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.

Comet assay to determine DNA damage induced by food deprivation in rats.

The aim of this work was to evaluate, by comet assay, the possible inducing of DNA lesions in peripheral blood mononuclear cells of rats subjected to acute or chronic food deprivation. Wistar male rats were subjected to 72 h of partial (50%), or total acute food deprivation, and then allowed to recover for different time periods (24, 48 and 72 h). In other experiments, comet scores were determined in peripheral blood mononuclear cells of rats subjected to chronic food deprivation (25% and 50%) for 50 days. Blood aliquots were obtained before, during and after food deprivation. Comet assay was carried out, the comet units photographed and scored (class 0 up to 3). Acute and chronic food-deprived rats presented peripheral blood mononuclear cells with DNA lesions (comet classes 1, 2 and 3) and a significant increase (p<0.05) in the number of comet units compared with its basal level. The increase was proportional to acute food deprivation time, but after being taken off, it progressively returned to basal level after 48 h (partial group) or 72 h (total group). Chronic food-deprived rats presented a progressive increase of comet score up to 5 days, and a decrease thereafter to reach a basal level. Possible mechanisms of DNA lesions are discussed.

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.

Analysis of genotoxic potentiality of stevioside by comet assay.

Stevioside is a natural non-caloric sweetener extracted from Stevia rebaudiana (Bertoni) leaves. It has been widely used in many countries, including Japan, Korea, China, Brazil and Paraguay, either as a substitute for sucrose in beverages and foods or as a household sweetener. The aim of this work was to study its genotoxic potentiality in eukaryotic cells. Wistar rats were treated with stevioside solution (4mg/mL) through oral administration (ad libitum) and the DNA-induced damage was evaluated using the single cell gel electrophoresis (comet assay). The results showed that treatment with stevioside generates lesions in peripheral blood, liver, brain and spleen cells in different levels, the largest effect being in liver. Therefore, these undesired effects must be better understood, once the data present here point to possible stevioside mutagenic properties.

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.

Genotoxicity of stannous chloride in yeast and bacteria.

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.

Genotoxic potentiality of aqueous extract prepared from Chrysobalanus icaco L. leaves.

Plants have been related to our lives, being used as medicine, regardless of scientific evidence of side effects. This work analyses the toxicological effects of Chrysobalanus icaco L. aqueous extract, used in different pathologies. It was studied through: (i) alteration of plasmid pUC 9.1 topology; (ii) survival of bacterial strains submitted, or not, to previous treatment with SnCl2; (iii) transformation efficiency of E. coli strain by the treatment with the plasmid pUC 9.1. In (i), the treatment of the plasmid resulted in DNA single-strand breaks (SSB). A decrease of the lethal effect induced by SnCl2 in presence of the extract was found, while no C. icaco bacterial survival reduction was observed. The transformation efficiency of the plasmid was also reduced. Results suggest that the extract could present a potential genotoxic effect, as demonstrated either by the induction of SSB in plasmid or in transformation efficiency experiments. Finally, it presents an antioxidant action.

Agarose gel electrophoresis system in the classroom: Detection of DNA strand breaks through the alteration of plasmid topology.

Good quality scientific teaching depends on the ability of researchers to translate laboratory experiments into high school and undergraduate classes, bridging the advanced and basic science with common knowledge. A fast-growing field in biomedical sciences is oxidative stress, which has been associated to several diseases, including cancer and neurodegenerative disorders. We suggest herein a simple methodology for exploring DNA damage as an introductory pathway to these themes. The potential of natural or artificial products to induce DNA strand breaks can be easily tested in supercoiled plasmids incubated with selected products followed by agarose gel electrophoresis. This is designed to detect single and double strand breaks caused by reactive oxygen species generated by the products being tested. The altered topology of the damaged plasmid migrates slowly in the gel, creating a new band. We further introduce the quantitation of supercoiled DNA forms using densitometry of the gel with a digital camera; the values can then be used to estimate the number of breaks per genome using Poisson distribution. The system is inexpensive, rapid, and does not need high-cost equipment and supplies and can be performed in high schools and undergraduate classes with a minimal structure.

Absence of mutagenic and citotoxic potentiality of senna (Cassia angustifolia Vahl.) evaluated by microbiological tests

Senna (Cassia angustifolia Vahl.) is widely used as laxative, but data from Ames test and animal and/or human studies with this agent have shown a mutagenic and carcinogenic potentiality. Using thee experimental models (bacterial inactivation test; bacterail mutagenisis assay-Mutoxitest; and growth Inhibition test, we investigated the toxicity of senna. Our data suggest an absence of mutagenic and citotoxic potentiality of senna.

Sena (Cassia angustifolia Vahl.) é uma espécie amplamente empregada como laxativa, mas dados mutagênicos realizados com teste de Ames e estudos animais e/ou em humanos com esse agente tem mostrado uma potencialidade mutagênica e carcinogênica. Usando três diferentes testes (inativação de bactérias; ensaio de mutagênese em bactérias - Mutoxitest; teste de inibição de crescimento), foi investigada a toxidade dessa planta. Nossos dados sugerem uma ausência da potencialidade mutagênica e citotoxicidade de sena.

Biological effects of stannous chloride, a substance that can produce stimulation or depression of the central nervous system.

It was demonstrated that tin, as stannous chloride (SnCl(2)), can facilitate the neuromuscular transmission by accelerating the transmitter release from the nerve terminals in the mouse. When this salt is injected into laboratory animals, it can produce stimulation or depression of the central nervous system. Because calcium (Ca(2+)) influx into the cytoplasm is indispensable to release the transmitter, it would be possible that SnCl(2) increases the Ca(2+) influx at the nerve terminals but not by blocking the K(+) channels. SnCl(2) is known to inhibit the immune response in rodents and to induce tumor generation in thyroid gland. There is no general agreement regarding its genotoxicity and it was discussed that the effects of this salt might depend on the physicochemical conditions and the route of its administration. SnCl(2) has been used in many sectors of human interest, such as food industry and nuclear medicine. This salt is directly administered to human beings endovenously, when it is used as a reducing agent to prepare 99mTc-radiopharmaceuticals which are also used for cerebral studies. SnCl(2) is capable to promote the generation of reactive oxygen species (ROS) that are responsible for the oxidative stress. Oxidative stress has been related with aging and other neurological diseases. So, it is relevant to evaluate other biological effects of SnCl(2). We decided to study these effects using Escherichia coli mutant strains, deficient in DNA repair genes, and supercoiled plasmid DNA. We evaluated the influence of medicinal plants, metal chelating agents, and ROS scavengers against the SnCl(2) deleterious effects. Our results show that SnCl(2) produced lesions in vitro as well as in vivo. This inactivation may be due to the production of ROS. We observed that the genotoxic effect of SnCl(2) was partly inhibited or disappeared, when the treatments were done in the presence of medicinal plants, metal chelating agents, and ROS scavengers. In conclusion, these findings suggest that the SnCl(2) biological effects may be associated with the generation of ROS. Moreover, we can speculate that ROS could be associated with the detrimental effects in the brain due to exogenous or endogenous metals.

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.

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

Evaluation of the cytotoxic and mutagenic potentiality of technetium-99m in Escherichi coli.

Since technetium-99m (99mTc) was introduced in medical research it has become one of the most employed radionuclides in nuclear medicine. 99mTc is ideal for routine use on the labeling of different radiopharmaceuticals due to its favorable characteristics. However, some biological effects have been described. These effects may be related to internal conversion electron and/or Auger electron emissions from 99mTc decay that present high linear energy transfer and can generate reactive oxygen species (ROS) in the medium. We evaluated in Escherichia coli K12S and Salmonella typhimurium TA102, both proficient in DNA repair, contribution of those decay emissions on the cytotoxicity induced by 99mTc, both either by generating lesions on DNA or by inducing alterations at membrane. We also studied the genotoxic and/or mutagenic potentiality of 99mTc, in Salmonella typhimurium, using the Ames test. The results showed that: i/ 99mTc is cytotoxic to the Escherichia coli K12S strains; ii/ this effect is related to the electrons (Auger and internal conversion) emissions, and iii/ the 99mTc is not mutagenic and/or genotoxic, when measured by Ames test.

Dna damage in peripheral blood nuclear cells assessed by comet assay from individuals submitted to scintigraphic examinations.

Stannous chloride (SnCl2) is employed as a reducing agent to obtain Technetium-99m-labelled radiophamaceuticals in nuclear medicine kits, being injected endovenously in humans. Toxic effects of these kits were not studied, thus making it important to evaluate their impact in humans. In this study, the toxic effects were evaluated from peripheral blood nuclear cells (PBNC) from patients who received radiopharmaceuticals obtained using such kits. The analyses included results performed by comet assay. DNA damage was visualized in PBNC samples collected within a time up to 2 hr, and 24 hr after radiopharmaceutical injection in the patients. Initially we observed an increase of comet signals, which subsequently were reduced to zero after 24 hr. The diminishing of comet amounts probably is associated with DNA repair of damaged cells or with the elimination by apoptosis of cells whose DNA are not repaired.

Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli.

Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences. Much is known about stannous chloride (SnCl(2)) toxicity, although, there is no general agreement regarding its genotoxicity. Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine. We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl(2) treatment. Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl(2). E. coli culture was treated simultaneously with SnCl(2) and a specific extract. Our results showed a reduction of the SnCl(2) effect on the survival of the cultures in presence of the crude extracts. The extract of M. ilicifolia showed the highest level of protection action against the SnCl(2) effect in comparison with the other extracts. This protector effect could due to the redox properties of these crude extracts. The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl(2) oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl(2) effect.

Assessment of the effect of Maytenus ilicifolia (espinheira santa) extract on the labeling of red blood cells and plasma proteins with technetium-99m.

We are trying to develop a model to assess properties of products utilized in popular medicine. Maytenus ilicifolia is used in herbal medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are employed in nuclear medicine. This labeling procedure depends on a reducing agent and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility of M. ilicifolia extract being capable to alter the labeling of blood elements with 99mTc. Blood was incubated with M. ilicifolia extract. Stannous chloride solution and Tc-99m were added. Blood was centrifuged and plasma (P) and blood cells (C) were isolated. Samples of P or C were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioactivity (%ATI) in C, IF-P and IF-C was calculated. The %ATI decreased on C from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-C from 80.0+/-3.4 to 12.6+/-4.8. Once in RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances of the M. ilicifolia extract could increase the valence these ions to stannic (+4). This fact would decrease the %ATI on blood elements and indicate the presence of oxidant agents in the M. ilicifolia extract.

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.

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.

Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with technetium-99m.

Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with 99mTc was studied. Stannous chloride and 99mTc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma.

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.