RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Syzygium cumini (L.) or Skeels (Myrtaceae) are widely used in Brazilian folk medicine to treat diabetes. AIM OF THE STUDY: The present study evaluated the functional capacity, biochemical parameters, oxidative stress and DNA damage from eight weeks of intervention with a crude hydroalcoholic extract of S. cumini leaves (EBH) and continuous aerobic training (TAC) in diabetic (D) rats. MATERIALS AND METHODS: A hydroalcoholic (50%) extract was prepared by ultrasound and phytochemical parameters (total phenols, total tannins and myricetin content) were analyzed. Thirty-seven male Wistar rats were divided into five groups: normoglycemic controls (CONT), diabetic controls (D-CONT), diabetics treated with extract (D+EBH), trained diabetic (D+TAC) and diabetics treated with extract and trained (D+EBH+TAC). Functional capacity was assessed with a maximum exercise capacity test; biochemical parameters with enzymatic kits; oxidative stress by superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and oxidized dichlorofluorescein (DCF), and the DNA damage by the comet assay. RESULTS: The D+TAC and D+EBH+TAC groups showed better functional capacity at the end of interventions. The D+EBH group showed glucose and triglyceride reduction, lowest DNA damage index in the blood, liver, kidney, heart, lung and gastrocnemius muscle, improved SOD levels in the liver, kidney and lung, improved CAT levels in the kidney and lower lipid peroxidation in all tissues studied, compared to the D-CONT group. The exercise (D+TAC) was effective in reducing triglycerides, improving SOD levels in the lung, reducing lipid peroxidation in all tissues studied and reducing the DCF oxidation in the kidney, in addition to protecting against DNA damage in the blood and heart. However, the additive effect of the intervention protocols when combined (EBH+TAC) was observed only in improving the gastrocnemius SOD levels. The phytochemical analyses showed a high content of phenols and the presence of myricetin glycosides. CONCLUSION: The findings in this study suggest a crude hydroalcoholic extract of S. cumini leaves has potential hypoglycemic, hypolipidemic and protective properties acting against oxidative stress and against DNA damage, probably due to its phenols and myricetin glycoside content and the antioxidant properties of these constituents. Moreover, exercise was suggested to have beneficial effects on diabetes, improving functional capacity, ameliorating blood triglyceride control and decreasing lipid peroxidation, but with no effects on ameliorating blood glucose levels. The association of intervention protocols presented an additive effect on the antioxidant SOD activity in the muscle cells of diabetic rats.

Asunto(s)

Diabetes Mellitus Experimental/tratamiento farmacológico , Myrtaceae/química , Condicionamiento Físico Animal/fisiología , Extractos Vegetales/farmacología , Hojas de la Planta/química , Syzygium/química , Animales , Antioxidantes/metabolismo , Brasil , Catalasa/metabolismo , Daño del ADN/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Medicina Tradicional/métodos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Ratas , Ratas Wistar , Estreptozocina/farmacología , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

Chromatin is thought to modulate access of repair proteins to DNA lesions, and may be altered by chromatin remodelers to facilitate repair. We investigated the participation of chromatin remodelers and DNA repair in 5-fluorouracil (5-FU) cytotoxicity in Saccharomyces cerevisiae. 5-FU is an antineoplastic drug commonly used in clinical settings. Among the several strains tested, only those with deficiencies in ATP-dependent chromatin remodeling (CR) and some histone acetyltransferases (HAT) exhibited sensitivity to 5-FU. CR and HAT double-mutants exhibited increased resistance to 5-FU in comparison to the wild-type mutant, but were still arrested in G2/M, as were the sensitive strains. The participation of Htz1p in 5-FU toxicity was also evaluated in single- and double-mutants of CR and HAT; the most significant effect was on cell cycle distribution. 5-FU lesions are repaired by different DNA repair machineries, including homologous recombination (HR) and post-replication repair (PRR). We investigated the role of CR and HAT in these DNA repair pathways. Deficiencies in Nhp10 and CR combined with deficiencies in HR or PRR increased 5-FU sensitivity; however, combined deficiencies of HAT, HR, and PRR did not. CRs are directly recruited to DNA damage and lead to chromatin relaxation, which facilitates access of HR and PRR proteins to 5-FU lesions. Combined deficiencies in HAT with defects in HR and PRR did not potentiate 5-FU cytotoxicity, possibly because they function in a common pathway.

Asunto(s)

Adenosina Trifosfato/metabolismo , Ensamble y Desensamble de Cromatina , Fluorouracilo/toxicidad , Histona Acetiltransferasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Ensamble y Desensamble de Cromatina/genética , Reparación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Relación Dosis-Respuesta a Droga , Fluorouracilo/metabolismo , Histona Acetiltransferasas/genética , Recombinación Homóloga , Pruebas de Sensibilidad Microbiana , Mutación , Saccharomyces cerevisiae/genética

RESUMEN

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organoselenium compounds. Studies have shown its interesting pharmacodinamic properties, as antioxidant, antimutagenic and antitumoral effects. Here we report the antigenotoxic properties of DPDS against tamoxifen (TAM)-induced oxidative DNA damage in MCF-7 cultured cell line. We determined the cytotoxicity by lactate dehydrogenase (LDH) leakage assay and evaluated oxidative DNA damage by modified comet assay employing the enzymes formamidopyrimidine DNA-glycosylase (Fpg) and endonuclease III (Endo III). Our results demonstrate that the cellular effects of DPDS appear to be complex and concentration-dependent. The present findings show that DPDS is not genotoxic (at concentrations lower than 2.0µmol/L) in MCF-7 cells, as observed in the modified comet assay. Moreover, DPDS protects against TAM-induced oxidative DNA damage, probably by its antioxidant activity, without interfering with its cytotoxicity. In this manner, the treatment with low concentrations of DPDS, a synthetic organoselenium compound, could be used as a potent antigenotoxic agent to prevent the risk of cancer induction triggered by tamoxifen hormone therapy. Thereby, more studies concerning the toxicity of DPDS and its structural derivatives are still necessary for future safe therapeutic application and development of novel chemopreventive compounds for combined therapy in breast cancer.

Asunto(s)

Antimutagênicos/farmacología , Antineoplásicos Hormonales/toxicidad , Derivados del Benceno/farmacología , Compuestos de Organoselenio/farmacología , Tamoxifeno/toxicidad , Antimutagênicos/administración & dosificación , Antimutagênicos/toxicidad , Antioxidantes/administración & dosificación , Antioxidantes/farmacología , Antioxidantes/toxicidad , Derivados del Benceno/administración & dosificación , Derivados del Benceno/toxicidad , Neoplasias de la Mama/patología , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Células MCF-7 , Compuestos de Organoselenio/administración & dosificación , Compuestos de Organoselenio/toxicidad , Estrés Oxidativo/efectos de los fármacos

RESUMEN

Piper gaudichaudianum Kunth is used in popular medicine as anti-inflamatory and against liver disorders. One of the most studied components of the plant is the essential oil for which chemical analysis revealed (E)-nerolidol as major compound. Recently, we have shown that P. gaudichaudianum essential oil possesses strong cytotoxic effects in mammalian V79 cells. The aim of this study was to analyze the cytotoxicity and mutagenicity of P. gaudichaudianum essential oil and nerolidol using Saccharomyces cerevisiae as model study. Treatment of the XV185-14c and N123 strains with essential oil and nerolidol led to cytotoxicity but did not induce mutagenicity. Our results revealed an important role of base excision repair (BER) as the ntg1, ntg2, apn1 and apn2 mutants showed pronounced sensitivity to essential oil and nerolidol. In the absence of superoxide dismutase (in sod1Δ mutant strain) sensitivity to the essential oil and nerolidol increased indicating that this oil and nerolidol are generating reactive oxygen species (ROS). The ROS production was confirmed by DCF-DA probing assay in Sod-deficient strains. From this, we conclude that the observed cytotoxicity to P. gaudichaudianum essential oil and nerolidol is mainly related to ROS and DNA single strand breaks generated by the presence of oxidative lesions.

Asunto(s)

Aceites Volátiles/farmacología , Piper/química , Aceites de Plantas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Sesquiterpenos/farmacología , Catalasa/genética , Citotoxinas/farmacología , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Pruebas de Mutagenicidad , Mutación , Aceites Volátiles/análisis , Aceites Volátiles/toxicidad , Aceites de Plantas/análisis , Aceites de Plantas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sesquiterpenos/toxicidad , Superóxido Dismutasa/genética

RESUMEN

Bio-electrospraying (BES) is a technique used for the processing of cells and can be applied to tissue engineering. The association of BES with scaffold production techniques has been shown to be an interesting strategy for the production of biomaterials with cells homogeneously distributed in the entire structure. Various studies have evaluated the effects of BES on different cell types. However, until the present moment, no studies have evaluated the impact of BES time on mesenchymal stem cells (MSC). Therefore, the aim of this work was to standardise the different parameters of BES (voltage, flow rate, and distance of the needle from the collecting plate) in relation to cell viability and then to evaluate the impact of BES time in relation to viability, proliferation, DNA damage, maintenance of plasticity and the immunophenotypic profile of MSC. Using 15 kV voltage, 0.46 ml/h flow rate and 4 cm distance, it was possible to form a stable and continuous jet of BES without causing a significant reduction in cell viability. Time periods between 15 and 60 min of BES did not cause alterations of viability, proliferation, plasticity, and immunophenotypic profile of the MSC. Time periods above 30 min of BES resulted in DNA damage; however, the DNA was able to repair itself within five hours. These results indicate that bio-electrospraying is an adequate technique for processing MSC which can be safely applied to tissue engineering and regenerative medicine.

RESUMEN

Kaurane diterpenes are considered important compounds in the development of new highly effective anticancer chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to the possibility that they induce secondary tumours in cancer patients. In this context, we evaluated the genotoxic and mutagenic potential of the natural diterpenoid kaurenoic acid (KA), i.e. (-)-kaur-16-en-19-oic acid, isolated from Xylopia sericeae St. Hill, using several standard in vitro and in vivo protocols (comet, chromosomal aberration, micronucleus and Saccharomyces cerevisiae assays). Also, an analysis of structure-activity relationships was performed with two natural diterpenoid compounds, 14-hydroxy-kaurane (1) and xylopic acid (2), isolated from X. sericeae, and three semi-synthetic derivatives of KA (3-5). In addition, considering the importance of the exocyclic double bond (C16) moiety as an active pharmacophore of KA cytotoxicity, we also evaluated the hydrogenated derivative of KA, (-)-kauran-19-oic acid (KAH), to determine the role of the exocyclic bond (C16) in the genotoxic activity of KA. In summary, the present study shows that KA is genotoxic and mutagenic in human peripheral blood leukocytes (PBLs), yeast (S. cerevisiae) and mice (bone marrow, liver and kidney) probably due to the generation of DNA double-strand breaks (DSB) and/or inhibition of topoisomerase I. Unlike KA, compounds 1-5 and KAH are completely devoid of genotoxic and mutagenic effects under the experimental conditions used in this study, suggesting that the exocyclic double bond (C16) moiety may be the active pharmacophore of the genetic toxicity of KA.

Asunto(s)

Diterpenos/química , Diterpenos/toxicidad , Mutágenos/toxicidad , Extractos Vegetales/toxicidad , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Pruebas de Mutagenicidad , Relación Estructura-Actividad

RESUMEN

We have investigated the chemical composition of Piper gaudichaudianum essential oil, as well as its cytotoxic, mutagenic and genotoxic effects in V79 cells. The chemical analyses showed that the major compounds are (E)-nerolidol (22.4%), alpha-humulene (16.5%), (E)-caryophyllene (8.9%) and bicyclogermacrene (7.4%). Dose-dependent cytotoxic effects were observed in V79 cells treated with essential oil by using clonal survival, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction assay (MTT) and trypan blue exclusion assay (TB), and a significant decrease in survival was observed at concentrations of 0.5 microg/mL and higher. The P. gaudichaudianum essential oil treatment caused DNA strand breaks in V79 cells at concentrations up to 2 microg/mL, as detected by the alkaline comet assay, but did not induce double-strand breaks, as verified by neutral comet assay. It induced a significant increase in the frequency of micronucleated cells at 4, 6 and 10 microg/mL. Moreover, P. gaudichaudianum essential oil significantly increased lipid peroxidation at doses of 0.5 microg/mL and higher, suggesting that the observed oxidant potential can be responsible, at least in part, for its cytotoxic and genotoxic effects.

Asunto(s)

Fibroblastos/efectos de los fármacos , Pulmón/efectos de los fármacos , Mutágenos/toxicidad , Aceites Volátiles/toxicidad , Piperaceae/química , Aceites de Plantas/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Cricetinae , Cricetulus , Daño del ADN , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/patología , Cromatografía de Gases y Espectrometría de Masas , Peroxidación de Lípido/efectos de los fármacos , Pulmón/patología , Malondialdehído/metabolismo , Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de Micronúcleos , Mutágenos/análisis , Aceites Volátiles/análisis , Hojas de la Planta/química , Aceites de Plantas/análisis , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

In order to clarify the molecular mechanisms of Sn(2+) genotoxicity, we evaluated the induction of strand breaks, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (Endo III) sensitive sites, and the interference with the repair of methyl methane sulfonate (MMS)-caused DNA damage in V79 Chinese hamster lung fibroblasts exposed to stannous chloride by comet assay. A concentration-related increase in the DNA damage induced by 2 h SnCl(2) treatment at a concentration range of 50-1,000 microM was observed (r = 0.993; P < 0.01). Significantly elevated DNA migration in relation to the control level was detected at doses 100, 500 and 1,000 microM in normal alkaline and at doses 500 and 1,000 microM in modified (with Fpg and Endo III) comet assay. Although 50 microM SnCl(2) concentration did not increase significantly the DNA migration by itself in comet assay, it was capable to inhibit the repair of MMS-induced DNA damage during the post-treatment period of 24 h. Our results demonstrate the genotoxic and comutagenic effects of stannous chloride in V79 cells. The inhibitory effect of Sn(2+) on repair of MMS-induced DNA damage suggests that this metal can also interfere in DNA repair systems thus contributing to increased mutation by shifting the balance from error-free to error-prone repair processes.

Asunto(s)

Roturas del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Metales Pesados/toxicidad , Mutágenos/toxicidad , Compuestos de Estaño/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Fibroblastos , Metilmetanosulfonato/toxicidad

RESUMEN

The genotoxic effect of two tanshinones isolated from roots of Hyptis martiussi Benth (Labiatae) was studied using V79 (Chinese hamster lung) cells by the alkaline comet assay and micronucleus test. Tanshinones were incubated with the cells at concentrations of 1, 3, 6 and 12 microg/mL for 3 h. Tanshinones were shown to be quite strongly genotoxic against V79 cells at all tested concentrations. The data obtained provide support to the view that tanshinones has DNA damaging activity in cultured V79 cells under the conditions of the assays.

Asunto(s)

Antioxidantes/uso terapéutico , Intoxicación por Tetracloruro de Carbono/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Flavonoides/uso terapéutico , Animales , Análisis Químico de la Sangre , Intoxicación por Tetracloruro de Carbono/patología , Catalasa/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hígado Graso/inducido químicamente , Hígado Graso/patología , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Peroxidación de Lípido/efectos de los fármacos , Hígado/patología , Masculino , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

The pharmacology of synthetic organoselenium compounds indicates that they can be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumor and anti-infectious agents, and immunomodulators. In this review, we focus on the effects of diphenyl diselenide (DPDS) in various biological model organisms. DPDS possesses antioxidant activity, confirmed in several in vitro and in vivo systems, and thus has a protective effect against hepatic, renal and gastric injuries, in addition to its neuroprotective activity. The activity of the compound on the central nervous system has been studied since DPDS has lipophilic characteristics, increasing adenylyl cyclase activity and inhibiting glutamate and MK-801 binding to rat synaptic membranes. Systemic administration facilitates the formation of long-term object recognition memory in mice and has a protective effect against brain ischemia and on reserpine-induced orofacial dyskinesia in rats. On the other hand, DPDS may be toxic, mainly because of its interaction with thiol groups. In the yeast Saccharomyces cerevisiae, the molecule acts as a pro-oxidant by depleting free glutathione. Administration to mice during cadmium intoxication has the opposite effect, reducing oxidative stress in various tissues. DPDS is a potent inhibitor of d-aminolevulinate dehydratase and chronic exposure to high doses of this compound has central effects on mouse brain, as well as liver and renal toxicity. Genotoxicity of this compound has been assessed in bacteria, haploid and diploid yeast and in a tumor cell line.

Asunto(s)

Animales , Ratones , Ratas , Antioxidantes/farmacología , Derivados del Benceno/farmacología , Compuestos de Organoselenio/farmacología , Porfobilinógeno Sintasa/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Derivados del Benceno/toxicidad , Modelos Biológicos , Pruebas de Mutagenicidad , Compuestos de Organoselenio/toxicidad

RESUMEN

The pharmacology of synthetic organoselenium compounds indicates that they can be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumor and anti-infectious agents, and immunomodulators. In this review, we focus on the effects of diphenyl diselenide (DPDS) in various biological model organisms. DPDS possesses antioxidant activity, confirmed in several in vitro and in vivo systems, and thus has a protective effect against hepatic, renal and gastric injuries, in addition to its neuroprotective activity. The activity of the compound on the central nervous system has been studied since DPDS has lipophilic characteristics, increasing adenylyl cyclase activity and inhibiting glutamate and MK-801 binding to rat synaptic membranes. Systemic administration facilitates the formation of long-term object recognition memory in mice and has a protective effect against brain ischemia and on reserpine-induced orofacial dyskinesia in rats. On the other hand, DPDS may be toxic, mainly because of its interaction with thiol groups. In the yeast Saccharomyces cerevisiae, the molecule acts as a pro-oxidant by depleting free glutathione. Administration to mice during cadmium intoxication has the opposite effect, reducing oxidative stress in various tissues. DPDS is a potent inhibitor of delta-aminolevulinate dehydratase and chronic exposure to high doses of this compound has central effects on mouse brain, as well as liver and renal toxicity. Genotoxicity of this compound has been assessed in bacteria, haploid and diploid yeast and in a tumor cell line.

Asunto(s)

Antioxidantes/farmacología , Derivados del Benceno/farmacología , Compuestos de Organoselenio/farmacología , Porfobilinógeno Sintasa/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Animales , Derivados del Benceno/toxicidad , Ratones , Modelos Biológicos , Pruebas de Mutagenicidad , Compuestos de Organoselenio/toxicidad , Ratas

RESUMEN

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Asunto(s)

Antioxidantes/metabolismo , Glutatión Peroxidasa/metabolismo , Estrés Oxidativo , Saccharomyces cerevisiae/enzimología , Superóxido Dismutasa/genética , Catalasa/metabolismo , Inducción Enzimática , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1

RESUMEN

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44 percent) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126 percent), while sod1delta had lower activity (77 percent) than the wild type. Glutathione levels in sod1delta were increased (200-260 percent) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167 percent) and sod2delta (225 percent) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Asunto(s)

Antioxidantes , Glutatión Peroxidasa , Estrés Oxidativo , Saccharomyces cerevisiae , Superóxido Dismutasa , Catalasa , Peróxido de Hidrógeno , Oxidación-Reducción , Especies Reactivas de Oxígeno , Saccharomyces cerevisiae , Superóxido Dismutasa

RESUMEN

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.

Asunto(s)

Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Daño del ADN , Reparación del ADN , Proteínas Fúngicas/química , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Fenotipo , Precursores del ARN/metabolismo , Empalme del ARN , Factores de Empalme de ARN , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Empalmosomas , Esporas Bacterianas , Temperatura , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta

RESUMEN

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.

Asunto(s)

Adenosina Trifosfatasas , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , ADN Helicasas/genética , Reparación del ADN/genética , ADN de Hongos/efectos de los fármacos , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Hongos/efectos de la radiación , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Mutágenos/toxicidad , Mutación , RecQ Helicasas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta/efectos adversos

RESUMEN

The Saccharomyces cerevisiae Sgs1 protein, together with Schizosaccharomyces pombe Rqh1 and the human Bloom and Werner proteins, is a DNA helicase of the Escherichia coli RecQ family. Mutation of SGS1 causes premature aging in yeast cells, including the accumulation of extrachromosomal rDNA circles. We have recently shown that Sgs1p interacts with the DNA repair Rad16p protein and is epistatic to Rad16p for UVC, 4-NQO and H2O2 lesions. Therefore we tested sgs1 strains containing mutations in the helicase and C-terminal domains. We demonstrate here that the helicase activity of the Sgs1 is important for most elements of the sgs1 mutation phenotype, including sensitivity to UVC, 4-NQO, H2O2, MMS and hydroxyurea.

Asunto(s)

ADN Helicasas/metabolismo , Reparación del ADN , Saccharomyces cerevisiae/enzimología , 4-Nitroquinolina-1-Óxido/farmacología , División Celular/efectos de los fármacos , División Celular/genética , División Celular/efectos de la radiación , ADN Helicasas/genética , Peróxido de Hidrógeno/farmacología , Hidroxiurea/farmacología , Mecloretamina/farmacología , Metilmetanosulfonato/farmacología , Mutágenos/farmacología , Mutación , Quinolonas/farmacología , RecQ Helicasas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Rayos Ultravioleta

RESUMEN

A putative tolerance, induced by heat shock (HS), to the lethal and mutagenic effects of 8-methoxypsoralen (8-MOP) photoaddition and hyperthermia was analyzed in Saccharomyces cerevisiae using the wild-type strain N123 and the isogenic DNA repair-deficient mutant pso3-1. In wild-type cells, the HS (38 degrees C for 1 h) did not modify either the survival or the mutation frequency observed after 8-MOP photoaddition, even though it conferred protection against the lethal effect of hyperthermia (50 degrees C). In the pso3-1 mutant, HS induced an increase of the survival, and a decrease of the mutation frequency, after 8-MOP photoaddition and it also protected against the lethal effect of hyperthermia. The responses induced by HS were specific for 8-MOP photoaddition, since they were not observed after 254 nm ultraviolet-light damage. These results indicate that the protection conferred by HS depends of the type of lesion, and operates through the induction of different repair processes. In the pso3-1 mutant, HS could channel the repair intermediates to and error-free repair pathway.

Asunto(s)

Genes Fúngicos/efectos de los fármacos , Calor , Metoxaleno/farmacología , Saccharomyces cerevisiae/crecimiento & desarrollo , Reparación del ADN/genética , Relación Dosis-Respuesta en la Radiación , Genes Fúngicos/efectos de la radiación , Cinética , Mutagénesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/efectos de la radiación , Rayos Ultravioleta