H2O2-induced mutagenesis.

Repair of DNA Lesions Induced by Hydrogen Peroxide in the Presence of Iron Chelators in Escherichia coli: Participation of Endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.

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.

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.