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At present, there are no medicinal plant extracts currently available for treatment and control of fasciolosis. The present work could provide, for the first study, conclusions on the in vitro fasciolicidal properties of the ethanol extract of Terminalia catappa L. (TcCE) leaves against adult Fasciola gigantica after incubation with RPMI-1640 medium containing the TcCE at various concentrations and times when compared with triclabendazole (TCZ). The relative motility and survival index values of the TcCE-treated flukes decreased at a more rapid rate than the TCZ-treated flukes. The death of the parasites was observed after exposed to TcCE at 3 h incubation with 400, 800 and 1000 µg mL-1, and at 6 h incubation in 100 and 200 µg mL-1. Vacuolization, blebbings and partial disruption on the parasites' tegument were observed by light microscopy. When examined by scanning electron microscopy, TcCE caused similar tegumental alterations in the parasites as those observed in TCZ treatment but with larger damage at comparative incubation periods, consisting of swelling, blebbing, disrupted blebs, loss of spines, leading to the erosion, lesion and eventual disruption of the total tegument. Therefore, the TcCE may exert its fasciolicidal effect against F. gigantica by initially causing the tegumental alteration.
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Antiplatelmínticos/farmacologia , Fasciola/efeitos dos fármacos , Extratos Vegetais/farmacologia , Terminalia/química , Animais , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Folhas de Planta/químicaRESUMO
Presently, no effective anthelmintic drugs have been used to treat and control paramphistomosis, a severe disease of ruminants. In this study, we have investigated the in vitro anthelmintic effect of the leaves of Terminalia catappa L. crude extract (TcCE) and albendazole (ABZ) on adult Fischoederius cobboldi after incubating the flukes in RPMI-1640 medium containing the TcCE at various doses and times. The TcCE-treated flukes at all dosages exhibited rapid decrease of motility, and the relative motility (RM) values were decreased sharply from start to 3 h. Worms were killed after 6 and 12 h of treatment with 1000, 1500 and 2000 µg mL(-1) as well as 500 µg mL(-1) of TcCE, respectively. By light microscopy examination, the flukes exhibited the earliest alteration in a limited area of the tegument. At scanning electron microscopy level, the flukes' tegument showed similar sequence of morphological alterations after treatment with ABZ and TcCE that consisted of swelling of ridges and folds, followed by blebbing and rupturing of the blebs, leading to the erosion, lesion and disruption of the tegument. Hence, in vivo studies should be performed to examine whether the TcCE may serve as a powerful anthelmintic drug for treatment of paramphistomosis.
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Anti-Helmínticos/farmacologia , Extratos Vegetais/farmacologia , Terminalia/química , Trematódeos/efeitos dos fármacos , Albendazol/farmacologia , Animais , Anti-Helmínticos/uso terapêutico , Búfalos/parasitologia , Bovinos , Microscopia Eletrônica de Varredura/veterinária , Movimento/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Rúmen/parasitologia , Trematódeos/fisiologia , Trematódeos/ultraestrutura , Infecções por Trematódeos/tratamento farmacológico , Infecções por Trematódeos/parasitologia , Infecções por Trematódeos/veterináriaRESUMO
Of global concern, environmental pollution adversely affects human health and socioeconomic development. The presence of environmental contaminants, especially bacterial, viral, and parasitic pathogens and their toxins as well as chemical substances, poses serious public health concerns. Nanoparticle-based biosensors are considered as potential tools for rapid, specific, and highly sensitive detection of the analyte of interest (both biotic and abiotic contaminants). In particular, there are several limitations of conventional detection methods for water-borne pathogens due to low concentrations and interference with various enzymatic inhibitors in the environmental samples. The increase of cells to detection levels requires long incubation time. This review describes current state of biosensor nanotechnology, the advantage over conventional detection methods, and the challenges due to testing of environmental samples. The major approach is to use nanoparticles as signal reporter to increase output rather than spending time to increase cell concentrations. Trends in future development of novel detection devices and their advantages over other environmental monitoring methodologies are also discussed.
Assuntos
Bactérias/citologia , Técnicas Biossensoriais/métodos , Monitoramento Ambiental/métodos , Poluentes Ambientais/toxicidade , Poluição Ambiental/análise , Nanopartículas/química , HumanosRESUMO
Due to the rapid advent in genomics technologies and attention to ecological risk assessment, the term "ecotoxicogenomics" has recently emerged to describe integration of omics studies (i.e., transcriptomics, proteomics, metabolomics, and epigenomics) into ecotoxicological fields. Ecotoxicogenomics is defined as study of an entire set of genes or proteins expression in ecological organisms to provide insight on environmental toxicity, offering benefit in ecological risk assessment. Indeed, Daphnia is a model species to study aquatic environmental toxicity designated in the Organization for Economic Co-operation and Development's toxicity test guideline and to investigate expression patterns using ecotoxicology-oriented genomics tools. Our main purpose is to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and relevant modes of toxicity in Daphnia magna. These approaches enable us to address adverse phenotypic outcomes linked to particular gene function(s) and mechanistic understanding of aquatic ecotoxicology as well as exploration of useful biomarkers. Furthermore, key challenges that currently face aquatic ecotoxicology (e.g., predicting toxicant responses among a broad spectrum of phytogenetic groups, predicting impact of temporal exposure on toxicant responses) necessitate the parallel use of other model organisms, both aquatic and terrestrial. By investigating gene expression profiling in an environmentally important organism, this provides viable support for the utility of ecotoxicogenomics.
Assuntos
Daphnia/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Daphnia/genética , Ecotoxicologia/métodos , Modelos Animais , Toxicogenética/métodosRESUMO
The industrial application of nanotechnology, particularly using zinc oxide (ZnO), has grown rapidly, including products such as cosmetics, food, rubber, paints, and plastics. However, despite increasing population exposure to ZnO, its potential genotoxicity remains controversial. The biological effects of nanoparticles depend on their physicochemical properties. Preparations with well-defined physico-chemical properties and standardized test methods are required for assessing the genotoxicity of nanoparticles. In this study, we have evaluated the genotoxicity of four kinds of ZnO nanoparticles: 20nm and 70nm size, positively or negatively charged. Four different genotoxicity tests (bacterial mutagenicity assay, in vitro chromosomal aberration test, in vivo comet assay, and in vivo micronucleus test, were conducted, following Organization for Economic Cooperation and Development (OECD) test guidelines with good laboratory practice (GLP) procedures. No statistically significant differences from the solvent controls were observed. These results suggest that surface-modified ZnO nanoparticles do not induce genotoxicity in in vitro or in vivo test systems.
Assuntos
Dano ao DNA , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Animais , Células Cultivadas , Ensaio Cometa/métodos , Cricetinae , Cricetulus , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testes para Micronúcleos , Ratos , Ratos Sprague-DawleyRESUMO
Exposure to cadmium (Cd) results in bioaccumulation and irreversible damage; this encourages an investigation of alternatives to address Cd toxicity, using natural compounds. Lysiphyllum strychnifolium, a well-known Thai medicinal plant, was investigated for its phytochemical compounds and corresponding bioactivities, including antioxidant and anti-cytogenotoxic effects against Cd toxicity in HEK293 renal and HDF dermal cell models. The crude extract of L. strychnifolium (LsCrude) was partitioned into four fractions, using sequential polarity solvents (hexane, dichloromethane, ethyl acetate, and water, denoted as LsH, LsD, LsE, and LsW, respectively). The extraction yields were 1.79 %, 5.08 %, 8.53 %, and 70.25 % (w/w), respectively. Phytochemical screening revealed the presence of tannins, alkaloids, and flavonoids in LsCrude and its fractions, except for LsH. LsE exhibited the highest concentrations of phenolics (286.83 ± 6.83 mg GAE/g extract) and flavonoids (86.36 ± 1.29 mg QE/g extract). Subsequent 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and ferric-reducing ability of plasma (FRAP) reducing powder assays demonstrated the high antioxidant capacity of LsCrude and its fractions. The lowest IC50 value (9.11 ± 0.43 µg/mL) in the DPPH assay corresponded to LsW, whereas the highest total FRAP value (6.06 ± 0.70 mg QE Eq./g dry mass) corresponded to LsE. MTT and alkaline comet assays revealed the lack of toxicity of the extracts, which were considered safe. Upon exposure to Cd at the CC50 level, HEK293 cells treated with LsE suppressed Cd-induced damage. HDF cells treated with LsCrude, LsD, or LsE attenuated Cd-induced damage. In the pre-treatment, LsD protected the HDF cells against Cd-mediated cytogenotoxicity. These anti-cytogenotoxic potentials are likely due to the antioxidant properties of the phytochemicals. Our findings highlight the cyto-geno-protective properties of L. strychnifolium stem extracts against Cd toxicity in HEK293 and HDF cells, and provide a novel approach for combating oxidative stress and DNA damage caused by environmental pollutants.
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Thioredoxin-1 (Trx1) is an antioxidant enzyme with a protective role in the removal of oxidative stress. We investigated the mechanism by which the redox modulator Trx1 affects base excision repair (BER) activity to understand the protective role of Trx1. We constructed a Trx1 knockdown system to demonstrate the specific mechanism of Trx1 shRNA cells compared with that in the wild type cells, leading to increased cellular susceptibility to a sublethal dose of BER-inducible toxicant, nitrosomethylurea (NMU). In addition, we observed a modulatory role of Trx1 in the BER pathway via the p53 downstream gene, growth arrest, and DNA-damage-inducible protein 45 α (Gadd45a). The protein level and function of p53, a Trx1 downstream gene, coincidently decreased in the Trx1 shRNA cells. Futhermore, Trx1 shRNA cells showed decreased Gadd45a expression and interaction of Gadd45a with apurinic/apyrimidinic endonuclease 1 (APE1) as well as APE activity. In conclusion, Trx1 might cooperate in the control of APE1 function by modulating the p53-mediated BER via the protein-protein interaction between Gadd45a and APE1, providing insight into the novel role of redox factor Trx1 in modulation of BER.
Assuntos
Reparo do DNA/fisiologia , Tiorredoxinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Técnicas de Silenciamento de Genes , Genes p53 , Humanos , Metilnitrosoureia/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tiorredoxinas/genéticaRESUMO
Biofertilizer as an amendment has growing awareness. Little attention has been paid to bioremediation potential of indigenous heavy-metal-resistant microbes, especially when isolated from long-term polluted soil, as a bioinoculant in biofertilizers. Biofertilizers are a type of versatile nutrient provider and soil conditioner that is cost-competitive and highly efficient with nondisruptive detoxifying capability. Herein, we investigated the effect of biofertilizers containing indigenous cadmium (Cd)-resistant microbial consortia on rice growth and physiological response. The Thai rice cultivar PSL2 (Oryza sativa L.) was grown in Cd-enriched soils amended with 3% biofertilizer. The composition of the biofertilizers' bacterial community at different taxonomic levels was explored using 16S rRNA gene Illumina MiSeq sequencing. Upon Cd stress, the test biofertilizer had maximum mitigating effects as shown by modulating photosynthetic pigment, MDA and proline content and enzymatic antioxidants, thereby allowing increased shoot and root biomass (46% and 53%, respectively) and reduced grain Cd content, as compared to the control. These phenomena might be attributed to increased soil pH and organic matter, as well as enriched beneficial detoxifiers, i.e., Bacteroidetes, Firmicutes and Proteobacteria, in the biofertilizers. The test biofertilizer was effective in alleviating Cd stress by improving soil biophysicochemical traits to limit Cd bioavailability, along with adjusting physiological traits such as antioxidative defense. This study first demonstrated that incorporating biofertilizer derived from indigenous Cd-resistant microbes could restrict Cd contents and consequently enhance plant growth and tolerance in polluted soil.
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Metal compounds such as arsenic, cadmium, chromium, cobalt, lead, mercury, and nickel are classified as carcinogens affecting human health through occupational and environmental exposure. However, the underlying mechanisms involved in tumor formation are not well clarified. Interference of metal homeostasis may result in oxidative stress which represents an imbalance between production of free radicals and the system's ability to readily detoxify reactive intermediates. This event consequently causes DNA damage, lipid peroxidation, protein modification, and possibly symptomatic effects for various diseases including cancer. This review discusses predominant modes of action and numerous molecular markers. Attention is paid to metal-induced generation of free radicals, the phenomenon of oxidative stress, damage to DNA, lipid, and proteins, responsive signal transduction pathways with major roles in cell growth and development, and roles of antioxidant enzymatic and DNA repair systems. Interaction of non-enzymatic antioxidants (carotenoids, flavonoids, glutathione, selenium, vitamin C, vitamin E, and others) with cellular oxidative stress markers (catalase, glutathione peroxidase, and superoxide dismutase) as well as certain regulatory factors, including AP-1, NF-κB, Ref-1, and p53 is also reviewed. Dysregulation of protective pathways, including cellular antioxidant network against free radicals as well as DNA repair deficiency is related to oncogenic stimulation. These observations provide evidence that emerging oxidative stress-responsive regulatory factors and DNA repair proteins are putative predictive factors for tumor initiation and progression.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinógenos/toxicidade , Metais/toxicidade , Neoplasias/induzido quimicamente , Oxirredutases/metabolismo , Animais , Biomarcadores Tumorais/genética , Carcinógenos/química , Humanos , Metais/química , Neoplasias/diagnóstico , Neoplasias/metabolismo , Estresse Oxidativo , Oxirredutases/genéticaRESUMO
The exponential growth of nanotechnology and the industrial production have raised concerns over its impact on human and environmental health and safety (EHS). Although there has been substantial progress in the assessment of pristine nanoparticle toxicities, their EHS impacts require greater clarification. In this review, we discuss studies that have assessed nanoparticle eco-genotoxicity in different test systems and their fate in the environment as well as the considerable confounding factors that may complicate the results. We highlight key mechanisms of nanoparticle-mediated genotoxicity. Then we discuss the reliability of endpoint assays, such as the comet assay, the most favored assessment technique because of its versatility to measure low levels of DNA strand breakage, and the micronucleus assay, which is complementary to the former because of its greater ability to detect chromosomal DNA fragmentation. We also address the current recommendations on experimental design, including environmentally relevant concentrations and suitable exposure duration to avoid false-positive or -negative results. The genotoxicity of nanoparticles depends on their physicochemical features and the presence of co-pollutants. Thus, the effect of environmental processes (e.g., aggregation and agglomeration, adsorption, and transformation of nanoparticles) would account for when determining the actual genotoxicity relevant to environmental systems, and assay procedures must be standardized. Indeed, the engineered nanoparticles offer potential applications in different fields including biomedicine, environment, agriculture, and industry. Toxicological pathways and the potential risk factors related to genotoxic responses in biological organisms and environments need to be clarified before appropriate and sustainable applications of nanoparticles can be established.
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Based on epidemiological studies, an International Agency for Research on Cancer Working Group determined that strong inorganic acid mists containing sulfuric acid are carcinogenic to human even though, sulfuric acid, per se, is not. Accumulative studies indicate that there is a link between chronic occupational exposure to sulfuric acid mists and an increased risk of laryngeal cancer. Unintended, acute exposure to sulfuric acid mists can cause corrosive damage to target tissues depending on the route of exposure. This review compares the toxicity and carcinogenicity of sulfuric acid mists compared to other strong inorganic acid mists. It also examines the routes and duration of exposure (short-term, prolonged, and long-term). In vivo evidence does not support or refute the carcinogenicity of sulfuric inorganic mists even though its co-carcinogenic or promoting potential has been considered. On the basis of existing evidence on sulfuric acid mist toxicity, we suggested a putative adverse outcome pathway (AOP) relevant to carcinogenicity caused by mists containing sulfuric acid. A possible key factor involved in sulfuric acid mist carcinogenesis is the genotoxic effects of low pH since it can increase instability in chromosomes and DNA. A putative AOP for sulfuric acid mist carcinogenicity would help generate better risk assessments and more accurate predictions regarding the risk of developing cancer due to prolonged exposure. Establishing an AOP would also be useful for future studies examining the carcinogenicity of other strong inorganic mists.
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Cadmium is a toxic metallic element that poses serious human health risks via consumption of contaminated agricultural products. The effect of mixtures of dicalcium phosphate and organic amendments, namely cow manure (MD) and leonardite (LD), on Cd and Zn uptake of three rice cultivars (KDML105, KD53, and PSL2) was examined in mesocosm experiments. Plant growth, Cd and Zn accumulation, and physicochemical properties of the test soils were investigated before and after plant harvest. Amendment application was found to improve soil physicochemical properties; in particular, soil organic matter content and nutrient (N, P, K, Ca, and Mg) concentrations increased significantly. The MD treatment was optimal in terms of increasing plant growth; the MD and LD treatments decreased soil Cd concentration by 3.3-fold and 1.6-fold, respectively. For all treatments, all rice cultivars accumulated greater quantities of Cd and Zn in roots compared with panicles and shoots. Among the three cultivars, RD53 accumulated the lowest quantity of Cd. Translocation factors (<0.28) and bioconcentration coefficients of roots (>1) indicate that the three rice cultivars are Cd excluders. Our results suggest that a mixture of organic and inorganic amendments can be used to enhance rice growth while reducing accumulation of heavy metals when grown in contaminated soil.
Assuntos
Cádmio/farmacocinética , Oryza/química , Poluentes do Solo/farmacocinética , Zinco/farmacocinética , Animais , Bovinos , Feminino , Esterco , SoloRESUMO
Cadmium contamination still occurs in some parts of the world, and its concentrations in the environment are monitored in most countries due to its adverse effects on human health. We herein established yeast (Saccharomyces cerevisiae) reporter assay strains carrying plasmids with the yeast JLP1, SEO1, and CUP1 promoters connected to the bacterial lacZ reporter gene. The strain carrying the high-copy number pESC-JLP1-lacZ reporter plasmid was more responsive to cadmium than strains with other reporter plasmids. This JLP1-lacZ reporter assay strain will be useful for monitoring cadmium contamination in environmental water and soil as a first screening tool preceding official instrumental analyses, because the assay is rapid, easy to handle, and has the ability to process a large number of samples at a low cost.
Assuntos
Cádmio/toxicidade , Dioxigenases/genética , Poluentes Ambientais/toxicidade , Óperon Lac , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Genes Fúngicos , Genes Reporter , Projetos Piloto , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Engineered nanoparticles (NPs) are widely used in many sectors, such as food, medicine, military, and sport, but their unique characteristics may cause deleterious health effects. Close attention is being paid to metal NP genotoxicity; however, NP genotoxic/carcinogenic effects and the underlying mechanisms remain to be elucidated. In this review, we address some metal and metal oxide NPs of interest and current genotoxicity tests in vitro and in vivo. Metal NPs can cause DNA damage such as chromosomal aberrations, DNA strand breaks, oxidative DNA damage, and mutations. We also discuss several parameters that may affect genotoxic response, including physicochemical properties, widely used assays/end point tests, and experimental conditions. Although potential biomarkers of nanogenotoxicity or carcinogenicity are suggested, inconsistent findings in the literature render results inconclusive due to a variety of factors. Advantages and limitations related to different methods for investigating genotoxicity are described, and future directions and recommendations for better understanding genotoxic potential are addressed.
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Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Óxido de Zinco/toxicidade , Animais , Artefatos , Biomarcadores/análise , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Testes de MutagenicidadeRESUMO
Heavy metals that are harmful to humans include arsenic, cadmium, chromium, lead, mercury, and nickel. Some metals or their related compounds may even cause cancer. However, the mechanism underlying heavy metal-induced cancer remains unclear. Increasing data show a link between heavy metal exposure and aberrant changes in both genetic and epigenetic factors via non-targeted multiple toxicogenomic technologies of the transcriptome, proteome, metabolome, and epigenome. These modifications due to heavy metal exposure might provide a better understanding of environmental disorders. Such informative changes following heavy metal exposure might also be useful for screening of biomarker-monitored exposure to environmental pollutants and/or predicting the risk of disease. We summarize advances in high-throughput toxicogenomic-based technologies and studies related to exposure to individual heavy metal and/or mixtures and propose the underlying mechanism of action and toxicant signatures. Integrative multi-level expression analysis of the toxicity of heavy metals via system toxicology-based methodologies combined with statistical and computational tools might clarify the biological pathways involved in carcinogenic processes. Although standard in vitro and in vivo endpoint testing of mutagenicity and carcinogenicity are considered a complementary approach linked to disease, we also suggest that further evaluation of prominent biomarkers reflecting effects, responses, and disease susceptibility might be diagnostic. Furthermore, we discuss challenges in toxicogenomic applications for toxicological studies of metal mixtures and epidemiological research. Taken together, this review presents toxicogenomic data that will be useful for improvement of the knowledge of carcinogenesis and the development of better strategies for health risk assessment.
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Carcinógenos/toxicidade , Metais Pesados/toxicidade , Mutagênicos/toxicidade , Neoplasias/genética , Animais , Exposição Ambiental/efeitos adversos , Genômica , Humanos , Neoplasias/induzido quimicamenteRESUMO
Cadmium and nickel have been classified as carcinogenic to humans by the World Health Organization's International Agency for Research on Cancer. Given their prevalence in the environment, the fact that cadmium and nickel may cause diseases including cancer even at low doses is a cause for concern. However, the exact mechanisms underlying the toxicological effects induced by low-dose exposure to cadmium and nickel remain to be elucidated. Furthermore, it has recently been recognized that integrative analysis of DNA, mRNA and proteins is required to discover biomarkers and signaling networks relevant to human toxicant exposure. In the present study, we examined the deleterious effects of chronic low-dose exposure of either cadmium or nickel on global profiling of DNA copy number variation, mRNA and proteins. Array comparative genomic hybridization, gene expression microarray and functional proteomics were conducted, and a bioinformatics tool, which predicted signaling pathways, was applied to integrate data for each heavy metal separately and together. We found distinctive signaling networks associated with subchronic low-dose exposure to cadmium and nickel, and identified pathways common to both. ACTB, HSP90AA1, HSPA5 and HSPA8, which are key mediators of pathways related to apoptosis, proliferation and neoplastic processes, were key mediators of the same pathways in low-dose nickel and cadmium exposure in particular. CASP-associated signaling pathways involving CASP3, CASP7 and CASP9 were observed in cadmium-exposed cells. We found that HSP90AA1, one of the main modulators, interacted with HIF1A, AR and BCL2 in nickel-exposed cells. Interestingly, we found that HSP90AA1 was involved in the BCL2-associated apoptotic pathway in the nickel-only data, whereas this gene interacted with several genes functioning in CASP-associated apoptotic signaling in the cadmium-only data. Additionally, JUN and FASN were main modulators in nickel-responsive signaling pathways. Our results provide valuable biomarkers and distinctive signaling networks that responded to subchronic low-dose exposure to cadmium and nickel.
Assuntos
Biomarcadores Tumorais/genética , Cádmio/efeitos adversos , Carcinógenos/farmacologia , Neoplasias do Colo/genética , Níquel/efeitos adversos , Mapas de Interação de Proteínas/efeitos dos fármacos , Toxicogenética/métodos , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Hibridização Genômica Comparativa , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Oligoelementos/efeitos adversos , Células Tumorais CultivadasRESUMO
For industrial applications, fermentation of ethanol at high temperature offers advantages such as reduction in cooling costs, reduced risk of microbial contamination and higher efficiency of fermentation processes including saccharification and continuous ethanol stripping. Three thermotolerant Saccharomyces cerevisiae isolates (C3723, C3751 and C3867) from Thai fruits were capable of growing and producing 38 g/L ethanol up to 41°C. Based on genetic analyses, these isolates were prototrophic and homothallic, with dominant homothallic and thermotolerant phenotypes. After short-term (30 min) and long-term (12 h) exposure at 37°C, expression levels increased for the heat stress-response genes HSP26, SSA4, HSP82, and HSP104 encoding the heat shock proteins small HSP, HSP70, HSP90 and the HSP100 family, respectively. In isolates C3723 and C3867, expression was significantly higher than that in reference isolates W303 and TISTR5606 for TPS1 encoding trehalose-6-phosphate synthase, NTH1 encoding neutral trehalase and GSY1 encoding glycogen synthase. The results suggested that continuous high expression of heat stress-response genes was important for the long-term, heat stress tolerance of these thermotolerant isolates.
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Frutas/microbiologia , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos , Temperatura Alta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Transcriptoma , Etanol/metabolismo , Fermentação , Glucosiltransferases/genética , Resposta ao Choque Térmico/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trealase/genética , Trealose/biossíntese , Trealose/metabolismoRESUMO
Use of thermotolerant strains is a promising way to reduce the cost of maintaining optimum temperatures in the fermentation process. Here we investigated genetically a Saccharomyces cerevisiae strain showing a high-temperature (41°C) growth (Htg(+)) phenotype and the result suggested that the Htg(+) phenotype of this Htg(+) strain is dominant and under the control of most probably six genes, designated HTG1 to HTG6. As compared with a Htg(-) strain, the Htg(+) strain showed a higher survival rate after exposure to heat shock at 48°C. Moreover, the Htg(+) strain exhibited a significantly high content of trehalose when cultured at high temperature and stronger resistance to Congo Red, an agent that interferes with cell wall construction. These results suggest that a strengthened cell wall in combination with increased trehalose accumulation can support growth at high temperature. The gene CDC19, encoding pyruvate kinase, was cloned as the HTG2 gene. The CDC19 allele from the Htg(+) strain possessed five base changes in its upstream region, and two base changes resulting in silent mutations in its coding region. Interestingly, the latter base changes are probably responsible for the increased pyruvate kinase activity of the Htg(+) strain. The possible mechanism leading to this increased activity and to the Htg(+) phenotype, which may lead to the activation of energy metabolism to maintain cellular homeostasis, is discussed.
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Proteínas de Ciclo Celular/metabolismo , Resposta ao Choque Térmico/fisiologia , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura Alta , Especificidade da EspécieRESUMO
XPG, a structure-specific DNA endonuclease responsible for the 3' incision of DNA lesions during nucleotide excision repair (NER), is associated with high risk of skin cancer as well as skeletal, neurological and developmental abnormalities when functionally defective. These observations have led to the model wherein the endonuclease activity of XPG is important for NER. Herein, we first demonstrate a sensitive assay of XPG cleavage activity using direct nuclear extracts as an XPG source. This method provided quantitative evaluation of the activity of endogenous XPG endonuclease derived from cells with high reproducibility. Our new assay takes advantage of 3'-end oligolabeling of the bubble-shaped substrate. Our results demonstrate efficient cleavage of the model substrate in two XPG wild-type cell lines (human fibroblasts and RKO colon cancer cells) in a time- and dose-dependent manner. In addition, XPG-deficient cells manifested lower cleavage activity relative to normal XPG cells, indicating that the incision activity of XPG was intrinsic in our methodology. It was also found that 7 mM MgCl2 and buffer pH 6.8 resulted in optimal endonucleolytic activity. Based on these results, our modified methodology has potential for quantitative monitoring of XPG cleavage activity in any cell type or tissue of interest.
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Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Nucleotídeos de Desoxiadenina/metabolismo , Endonucleases/deficiência , Fibroblastos/enzimologia , Humanos , Proteínas Nucleares/deficiência , Radioisótopos de Fósforo , Fatores de Transcrição/deficiênciaRESUMO
Thioredoxin reductase 1 (Trr1) is an antioxidant and redox regulator that functions in governing the cellular redox state and survival against oxidative insults in mammals. However, this selenoprotein is also overexpressed in various forms of malignant cancers, leading to the hypothesis that Trr1 may be a potential target for cancer therapy. A quinone anti-cancer drug, mitomycin C (MMC), has been clinically used in the treatment of several types of tumors, including those of the colon. MMC exerts its activity via ROS induction and further results in DNA cross-linkage. To evaluate the significant role of Trr1 in MMC resistance in human colon cancer (RKO) cells, specific reduction in the expression of Trr1 was achieved using short-hairpin RNA (shRNA)-based interference. Our results showed that stable Trr1 shRNA knockdown manifested higher cellular susceptibility to MMC in comparison to that in wild-type cells. In addition, increased intracellular ROS accumulation appeared in the Trr1 shRNA knockdown cells compared to the RKO wild-type cells, in proportion to a relatively higher fraction of the DNA damage reporter protein phosphorylated histone 'γ-H2AX'. Notably, a neutral comet assay demonstrated that DNA double-strand breaks were highly induced in the Trr1-deficient cancer cells in the presence of MMC, presumably stimulating cancer cell death. Our results also revealed that MMC-induced apoptosis was associated with enhancement of oxidative damage to DNA. These results suggest that the specific knockdown of Trr1 expression via shRNA vector interference technology may be a potent molecular strategy by which to enhance the effectiveness of MMC-mediated killing in human colon cancer cells, through acceleration of double-strand DNA damage-oxidative stress as a trigger for apoptosis. This implies that Trr1 may be a prime target for enhancing the effectiveness of MMC chemotherapy in combination with specific RNA interference.