RESUMEN
The present study aimed to investigate the in vitro mutagenic activity of Origanum majorana essential oil. The most abundant compounds identified by GC-MS were γ-terpinene (25.73%), α-terpinene (17.35%), terpinen-4-ol (17.24%), and sabinene (10.8%). Mutagenicity was evaluated by the Salmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 Salmonella typhimurium strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 µL/plate in the absence of S9 mix and higher than 0.08 µL/plate in the presence of S9 mix and no gene mutation increase was observed. For the in vitro mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 µg/mL. Moreover, when tested in noncytotoxic concentrations, O. majorana essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that O. majorana essential oil is not mutagenic at the concentrations tested in the Salmonella/microsome and micronucleus assays.
Asunto(s)
Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Aceites Volátiles/farmacología , Origanum/química , Animales , Células Cultivadas , Cricetinae , Fibroblastos/efectos de los fármacos , Microsomas/efectos de los fármacos , Mutágenos , Salmonella typhimurium/efectos de los fármacosRESUMEN
The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 µM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 µM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 µM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 µM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 µM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells.
Asunto(s)
Antimutagênicos/farmacología , Derivados del Benceno/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Mutágenos/farmacología , Compuestos Organometálicos/farmacología , Animales , Biomarcadores , Catalasa/metabolismo , Línea Celular , Ensayo Cometa , Cricetinae , L-Lactato Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Pulmón , Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de Mutagenicidad , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Thiazacridine derivatives (ATZD) are a novel class of cytotoxic agents that combine an acridine and thiazolidine nucleus. In this study, the cytotoxic action of four ATZD were tested in human colon carcinoma HCT-8 cells: (5Z)-5-acridin-9-ylmethylene-3-(4-methylbenzyl)-thiazolidine-2,4-dione - AC-4; (5ZE)-5-acridin-9-ylmethylene-3-(4-bromo-benzyl)-thiazolidine-2,4-dione - AC-7; (5Z)-5-(acridin-9-ylmethylene)-3-(4-chloro-benzyl)-1,3-thiazolidine-2,4-dione - AC-10; and (5ZE)-5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2,4-dione - AC-23. All of the ATZD tested reduced the proliferation of HCT-8 cells in a concentration- and time-dependent manner. There were significant increases in internucleosomal DNA fragmentation without affecting membrane integrity. For morphological analyses, hematoxylin-eosin and acridine orange/ethidium bromide were used to stain HCT-8 cells treated with ATZD, which presented the typical hallmarks of apoptosis. ATZD also induced mitochondrial depolarisation and phosphatidylserine exposure and increased the activation of caspases 3/7 in HCT-8 cells, suggesting that this apoptotic cell death was caspase-dependent. In an assay using Saccharomyces cerevisiae mutants with defects in DNA topoisomerases 1 and 3, the ATZD showed enhanced activity, suggesting an interaction between ATZD and DNA topoisomerase enzyme activity. In addition, ATZD inhibited DNA topoisomerase I action in a cell-free system. Interestingly, these ATZD did not cause genotoxicity or inhibit the telomerase activity in human lymphocyte cultures at the experimental levels tested. In conclusion, the ATZD inhibited the DNA topoisomerase I activity and induced tumour cell death through apoptotic pathways.
Asunto(s)
Acridinas/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Fragmentación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Tiazolidinedionas/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Ensayo Cometa , Humanos , Microscopía Fluorescente , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismoRESUMEN
Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Delta) and CuZnSOD/MnSOD (sod1Deltasod2Delta). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.
Asunto(s)
Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutasa/deficiencia , Vitamina A/farmacología , Vitaminas/farmacología , Catalasa/metabolismo , Proteínas Fúngicas/genética , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Superóxido Dismutasa/genéticaRESUMEN
Biflorin is a natural quinone isolated from Capraria biflora L. Previous studies demonstrated that biflorin inhibits in vitro and in vivo tumor cell growth and presents potent antioxidant activity. In this paper, we report concentration-dependent cytotoxic, genotoxic, antimutagenic, and protective effects of biflorin on Salmonella tiphymurium, yeast Saccharomyces cerevisiae, and V79 mammalian cells, using different approaches. In the Salmonella/microsome assay, biflorin was not mutagenic to TA97a TA98, TA100, and TA102 strains. However, biflorin was able to induce cytotoxicity in haploid S. cerevisiae cells in stationary and exponential phase growth. In diploid yeast cells, biflorin did not induce significant mutagenic and recombinogenic effects at the employed concentration range. In addition, the pre-treatment with biflorin prevented the mutagenic and recombinogenic events induced by hydrogen peroxide (H(2)O(2)) in S. cerevisiae. In V79 mammalian cells, biflorin was cytotoxic at higher concentrations. Moreover, at low concentrations biflorin pre-treatment protected against H(2)O(2)-induced oxidative damage by reducing lipid peroxidation and DNA damage as evaluated by normal and modified comet assay using DNA glycosylases. Our results suggest that biflorin cellular effects are concentration dependent. At lower concentrations, biflorin has significant antioxidant and protective effects against the cytotoxicity, genotoxicity, mutagenicity, and intracellular lipid peroxidation induced by H(2)O(2) in yeast and mammalian cells, which can be attributed to its hydroxyl radical-scavenging property. However, at higher concentrations, biflorin is cytotoxic and genotoxic.
Asunto(s)
Antimutagênicos/toxicidad , Antineoplásicos/toxicidad , Helechos/química , Naftoquinonas/toxicidad , Animales , Antimutagênicos/química , Antimutagênicos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Línea Celular , Ensayo Cometa , Ensayos de Selección de Medicamentos Antitumorales , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/aislamiento & purificación , Depuradores de Radicales Libres/toxicidad , Naftoquinonas/química , Naftoquinonas/aislamiento & purificación , Saccharomyces cerevisiae/efectos de los fármacos , Salmonella/efectos de los fármacosRESUMEN
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/toxicidadRESUMEN
Tellurium is a rare element that has been regarded as a toxic, nonessential element, and its biological role is not clearly established. In addition, the biological effects of elemental tellurium and some of its organic and inorganic derivatives have been studied, leading to a set of interesting and promising applications. Diphenyl ditelluride (DPDT), an organic tellurium derivate, showed antioxidant, antigenotoxic, antimutagenic, and anticancer properties. The antioxidant and prooxidant properties of DPDT are complex and depend on experimental conditions, which may explain the contradictory reports of these properties. In addition, DPDT may exert its effects through different pathways, including distinct ones to those responsible for chemotherapy resistance phenotypes: transcription factors, membrane receptors, adhesion, structural molecules, cell cycle regulatory components, and apoptosis pathways. This review aims to present recent advances in our understanding of the biological effects, therapeutic potential, and safety of DPDT treatment. Moreover, original results demonstrating the cytotoxic effects of DPDT in different mammalian cell lines and systems biology analysis are included, and emerging approaches for possible future applications are inferred.
Asunto(s)
Antioxidantes/uso terapéutico , Derivados del Benceno/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Telurio/química , Antioxidantes/farmacología , Derivados del Benceno/farmacología , Humanos , Compuestos Organometálicos/farmacología , Oxidación-ReducciónRESUMEN
PHARMACOLOGY RELEVANCE: Baccharis trinervis (Lam, Persoon) leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptic. AIM OF THE STUDY: To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, and to examine the mutagenic activity in Salmonella typhimurium. MATERIAL AND METHODS: Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF), and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3h with extracts and fractions. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)-2.5-biphenyl tetrazolium bromide reduction assay (MTT). Genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia. RESULTS: Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC (AEC>AEB). The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries in negative mode. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference in relation to the control. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay. CONCLUSIONS: Cytotoxic and genotoxic effects were demonstrated in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed.
Asunto(s)
Baccharis/química , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Flavonoides/análisis , Mutágenos/farmacología , Extractos Vegetales/toxicidad , Animales , Brasil , Colombia , Ensayo Cometa , Cricetulus , Relación Dosis-Respuesta a Droga , Pruebas de Micronúcleos , Microsomas/efectos de los fármacos , Hojas de la Planta/químicaRESUMEN
OBJECTIVES: To evaluate the antioxidant status and repair capacity in breast cancer patients as well as the relationship between these parameters and expression of critical proteins in breast cancer tissue. DESIGN AND METHODS: Blood samples were obtained from 25 female breast cancer patients and 19 healthy women. The antioxidant status was determined by the concentration of thiobarbituric-reactive substances (TBARS) and activity of superoxide dismutase (SOD) and catalase (CAT). The basal DNA damage and repair capacity in lymphocytes were evaluated by comet assay. The expression of p53, c-erbB2, Ki-67, estrogen receptor (ER) and progesterone receptor (PR) in cancer tissue was detected by immunohistochemical staining. RESULTS: The breast cancer patients presented significantly elevated endogenous DNA damage in lymphocytes and lower susceptibility to DNA damage induced by H(2)O(2) when compared to the control group. There is a negative correlation between TBARS and sensitivity to peroxide induced DNA damage in patients. The percentage of residual damage after H(2)O(2) treatment followed by 3h of post-incubation is significantly higher in patients and also correlates positively with SOD activity, ER and PR expression and negatively with the basal DNA damage. CONCLUSIONS: Our results demonstrate low repair capacity in lymphocytes of breast cancer patients and suggest that the regulation of DNA repair is sensitive to cellular redox state and can be modulated by ER/PR status.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Superóxido Dismutasa/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Catalasa/metabolismo , Ensayo Cometa , Daño del ADN , Reparación del ADN , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , Antígeno Ki-67/análisis , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Persona de Mediana Edad , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Proteína p53 Supresora de Tumor/análisisRESUMEN
In cancer treatment, chemotherapy is one of the main strategies used. The knowledge of the cellular and molecular characteristics of tumors allows the use of more specific drugs, making the removal of tumors more efficient. Among the drugs of choice in these treatments, topoisomerase inhibitors are widely used against different types of tumors. Topoisomerases are enzymes responsible for maintaining the structure of DNA, altering its topological state temporarily during the processes of replication and transcription, in order to avoid supercoiling and entanglements at the double helix. The DNA damage formed as a result of topoisomerase inhibition can be repaired by DNA repair mechanisms. Thus, DNA repair pathways can modulate the effectiveness of chemotherapy. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways involved in the removal of double strand breaks (DSBs); while nucleotide excision repair (NER) is mainly characterized by the removal of lesions that lead to significant structural distortions in the DNA double helix. Evidence has shown that DSBs are the main type of damage resulting from the inhibition of the DNA topoisomerase II enzyme, and therefore the involvement of HR and NHEJ pathways in the repair process is well established. However, some topoisomerase II inhibitors induce other types of lesions, like DNA adducts, interstrand crosslinks and reactive oxygen species, and studies have shown that other DNA repair pathways might be participating in removing injury induced by these drugs. This review aims to correlate the involvement of proteins from different DNA repair pathways in response to these drugs, with an emphasis on NER.
Asunto(s)
Daño del ADN/efectos de los fármacos , Reparación del ADN , ADN-Topoisomerasas/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Animales , Aductos de ADN , Humanos , Oxidación-Reducción , Especies Reactivas de Oxígeno , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/químicaRESUMEN
Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 µg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.
RESUMEN
The diphenyl ditelluride (DPDT) is a prototype for the development of new biologically active molecules. In previous studies, DPDT showed an elevated cytotoxicity in Chinese hamster fibroblast (V79) cells but the mechanisms for reduction of cell viability still remain unknown. DPDT showed mutagenic properties by induction of frameshift mutations in bacterium Salmonella typhimurium and yeast Saccharomyces cerevisiae. This organotelluride also induced DNA strand breaks in V79 cells. In this work, we investigated the mechanism of DPDT cytotoxicity by evaluating the effects of this compound on cell cycle progression, apoptosis induction and topoisomerase I inhibition. Significant decrease of V79 cell viability after DPDT treatment was revealed by MTT assay. Morphological analysis showed induction of apoptosis and necrosis by DPDT in V79 cells. An increase of caspase 3/7 activity confirmed apoptosis induction. The cell cycle analysis showed an increase in the percentage of V79 cells in S phase and sub-G1 phase. The yeast strain deficient in topoisomerase I (Topo I) showed higher tolerance to DPDT compared with the isogenic wild-type strain, suggesting that the interaction with this enzyme could be involved in DPDT toxicity. The sensitivity to DPDT found in top3∆ strain indicates that yeast topoisomerase 3 (Top3p) could participate in the repair of DNA lesions induced by the DPDT. We also demonstrated that DPDT inhibits human DNA topoisomerase I (Topo I) activity by DNA relaxation assay. Therefore, our results suggest that the DPDT-induced cell cycle arrest and reduction in cell viability could be attributed to interaction with topoisomerase I enzyme.
Asunto(s)
Apoptosis/efectos de los fármacos , Derivados del Benceno/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Inhibidores de Topoisomerasa I/toxicidad , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Necrosis/inducido químicamente , Saccharomyces cerevisiae/efectos de los fármacosAsunto(s)
Enfermedades Cardiovasculares , Estudios de Cohortes , Humanos , Prevalencia , Factores de RiesgoRESUMEN
By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal ß-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.
Asunto(s)
Daño del ADN , Endodesoxirribonucleasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/genética , Metoxaleno/farmacología , Proteína Recombinante y Reparadora de ADN Rad52/genética , Saccharomyces cerevisiae , Técnicas del Sistema de Dos Híbridos , Rayos UltravioletaRESUMEN
Tin or stannous (Sn(2+)) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn(2+) up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn(2+) by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25mM Sn(2+) concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn(2+) concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1h exposure to SnCl(2). The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn(2+) uptake. Moreover, our qRT-PCR data showed that Sn(2+) treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1, YAP1, and APN1.
Asunto(s)
Proteínas Portadoras/genética , Contaminantes Ambientales/toxicidad , Estaño/toxicidad , Levaduras/genética , Adaptación Fisiológica , Proteínas Portadoras/metabolismo , Contaminantes Ambientales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Estaño/metabolismo , Levaduras/efectos de los fármacos , Levaduras/metabolismoRESUMEN
This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well-known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ-irradiation). Third-instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ-irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose-related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment.
Asunto(s)
Ensayo Cometa , Drosophila melanogaster/citología , Pruebas de Mutagenicidad/métodos , Animales , Daño del ADN , Relación Dosis-Respuesta a Droga , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/efectos de la radiación , Metanosulfonato de Etilo/toxicidad , Rayos gamma , Hemocitos/efectos de los fármacos , Hemocitos/efectos de la radiación , Larva/citología , Larva/efectos de los fármacos , Larva/efectos de la radiación , Mutágenos/toxicidad , Dicromato de Potasio/toxicidad , Dosis de RadiaciónRESUMEN
Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gammaH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIalpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions.
Asunto(s)
Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , ADN/metabolismo , Doxorrubicina/farmacología , Mutación , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , ADN/genética , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Humanos , ARN Interferente Pequeño/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/genéticaRESUMEN
Ultraviolet radiation is known to cause adverse effects to aquatic species and aquatic environments. The fish Colossoma macropomum (tambaqui) and Arapaima gigas (pirarucu) live in the Amazon basin, near the Equator, and thus receive high intensity of ultraviolet radiation. Deforestation further aggravates the situation by reducing shade at ground level. The aim of this study was to evaluate the genotoxic effects of UVA and UVB radiation on erythrocytes of tambaqui and pirarucu fish using Micronuclei test and Comet assay. Our study showed that UV radiation caused DNA damage in both species as detected by Comet assay. In addition, there were differences in response to genotoxicity between both species, which are possibly related to their evolutionary history. Tambaqui fish exposed to ultraviolet radiation for different periods presented clear dose-response in DNA damage profile. Significant damage repair was observed 24h after cessation of ultraviolet radiation exposure. At the test conditions used, no significant increase in micronucleated cells was observed in tambaqui and pirarucu fish. Tambaqui proved to be more sensitive to ultraviolet radiation than Pirarucu, as detected by Comet assay, showing statistically higher baseline DNA damage. The present results demonstrated that alkaline Comet assay was very sensitive for detecting the UV-induced genotoxicity during the short exposure period in our study. In addition, the present study also suggests that tambaqui and pirarucu fish are useful sentinel organisms, as their UV sensitivity allows them to be effective monitors of biological hazards in the Amazon region.
Asunto(s)
Daño del ADN , Reparación del ADN , Peces , Rayos Ultravioleta , Animales , Ensayo Cometa , Eritrocitos/efectos de la radiación , Peces/genética , Pruebas de MutagenicidadRESUMEN
The effect of dietary diphenyl diselenide (1 ppm) on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis was examined in female Wistar rats. Beginning at 5 weeks of age, the animals were fed with either control or diphenyl-diselenide-supplied diets until the end of the study (210 days). At 50 days of age, mammary tumor was induced by the administration of three doses of NMU (50 mg/kg body wt, intraperitoneally) once a week for 3 weeks. In experimental trials, latency to tumor onset was extended in rats fed with diet supplemented with diphenyl diselenide (P < 0.05). The incidence and frequency of tumors were significantly small in animals supplemented with diphenyl diselenide. However, the multiplicity of tumors was not altered by dietary diphenyl diselenide. Diphenyl diselenide supplementation also restored superoxide dismutase (SOD) activity and vitamin C levels altered in the NMU group (P < 0.05). Our results suggest that diphenyl diselenide can be considered a chemopreventive agent, even when supplemented at a relatively low concentration.
Asunto(s)
Anticarcinógenos , Derivados del Benceno/farmacología , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Neoplasias Mamarias Animales/inducido químicamente , Neoplasias Mamarias Animales/prevención & control , Metilnitrosourea/toxicidad , Compuestos de Organoselenio/farmacología , Animales , Ácido Ascórbico/metabolismo , Biomarcadores , Peso Corporal/efectos de los fármacos , Ensayo Cometa , Dieta , Femenino , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
The hsp60 expression pattern and catalase activity in the freshwater planarian Dugesia schubarti exposed to copper under laboratory conditions were investigated. In the hsp60 induction experiments, planarians were exposed to a range of copper concentrations (0-960 microgCu/L) for 4 or 24h, to concentrations of 50 or 100 microgCu/L for 2, 4, 8, and 24h at 19 degrees C, and to heat shock at 27 degrees C for 24h. The concentrations of hsp60 in whole-body homogenates were determined immunochemically by Western blotting. Stress protein induction was detected only after 24h treatment at 27 degrees C. The tissue concentration of hsp60 remained unaltered in Cu-exposed planarians under the experimental conditions used. Catalase activity was significantly induced at concentrations of 40, 80, and 160 microgCu/L after 24h exposure. Our results suggest that catalase levels in planarians could represent biomarkers of interest for the estimation of copper effects in freshwater ecosystems.