RESUMO
DNA repair pathways, cell cycle checkpoints, and redox protection systems are essential factors for securing genomic stability. The aim of the present study was to analyze the effect of Ilex paraguariensis (Ip) infusion and one of its polyphenolic components rutin on cellular and molecular damage induced by ionizing radiation. Ip is a beverage drank by most inhabitants of Argentina, Paraguay, Southern Brazil, and Uruguay. The yeast Saccharomyces cerevisiae (SC7Klys 2-3) was used as the eukaryotic model. Exponentially growing cells were exposed to gamma rays (γ) in the presence or absence of Ip or rutin. The concentrations used simulated those found in the habitual infusion. Surviving fractions, mutation frequency, and DNA double-strand breaks (DSB) were determined after treatments. A significant increase in surviving fractions after gamma irradiation was observed following combined exposure to γ+R, or γ+Ip. Upon these concomitant treatments, mutation and DSB frequency decreased significantly. In the mutant strain deficient in MEC1, a significant increase in γ sensitivity and a low effect of rutin on γ-induced chromosomal fragmentation was observed. Results were interpreted in the framework of a model of interaction between radiation-induced free radicals, DNA repair pathways, and checkpoint controls, where the DNA damage that induced activation of MEC1 nodal point of the network could be modulated by Ip components including rutin. Furthermore, ionizing radiation-induced redox cascades can be interrupted by rutin potential and other protectors contained in Ip.
Assuntos
Antimutagênicos/farmacologia , Ilex paraguariensis/química , Extratos Vegetais/farmacologia , Rutina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Cromatografia Líquida , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gama , Espectrometria de Massas , Mutagênese , Taxa de Mutação , Proteção Radiológica/métodos , Reprodutibilidade dos TestesRESUMO
DNA repair pathways, cell cycle checkpoints, and redox protection systems are essential factors for securing genomic stability. The aim of the present study was to analyze the effect of Ilex paraguariensis (Ip) infusion and one of its polyphenolic components rutin on cellular and molecular damage induced by ionizing radiation. Ip is a beverage drank by most inhabitants of Argentina, Paraguay, Southern Brazil, and Uruguay. The yeast Saccharomyces cerevisiae (SC7Klys 2-3) was used as the eukaryotic model. Exponentially growing cells were exposed to gamma rays (γ) in the presence or absence of Ip or rutin. The concentrations used simulated those found in the habitual infusion. Surviving fractions, mutation frequency, and DNA double-strand breaks (DSB) were determined after treatments. A significant increase in surviving fractions after gamma irradiation was observed following combined exposure to γ+R, or γ+Ip. Upon these concomitant treatments, mutation and DSB frequency decreased significantly. In the mutant strain deficient in MEC1, a significant increase in γ sensitivity and a low effect of rutin on γ-induced chromosomal fragmentation was observed. Results were interpreted in the framework of a model of interaction between radiation-induced free radicals, DNA repair pathways, and checkpoint controls, where the DNA damage that induced activation of MEC1 nodal point of the network could be modulated by Ip components including rutin. Furthermore, ionizing radiation-induced redox cascades can be interrupted by rutin potential and other protectors contained in Ip.
Assuntos
Rutina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Extratos Vegetais/farmacologia , Antimutagênicos/farmacologia , Ilex paraguariensis/química , Proteção Radiológica/métodos , Espectrometria de Massas , DNA Fúngico/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Reprodutibilidade dos Testes , Cromatografia Líquida , Mutagênese , Reparo do DNA , Relação Dose-Resposta à Radiação , Quebras de DNA de Cadeia Dupla , Taxa de Mutação , Raios gamaRESUMO
Photodynamic therapy (PDT) is a promising method for localized and specific inactivation of fungi and bacteria. A nontoxic light-sensitive compound is taken up by cells, which are then exposed selectively to light, which activates toxicity of the compound. We investigated the potential of sublethal PDT using light-sensitive curcumin (CUR) in combination with blue (455 nm) light to promote reactive oxygen species (ROS) formation in the form of singlet oxygen and DNA damage of Candida albicans. Surprisingly, CUR-mediated PDT but also light alone caused significantly longer comet tails, an indication of DNA damage of C. albicans when compared with the negative control. The intracellular ROS production was also significantly higher for the group treated only with light. However, PDT compared to blue light alone significantly slowed DNA repair. Comet tails decreased during 30 min visualized as a 90% reduction in length in the absence of light for cells treated with light alone, while comet tails of cells treated with PDT only diminished in size about 45%. These results indicate that complex mechanisms may result in PDT in a way that should be considered when choosing the photosensitive compound and other aspects of the treatment design.
Assuntos
Candida albicans/efeitos dos fármacos , Curcumina/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Fúngico/efeitos dos fármacos , Luz , Mutagênicos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Candida albicans/efeitos da radiação , Ensaio Cometa , Dano ao DNA/efeitos da radiação , DNA Fúngico/efeitos da radiação , Fotoquimioterapia/métodos , Espécies Reativas de Oxigênio/análiseRESUMO
Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.
Assuntos
Raios gama , Sporothrix/efeitos da radiação , Animais , DNA Fúngico/efeitos da radiação , Vacinas Fúngicas/química , Vacinas Fúngicas/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Sporothrix/crescimento & desenvolvimento , Sporothrix/metabolismo , Sporothrix/patogenicidade , Esporotricose/microbiologia , Esporotricose/terapia , Vacinas Atenuadas/química , Vacinas Atenuadas/efeitos da radiaçãoRESUMO
Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m(-2). CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m(-2)) of UVB radiation.
Assuntos
Aspergillus fumigatus/efeitos da radiação , Aspergillus nidulans/efeitos da radiação , Metarhizium/efeitos da radiação , Dímeros de Pirimidina/análise , Dímeros de Pirimidina/efeitos da radiação , Dano ao DNA , DNA Fúngico/efeitos da radiação , Relação Dose-Resposta à Radiação , Metarhizium/genética , Pigmentação/genética , Esporos Fúngicos/efeitos da radiação , Raios UltravioletaRESUMO
Paracoccidioides brasiliensis is a thermally dimorphic fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans with high prevalence in Latin America. Until now no vaccine has been reported. Ionizing radiation can be used to attenuate pathogens for vaccine development and we have successfully attenuated yeast cells of P. brasiliensis by gamma irradiation. The aim of the present study was to examine at ultrastructural level the effects of gamma irradiation attenuation on the morphology of P. brasiliensis yeast cells. Paracoccidioides brasiliensis (strain Pb-18) cultures were irradiated with a dose of 6.5 kGy. The irradiated cells were examined by scanning and also transmission electron microscopy. When examined 2 h after the irradiation by scanning electron microscopy, the 6.5 kGy irradiated cells presented deep folds or were collapsed. These lesions were reversible since when examined 48 h after irradiation the yeast had recovered the usual morphology. The transmission electron microscopy showed that the irradiated cells plasma membrane and cell wall were intact and preserved. Remarkable changes were found in the nucleus that was frequently in a very electrondense form. An extensive DNA fragmentation was produced by the gamma irradiation treatment.
Assuntos
Membrana Celular/efeitos da radiação , Parede Celular/efeitos da radiação , DNA Fúngico/efeitos da radiação , Raios gama , Paracoccidioides/efeitos da radiação , Paracoccidioides/ultraestrutura , Membrana Celular/ultraestrutura , Parede Celular/ultraestrutura , Fragmentação do DNA , Vacinas Fúngicas , Paracoccidioides/citologia , Paracoccidioidomicose/microbiologia , Vacinas AtenuadasRESUMO
Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/fisiologia , DNA Fúngico/fisiologia , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Dano ao DNA/fisiologia , Reparo do DNA/efeitos da radiação , Genes cdc/fisiologia , Genes cdc/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiaçãoRESUMO
The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.
Assuntos
Adenosina Trifosfatases , DNA Helicases/metabolismo , Reparo do DNA , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , DNA Helicases/genética , Reparo do DNA/genética , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Mutagênicos/toxicidade , Mutação , RecQ Helicases , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-Híbrido , Raios Ultravioleta/efeitos adversosAssuntos
Mutagênese , Micologia/métodos , Saccharomyces cerevisiae/genética , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Resistência Microbiana a Medicamentos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos/efeitos da radiação , Mutagênicos/farmacologia , Consumo de Oxigênio/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , TemperaturaRESUMO
The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H2O2 and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.