José Carlos Pelielo de Mattos

Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation.

Stannous ion (Sn) has been employed in nuclear medicine and in food industry. We described that Stannous Chloride (SnCl2) inactivation effect in Escherichia coli is mediated by a Fenton-like reaction. The effect of SnCl2 was studied through: (i) the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; and (ii) the transformation efficiency of an wild type E. coli strain. Treatment of plasmid DNA pUC 9.1 with SnCl2, at pH 7.4, results in DNA single-strand breaks (SSB), in a dose-dependent manner. Addition of sodium benzoate partly inhibited the DNA damage, while EDTA completely abolishes DNA-SSB. Furthermore, the ability of the plasmid to transform E. coli was reduced. At pH 1.3, SnCl2 exerts a protective effect on plasmid against HCI depurination. Our results suggest the generation of ROS, such as *OH by a Fenton-like reaction, close to the site of the lesions due to a possible complexation of stannous ion to DNA.

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.

Boldine action against the stannous chloride effect.

Peumus boldus extract has been used in popular medicine in the treatment of biliar litiase, hepatic insufficiency and liver congestion. Its effects are associated to the substance boldine that is present in its extract. In the present work, we evaluated the influence of boldine both in: (i) the structural conformation of a plasmid pUC 9.1 through gel electrophoresis analysis; and in (ii) the survival of the strain of Escherichia coli AB1157 submitted to reactive oxygen species (ROS), generated by a Fenton like reaction, induced by stannous chloride. Our results show a reduction of the lethal effect induced by stannous chloride on the survival of the E. coli culture in the presence of boldine. The supercoiled form of the plasmid is not modified by stannous chloride in the presence of boldine. We suggest that the protection induced by boldine could be explained by its anti-oxidant mechanism. In this way, the boldine could be reacting with stannous ions, protecting them against the oxidation and, consequently, avoiding the generation of ROS.

Assessment of DNA damage induced by extracts, fractions and isolated compounds of Davilla nitida and Davilla elliptica (Dilleniaceae)

Davilla nitida and Davilla elliptica (Dilleniaceae) are plants that occur predominantly in the cerrado region of South America. They are used in popular medicine to treat stomach diseases, diarrhea and swelling, particularly of the lymph nodes and testicles. Chemical investigation of these two plant species led to the identification of the compounds myricetin-3-O-α-l-rhamnoside (myricitrin), quercetin-3-O-α-l-rhamnoside (quercitrin), myricetin, quercetin and gallic acid derivatives in the leaves of D. nitida and D. elliptica. Therefore, it was concluded that the two species of Davilla possess qualitatively similar chemical profiles. In the present study, the mutagenic and genotoxic potential of these plants and of their isolated compounds was tested in the Salmonella typhimurium assay (Ames test) with strains TA100, TA98, TA102 and TA97a, in the micronucleus test with peripheral blood cells of mice treated in vivo, and in plasmid DNA to analyze DNA strand-breaks. In the assessment of mutagenic potential by the Ames test, extracts from both plant species and a D. nitida ethyl-acetate fraction induced positive responses. On the other hand, none of the extracts showed genotoxic activity in the mouse cells. In the presence of metal ion, D. nitida and D. elliptica aqueous and ethyl-acetate fractions, as well as their isolated compounds, induced single- and double-strand-breaks in plasmid DNA in a cell-free system.

Interaction of stannous chloride leads to alteration in DNA, triphosphate nucleotides and isolated bases

Stannous chloride (SnCl2) is a reducing chemical agent used in several man-made products. SnCl2 can generate reactive oxygen species (ROS); therefore, studies have been carried out in order to better understand its damaging action in biological systems. In this work, calf thymus DNA, triphosphate nucleotides and isolated bases were incubated with SnCl2 and the results were analyzed through UV spectrophotometry. The presence of stannous ions altered the absorption spectra of all three isolates. The amount of stannous ions associated to DNA was measured by atomic absorption spectrophotometry. Data showed that more than 40% of the initial SnCl2 concentration was present in the samples. Our results are in accordance with the damaging potential of this salt and present evidence that stannous ions can complex with DNA, inducing ROS in its vicinity, which may be responsible for the observed lesions. (Mol Cell Biochem xxx: 173–179, 2005)

Cellular inactivation induced by a radiopharmaceutical kit : role of stannous chloride

Stannous chloride (SnCl2) has been used in many sectors of human activities such as food manufacturing and in nuclear medicine to produce radiopharmaceuticals labeled with technetium-99m (99mTc). Due to its importance and genotoxic potentiality, we decided to evaluate the biological effect induced by a nuclear medicine kit, which includes SnCl2, in association with glucoheptonic acid (GHA) which is employed for brain and renal scintigraphies. These studies were carried out with the Escherichia coli AB1157 strain and the deoxyribonucleic acid (DNA) plasmid pUC 9.1. The experiments, with different concentrations of SnCl2 and GHA, show an inverse relationship between both agents. When the GHA concentration was increased, the cellular inactivation induced by SnCl2 was reduced, as measured by the number of viable cells. Moreover, GHA protects the DNA molecule against the damage induced by SnCl2.

Evaluation of Deoxyribonucleic Acid Toxicity Induced by the Radiopharmaceutical 99mTechnetium-Methylenediphosphonic Acid and by Stannous Chloride in Wistar Rats

Radiopharmaceuticals are employed in patient diagnostics and disease treatments. Concerning the diagnosis aspect, technetium-99m (99mTc) is utilized to label radiopharmaceuticals for single photon computed emission tomography (SPECT) due to its physical and chemical characteristics. 99mTc fixation on pharmaceuticals depends on a reducing agent, stannous chloride (SnCl2) being the most widely-utilized. The genotoxic, clastogenic and anegenic properties of the 99mTc-MDP(methylene diphosphonate used for bone SPECT) and SnCl2 were evaluated in Wistar rat blood cells using the Comet assay and micronucleus test. The experimental approach was to endovenously administer NaCl 0.9% (negative control), cyclophosphamide 50 mg/kg b.w. (positive control), SnCl2 500 μg/mL or 99mTc-MDP to animals and blood samples taken immediately before the injection, 3, and 24 h after (in the Comet assay) and 36 h after, for micronucleus test. The data showed that both SnCl2 and 99mTc-MDP-induced deoxyribonucleic acid (DNA) strand breaks in rat total blood cells, suggesting genotoxic potential. The 99mTc-MDP was not able to induce a significant DNA strand breaks increase in in vivo assays. Taken together, the data presented here points to the formation of a complex between SnCl2 in the radiopharmaceutical 99mTc-MDP, responsible for the decrease in cell damage, compared to both isolated chemical agents. These findings are important for the practice of nuclear medicine.

Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

Stannous chloride (SnCl2) and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed by SnCl2 treatment. E. coli BER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i) The nfo mutant was the most sensitive to SnCl2; (ii) lethal synergistic effect was observed after UVA pre-illumination, plus SnCl2 incubation, the nfo mutant being the most sensitive; (iii) wild type and nfo mutants, transformed with pBW21 plasmid (nfo+) had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i) UVA induced DNA breaks and fpg mutant was the most sensitive; (ii) SnCl2-induced DNA strand breaks were higher than those from UVA and nfo mutant had the slowest repair kinetics; (iii) UVA + SnCl2 promoted an increase in DNA breaks than SnCl2 and, again, nfo mutant displayed the slowest repair kinetics. In summary, Nfo protects E. coli cells against damage induced by SnCl2 and UVA + SnCl2.

Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

ABSTRACT Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. Keywords: Alkaline gel electrophoresis, Escherichia coli , base excision repair, stannous chloride, DNA strand breaks, reactive oxygen species