RESUMO
Reactive oxygen species (ROS)-induced cell apoptosis has emerged as an efficient strategy for cancer therapy. However, tumor hypoxia and insufficient amounts of endogenous hydrogen peroxide (H2O2) in the tumor microenvironment are currently the main limitations of photodynamic therapy (PDT) and chemodynamic therapy (CDT). Moreover, the glutathione (GSH) scavenging effect on ROS further hinders the efficiency of ROS-mediated therapy. Here, a CaO2-based nanosystem (named as CF@CO@HC) with ROS self-amplification and GSH-depletion abilities was developed by a bottom-up approach. This hybrid nanoparticle consisted of a photosensitizer-doped calcium peroxide (CaO2) core (CaO2-FM), a hybrid organosilica framework (Cu-ONS) incorporated with Fenton reagents (Cu2+) and tetrasulfide groups, and a local hydrophobic cage (HC) shell. The photosensitizer was fluorescein derivative 4-FM with a thermally activated delayed fluorescence (TADF) property. The HC shell was built to protect the CaO2 and the photosensitizer from being attacked by water. Upon being internalized into cancer cells, the nanosystem was decomposed through the reduction reactions of Cu2+ and the tetrasulfide bond-doped silica shell by GSH, thus releasing Cu+ for Cu+-mediated CDT. Meanwhile, the exposed CaO2-FM can react with H2O to liberate photosensitizer 4-FM and generate H2O2 and O2 to overcome barriers in CDT and PDT. Thus, our study provided an open-source and reduced-expenditure strategy via GSH depletion and ROS self-amplification behaviors for ROS generation and significantly achieved an improved synergistic PDT/CDT for cancers.
Assuntos
Nanopartículas , Fotoquimioterapia , Linhagem Celular Tumoral , Glutationa/química , Gastos em Saúde , Peróxido de Hidrogênio/química , Nanopartículas/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de OxigênioRESUMO
Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnosis of ocular diseases, tear fluid has unique advantages. Although numerous analytical methods exist for the measurement of different types of biomolecules in tear fluid, few have been reported for comprehensive understanding of oxidative stress-related thiol redox signaling. In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine a panel of twelve metabolites that systematically covered several thiol metabolic pathways. With optimization of MS/MS parameters and HPLC mobile phases, this method was sensitive (LOQ as low as 0.01 ng/ml), accurate (80-125% spike recovery) and precise (<10% RSD). This LC-MS/MS method combined with a simple tear fluid collection with Schirmer test strip followed by ultrafiltration allowed the high-throughput analysis for efficient determination of metabolites associated with thiol redox signaling in human tear fluids. The method was then applied to a small cohort of tear fluids obtained from healthy individuals. The method presented here provides a new technique to facilitate future work aiming to determine the complex thiol redox signaling in tear fluids for accurate assessment and diagnosis of ocular diseases.
Assuntos
Biomarcadores/química , Compostos de Sulfidrila/química , Lágrimas/química , Cromatografia Líquida de Alta Pressão , Glutationa/química , Humanos , Limite de Detecção , Oxirredução , Estresse Oxidativo , Espectrometria de Massas em TandemRESUMO
A simple and selective 'turn-on' fluorescence sensor have been developed for the determination of xanthine (XA) based on glutathione (GSH) capped copper nanoclusters (CuNCs) as the fluorescent probe. The proposed sensor possess several advantages such as sensitivity, short analysis time and requires no sample pretreatment. The conditions for the performances of the sensor have been optimized and good linear relationship was obtained between concentration and relative fluorescence intensity in the concentration range 9.0[Formula: see text]10-3 M to 8.0[Formula: see text]10-5 M with a detection limit 6.0[Formula: see text]10-6 M. The mechanism behind the fluorescence enhancement may be ascribed to the binding of XA on the surface of GSH CuNCs. The sensor have been successfully applied to determine XA in spiked physiological samples.
Assuntos
Complexos de Coordenação/química , Cobre/química , Corantes Fluorescentes/química , Glutationa/química , Xantina/análise , Complexos de Coordenação/economia , Cobre/economia , Análise Custo-Benefício , Corantes Fluorescentes/economia , Glutationa/economia , Tamanho da Partícula , Espectrometria de Fluorescência/economia , Propriedades de Superfície , Xantina/economiaRESUMO
Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.
Assuntos
Glutarredoxinas/química , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos/genética , Catálise , Domínio Catalítico/genética , Dissulfetos/química , Ativação Enzimática , Ensaios Enzimáticos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/química , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica em alfa-Hélice , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Due to the high toxicity of currently used chemotherapeutics, novel methods of cancer treatment are needed. Gold nanoparticles (AuNPs) seem to be an interesting alternative due to penetration through biological membranes and systemic barriers. AuNPs as carriers of chemotherapeutics allow for reduced concentrations whilst maintaining the expected effect, and thus reducing the costs of therapy and adverse effects. We synthesized AuNPs stabilized with reduced glutathione (GSH) and conjugated with doxorubicin (DOX), gemcitabine (GEM) or cytarabine (CTA). This is the first study in which cytarabine-AuNPs were synthesized and characterized. Transmission electron microscopy (TEM), thermogravimetric analysis (TGA), nuclear magnetic resonance spectroscopy (NMR) and high-performance liquid chromatography (HPLC) were used to chemically characterize obtained nanoparticles. Antitumor activity and safety of application were assessed by MTT assay in in vitro model (human osteosarcoma cells -143B, human osteoblast- hFOB1.19, breast cancer cells - MCF7, breast epithelial cells - MCF10A, pancreatic cancer cells - PANC-1, and pancreatic cells - hTERT-HPNE cells). We have shown that cellular response varies according to the type and concentration of AuNPs. At some concentrations, we were able to show selective cytotoxicity of our AuNPs conjugates only to cancer cell lines. Synthesized nanoparticles were more cytotoxic to tumor cell lines than chemotherapeutics alone.
Assuntos
Glutationa/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/tratamento farmacológico , Cromatografia Líquida de Alta Pressão , Citarabina/química , Citarabina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Glutationa/química , Ouro/efeitos adversos , Humanos , Células MCF-7 , Nanopartículas Metálicas/efeitos adversos , Microscopia Eletrônica de Transmissão , Neoplasias/genética , Neoplasias/patologia , Osteoblastos/efeitos dos fármacos , Telomerase/química , GencitabinaRESUMO
An adequate level of low molecular weight thiols (LMW-SH, especially glutathione (GSH)) protects cellular macromolecules against toxic agents, and is used as a sensitive biomarker of exposure to toxic compounds. During sample collection, storage and preparation, non-enzymatic and enzymatic oxidation of LMW-SH can occur leading to analytical inaccuracy. The aim of this study was to optimize a fast and reliable screening method for the determination of LMW-SH, mainly GSH, in blood and plasma samples as well as to investigate the impact of storage conditions on the LMW-SH stability. Based on our results, the described spectrophotometric method allows fast and reliable determination of LMW-SH in blood and plasma samples. Results on incubation of samples at 37 °C imply that synthesis of LMW-SH (probably GSH) as well as dynamic interexchange among various thiols forms can be induced in blood cells in in vitro conditions. Importantly, the level of LMW-SH in blood and plasma stored at -20 °C was constant, indicating that they can be stored at -20 °C for at least 30 days. Therefore, the method is suitable for assessment of LMW-SH in long-term human biomonitoring as well as environmental field studies, especially those involving a large number of samples such as epidemiological studies.
Assuntos
Monitoramento Biológico/métodos , Compostos de Sulfidrila/sangue , Biomarcadores/sangue , Biomarcadores/química , Glutationa/sangue , Glutationa/química , Humanos , Peso Molecular , Oxirredução , Manejo de Espécimes , Compostos de Sulfidrila/química , TemperaturaRESUMO
The aim of the present study was to predict the effect of inter-individual and inter-ethnic human kinetic variation on the sensitivity towards acute liver toxicity of lasiocarpine in the Chinese and the Caucasian population, and to derive chemical specific adjustment factors (CSAFs) by integrating variation in the in vitro kinetic constants Vmax and Km, physiologically based kinetic (PBK) modelling and Monte Carlo simulation. CSAFs were derived covering the 90th and 99th percentile of the population distribution of pyrrole glutathione adduct (7-GS-DHP) formation, reflecting bioactivation. The results revealed that in the Chinese population, as compared to the Caucasian population, the predicted 7-GS-DHP formation at the geometric mean, the 90th and the 99th percentile were 2.1-, 3.3- and 4.3-fold lower respectively. The CSAFs obtained using the 99th percentile values were 8.3, 17.0 and 19.5 in the Chinese, the Caucasian population and the two populations combined, respectively, while the CSAFs were generally 3.0-fold lower at the 90th percentile. These results indicate that when considering the formation of 7-GS-DHP the Caucasian population may be more sensitive towards acute liver toxicity of lasiocarpine, and further point out that the default safety factor of 3.16 for inter-individual human kinetic differences may not be sufficiently protective. Altogether, the results obtained demonstrate that integrating PBK modelling with Monte Carlo simulations using human in vitro data is a powerful strategy to quantify inter-individual variations in kinetics, and can be used to refine the human risk assessment of pyrrolizidine alkaloids.
Assuntos
Povo Asiático , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Biológicos , Alcaloides de Pirrolizidina/farmacocinética , População Branca , Animais , Doença Hepática Induzida por Substâncias e Drogas/etnologia , Simulação por Computador , Glutationa/química , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Método de Monte Carlo , Alcaloides de Pirrolizidina/toxicidade , Medição de Risco/métodosRESUMO
Uncapped and Glutathione capped Cupric oxide nanospheres were synthesized by the interaction of Berberis lycium (Bl) root extract with corresponding salt solution. CuO nanospheres were best optimized by mixing 2% Bl extract solution with 1 mM CuSO4·5H2O (pH 11, 90 °C) Reduced glutathione (0.25 mM) in solution form was added in respective emulsion after 24 h. Synthesis of nanospheres was ensured by distinct surface plasmonic resonance peaks shown by CuO (370-420 nm). Addition of glutathione resulted in sharp blue shift and lowered absorbance values in UV spectra suggesting the decrease in nanoparticles' size and concentration. Average particle sizes as deduced with XRD were found to be 18.52 and 16.57 nm for CuO and GSH-CuO nanospheres respectively. Additive based stability assessment of synthesized nanospheres revealed CuO and GSH-CuO nanospheres to be highly stable in the presence of Catechin hydrate among various tested chemical compounds while ascorbic acid appeared as a strong destabilizing agent. TMB was oxidized by H2O2 in the presence of synthesized enzymes likewise horseradish peroxidase; though exhibited moderate results. Glutathione stabilized cupric oxide nanospheres exhibited the potential to be modulated further into efficient nanozymes as these showed better affinity towards chromogenic substrate TMB (Km value 0.32 mM) and better catalytic efficiency (0.075 mM-1 s-1) compared to uncapped CuO nanomimetics (1.6 mM, 0.033 mM-1 s-1). All of the tested additives served as inhibitors to the peroxidase mimicking potential of CuO and GSH-CuO nanozymes.
Assuntos
Técnicas Biossensoriais/métodos , Cobre/química , Glutationa/química , Nanopartículas Metálicas/química , Nanosferas/química , Peróxido de Hidrogênio/química , Peroxidase/metabolismoRESUMO
Rac1 is a member of the family of small Rho GTPases that are molecular switches governing a variety of fundamental cellular processes, such as cell growth and motility. Its subcellular location and activity are regulated by several posttranslational modifications. S-glutathionylation, the adduction of glutathione to cysteine residues in Rac1, is a redox-dependent thiol modification and is generally associated with oxidative/nitrosative stress, representing a novel mechanism of GTPase regulation. Here, we describe the use of biotin-labeled glutathione to monitor intracellular glutathionylated Rac1 in response to exogenous stimuli.
Assuntos
Biotina , Glutationa , Processamento de Proteína Pós-Traducional , Coloração e Rotulagem/métodos , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Biotina/química , Biotina/metabolismo , Células COS , Chlorocebus aethiops , Glutationa/química , Glutationa/metabolismoRESUMO
Glutathione (GLU) is a peptidic thiol that plays important anti-oxidant roles in organisms and that occurs in both freshwater and seawater, where it can undergo both bio- and photodegradation. Recent results have elucidated the role played by OH, 1O2, H2O2 and other yet unidentified transients in GLU photochemistry, but very little is known of the role of CO3-. This is an important gap because CO3- is usually very reactive towards electron-rich compounds including thiols and mercaptans. Very little is also known on the environmental importance of the reaction between GLU and Br2-, which could account for the literature finding that GLU phototransformation is enhanced in simulated seawater compared to freshwater. By means of a photochemical model approach based on the APEX software (Aqueous Photochemistry of Environmentally-occurring Xenobiotics), here we provide an assessment of the role that several photoreactants, including most notably CO3- and Br2-, have in the photodegradation of GLU (both the whole substance and the separate neutral and mono-anionic species) under representative fresh- and saltwater conditions. Our model suggests that CO3- would dominate the photodegradation of GLU in low-DOC and high-pH freshwater, which are the only freshwater conditions that really ensure GLU photodegradation to be competitive with biotransformation. This result supports the potential key importance of CO3- in the environmental photochemistry of GLU. In surface seawater and in brackish water, GLU phototransformation might be dominated by the Br2- reaction (the role of additional halogen species such as Cl2- and ClBr- is still unknown).
Assuntos
Brometos/química , Carbonatos/química , Água Doce/química , Glutationa/química , Fotólise , Água do Mar/química , Poluentes Químicos da Água/química , Glutationa/análise , Luz Solar , Poluentes Químicos da Água/análiseRESUMO
The present study aimed to evaluate the responses of different biochemicals parameters associated with environmental pollution in the digestive gland of the gastropod mollusc Stramonita haemastoma. Physiochemical parameters and trace metal elements (Copper (Cu), Zinc (Zn), Chromium (Cr), Cadmium (Cd) and Lead (Pb)) were measured in seawater. Spatiotemporal variations in reduced glutathione (GSH), malondialdehyde (MDA) and metallothionein (Mt) as well as the specific activities of glutathione S-transferase (GST) and catalase (CAT) were evaluated in digestive gland of this species during a one-year period in 2013-2014. Samples collection was conducted at three sites. The results obtained showed seasonal fluctuations in GST and CAT activities and in the rate of Mt content. In addition, intersite variations in GSH, MDA, Mt and CAT were recorded in individuals. Also, trace metal elements concentrations determined by season in the digestive gland revealed spatial and temporal variations for Cu and Zn but they are below the limit of detection for Cd and Pb. The highest values were generally recorded in spring for Cu and in winter for Zn. In this first regional study using in S. haemastoma as a model, the biomarkers measured were seen to be inducible parameters to evaluate the health state of the organism and the overall quality of the study sites.
Assuntos
Catalase/química , Biomarcadores Ambientais/fisiologia , Glutationa/química , Malondialdeído/química , Metalotioneína/química , Metais/efeitos adversos , Oligoelementos/química , Mar MediterrâneoRESUMO
Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.
Assuntos
Cisplatino/metabolismo , Cobre/metabolismo , Glutationa/metabolismo , Metalochaperonas/metabolismo , Platina/metabolismo , Enxofre/metabolismo , Sítios de Ligação , Cisplatino/química , Cobre/química , Proteínas de Transporte de Cobre , Glutationa/química , Metalochaperonas/química , Metalochaperonas/isolamento & purificação , Chaperonas Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Método de Monte Carlo , Oxirredução , Platina/química , Enxofre/químicaRESUMO
The genetically encoded probes roGFP2-Orp1 and Grx1-roGFP2 have been designed to be selectively oxidized by hydrogen peroxide (H2O2) and glutathione disulfide (GSSG), respectively. Both probes have demonstrated such selectivity in a broad variety of systems and conditions. In this study, we systematically compared the in vitro response of roGFP2, roGFP2-Orp1 and Grx1-roGFP2 to increasing amounts of various oxidant species that may also occur in biological settings. We conclude that the previously established oxidant selectivity is highly robust and likely to be maintained under most physiological conditions. Yet, we also find that hypochlorous acid, known to be produced in the phagocyte respiratory burst, can lead to non-selective oxidation of roGFP2-based probes at concentrations ≥2µM, in vitro. Further, we confirm that polysulfides trigger direct roGFP2 responses. A side-by-side comparison of all three probes can be used to reveal micromolar amounts of hypochlorous acid or polysulfides.
Assuntos
Dissulfeto de Glutationa/química , Proteínas de Fluorescência Verde/genética , Peróxido de Hidrogênio/isolamento & purificação , Oxidantes/química , Ácido Peroxinitroso/metabolismo , Glutarredoxinas/química , Glutationa/química , Glutationa/metabolismo , Dissulfeto de Glutationa/isolamento & purificação , Proteínas de Fluorescência Verde/química , Peróxido de Hidrogênio/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Oxirredução , Ácido Peroxinitroso/química , Fagócitos/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Acrylamide (AA), a neurotoxin and a potential carcinogen, has been found in various thermally processed foods such as potato chips, biscuits, and coffee. Simple, cost-effective, and sensitive methods for the rapid detection of AA are needed to ensure food safety. Herein, a novel colorimetric method was proposed for the visual detection of AA based on a nucleophile-initiated thiol-ene Michael addition reaction. Gold nanoparticles (AuNPs) were aggregated by glutathione (GSH) because of a ligand-replacement, accompanied by a color change from red to purple. In the presence of AA, after the thiol-ene Michael addition reaction between GSH and AA with the catalysis of a nucleophile, the sulfhydryl group of GSH was consumed by AA, which hindered the subsequent ligand-replacement and the aggregation of AuNPs. Therefore, the concentration of AA could be determined by the visible color change caused by dispersion/aggregation of AuNPs. This new method showed high sensitivity with a linear range from 0.1 µmol L(-1) to 80 µmol L(-1) and a detection limit of 28.6 nmol L(-1), and especially revealed better selectivity than the fluorescence sensing method reported previously. Moreover, this new method was used to detect AA in potato chips with a satisfactory result in comparison with the standard methods based on chromatography, which indicated that the colorimetric method can be expanded for the rapid detection of AA in thermally processed foods.
Assuntos
Acrilamida/análise , Acrilamida/química , Colorimetria/métodos , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Solanum tuberosum , Compostos de Sulfidrila/química , Colorimetria/economia , Análise Custo-Benefício , Análise de Alimentos/economia , Manipulação de Alimentos , Glutationa/química , Ouro/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Nanopartículas Metálicas/química , Fatores de TempoRESUMO
The first water soluble maleimide bearing NIR BF2-azadipyrromethene (NIR-AZA) fluorochrome has been synthesised which is capable of rapid thiol conjugations in water with peptides such as glutathione, the cell penetrating peptide (CPP) C(ß-A)SKKKKTKV-NH2 and a thiol substituted cRGD. NIR fluorescence imaging showed rapid cellular delivery of the CPP conjugate and effective in vivo tumour localization for the cRGD conjugate.
Assuntos
Compostos Aza/síntese química , Corantes Fluorescentes/síntese química , Raios Infravermelhos , Maleimidas/química , Porfobilinogênio/análogos & derivados , Animais , Compostos Aza/química , Compostos Aza/farmacocinética , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Glutationa/química , Glutationa/farmacocinética , Células HeLa , Humanos , Maleimidas/farmacocinética , Camundongos , Estrutura Molecular , Neoplasias Experimentais/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Porfobilinogênio/química , Porfobilinogênio/farmacocinética , Compostos de Sulfidrila/químicaRESUMO
We developed a Bayesian method to extract macromolecular structure information from sparse single-molecule x-ray free-electron laser diffraction images. The method addresses two possible scenarios. First, using a "seed" structural model, the molecular orientation is determined for each of the provided diffraction images, which are then averaged in three-dimensional reciprocal space. Subsequently, the real space electron density is determined using a relaxed averaged alternating reflections algorithm. In the second approach, the probability that the "seed" model fits to the given set of diffraction images as a whole is determined and used to distinguish between proposed structures. We show that for a given x-ray intensity, unexpectedly, the achievable resolution increases with molecular mass such that structure determination should be more challenging for small molecules than for larger ones. For a sufficiently large number of recorded photons (>200) per diffraction image an M^{1/6} scaling is seen. Using synthetic diffraction data for a small glutathione molecule as a challenging test case, successful determination of electron density was demonstrated for 20000 diffraction patterns with random orientations and an average of 82 elastically scattered and recorded photons per image, also in the presence of up to 50% background noise. The second scenario is exemplified and assessed for three biomolecules of different sizes. In all cases, determining the probability of a structure given set of diffraction patterns allowed successful discrimination between different conformations of the test molecules. A structure model of the glutathione tripeptide was refined in a Monte Carlo simulation from a random starting conformation. Further, effective distinguishing between three differently arranged immunoglobulin domains of a titin molecule and also different states of a ribosome in a tRNA translocation process was demonstrated. These results show that the proposed method is robust and enables structure determination from sparse and noisy x-ray diffraction images of single molecules spanning a wide range of molecular masses.
Assuntos
Substâncias Macromoleculares/química , Difração de Raios X/métodos , Algoritmos , Teorema de Bayes , Simulação por Computador , Conectina/química , Elétrons , Glutationa/química , Funções Verossimilhança , Modelos Químicos , Método de Monte Carlo , Fótons , Probabilidade , RNA de Transferência/química , Ribossomos/química , Translocação Genética , VácuoRESUMO
The new mixed ligand silver(I) complex of formula [AgI(TPP)2(MBZT)] (1) was obtained by reacting 2-mercapto-benzothiazole (MBZT) with triphenylphosphine (TPP). The complex was characterized by m.p., vibrational spectroscopy (FT-IR), (1)H NMR, UV-vis, ESI-MS spectroscopic techniques and its structure was confirmed by X-ray crystallography. Mixed ligand complexes of silver(I) iodide with thiones and phosphines are very rare in the literature and to the best of our knowledge compound 1 is the first of this kind exhibiting significant biological effects. Complex 1 was evaluated for its in vitro cytotoxic activity (cell viability) under irradiation with UV light and without irradiation against human cancer cell lines: MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative), Caki-1 (renal), A549 (lung), OAW-42 (ovarian), HeLa (cervical) and additionally against the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line (MTSV17) with SRB assay. The results showed that 1 mediates a strong cytotoxic response to the tested normal and cancer cell lines. It exhibits equal activity against MDA-MB-231 cells where estrogen receptors (ERs) are devoid with the one against MCF-7 where ERs are present. Molecular docking studies have shown that 1 is docked in the different pocket than that of the ERs modulators. The binding affinity of 1 towards the intracellular molecules DNA and lipoxygenase (LOX) was studied for the evaluation of the mechanism of its cytostasis. The binding constant (Kb) of 1 towards CT-DNA was calculated by UV-Vis and fluorescent spectra suggesting intercalation or electrostatic interactions of 1 into DNA. Docking studies on DNA-complex interactions confirm the binding of 1. Moreover, the influence of complex 1 on the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied. In addition, since the deactivation of cisplatin caused by glutathione, seems to be an important determinant of its cytotoxic effects, the reaction of 1 with glutathione (GSH) was investigated by UV-absorption spectroscopy.
Assuntos
Antineoplásicos/farmacologia , DNA/química , Glutationa/química , Iodetos/química , Lipoxigenase/química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Compostos de Prata/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa/metabolismo , Células HeLa , Humanos , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Lipoxigenase/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Organometálicos/síntese química , Relação Estrutura-Atividade , Raios UltravioletaRESUMO
A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2×10(-10)M and a linear range from 10(-9) to 10(-8)M is reported. For the most useful analytical concentration of quantum dots, 1160µg/ml, a 1/Ksv value of 11µM Cu(2+) was determined. The method is based on the interaction of Cu(2+) with glutathione-capped CdTe quantum dots (CdTe-GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe-GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu(2+) quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu(2+) quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu(2+)-mediated QD fluorescence quenching was associated with nanoparticle decomposition.
Assuntos
Cobre/análise , Escherichia coli/citologia , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Transporte Biológico , Biomimética , Compostos de Cádmio/química , Células Cultivadas , Cobre/química , Cobre/metabolismo , Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Glutationa/química , Limite de Detecção , Espectrometria de Fluorescência/economia , Telúrio/química , Fatores de Tempo , Água/químicaRESUMO
Disinfection by-products (DBP) formed from natural organic matter and disinfectants like chlorine and chloramine may cause adverse health effects. Here, we evaluate how the quantity and quality of natural organic matter and other precursors influence the formation of DBPs during chlorination and chloramination using a comprehensive approach including chemical analysis of regulated and emerging DBPs, total organic halogen quantification, organic matter characterisation and bioanalytical tools. In vitro bioassays allow us to assess the hazard potential of DBPs early in the chain of cellular events, when the DBPs react with their molecular target(s) and activate stress response and defence mechanisms. Given the reactive properties of known DBPs, a suite of bioassays targeting reactive modes of toxic action including genotoxicity and sensitive early warning endpoints such as protein damage and oxidative stress were evaluated in addition to cytotoxicity. Coagulated surface water was collected from three different drinking water treatment plants, along with reverse osmosis permeate from a desalination plant, and DBP formation potential was assessed after chlorination and chloramination. While effects were low or below the limit of detection before disinfection, the observed effects and DBP levels increased after disinfection and were generally higher after chlorination than after chloramination, indicating that chlorination forms higher concentrations of DBPs or more potent DBPs in the studied waters. Bacterial cytotoxicity, assessed using the bioluminescence inhibition assay, and induction of the oxidative stress response were the most sensitive endpoints, followed by genotoxicity. Source waters with higher dissolved organic carbon levels induced increased DBP formation and caused greater effects in the endpoints related to DNA damage repair, glutathione conjugation/protein damage and the Nrf2 oxidative stress response pathway after disinfection. Fractionation studies indicated that all molecular weight fractions of organic carbon contributed to the DBP formation potential, with the humic rich fractions forming the greatest amount of DBPs, while the low molecular weight fractions formed more brominated DBPs due to the high bromide to organic carbon ratio. The presence of higher bromide concentrations also led to a higher fraction of brominated DBPs as well as proportionally higher effects. This study demonstrates how a suite of analytical and bioanalytical tools can be used to effectively characterise the precursors and formation potential of DBPs.
Assuntos
Desinfetantes/análise , Desinfetantes/química , Testes de Mutagenicidade/métodos , Purificação da Água/métodos , Animais , Bioensaio , Brometos/química , Fracionamento Químico , Desinfetantes/toxicidade , Desinfecção , Água Potável/análise , Água Potável/química , Glutationa/química , Halogenação , Halogênios , Medições Luminescentes , Peso Molecular , Estresse Oxidativo , Proteínas/química , Proteínas/metabolismo , Ratos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/químicaRESUMO
A novel, robust and fast ultra-high performance liquid chromatography-MS method has been developed for the simultaneous quantification of reduced glutathione (GSH) and oxidised glutathione (GSSG) in grape juice, wine and model wine solution. Sample preparation is minimal and does not require derivatisation. The method has very good performance in terms of sensitivity and selectivity. The limit of detection was 0.002 and 0.001 mg L(-1) for GSH and GSSG, respectively. The amount of GSH and GSSG released by commercial glutathione-enriched inactivated dry yeast preparations (GSH-IDYs) into a model solution was assessed. Significant differences in the amount of GSH and/or GSSG released into a model wine by different GSH-IDYs were observed, with ethanol influencing this release under certain conditions. The GSH and GSSG levels in grape juice fermentations supplemented with GSH-IDY were also assessed in relation to different addition times during fermentation. GSH-IDY addition can lead to elevated wine GSH levels, provided the supplementation is done early during alcoholic fermentation.