RESUMO
Because cadmium might interact with proteins and, thus, exert toxicity in organisms, it is vital to understand the molecular mechanism of the interaction between cadmium and biologically relevant proteins as well as the structural and functional changes in these proteins. In this study, the interaction between α-chymotrypsin (α-ChT) and cadmium chloride (CdCl2 ) was investigated by performing enzyme activity determinations, multispectroscopic measurements, isothermal titration calorimetry, and molecular docking studies. It was demonstrated that CdCl 2 binds to α-ChT mainly via electrostatic forces with (21.0 ± 0.982) binding sites, leading to the increase of α-helix and the decrease of ß-sheet. The interaction between CdCl 2 and α-ChT loosened the protein skeleton and increased the molecular volume of α-ChT. CdCl 2 first binds to the interface of α-ChT and then interacts with the key residues His 57 or Asp 102 or both in the active sites, leading to the activity inhibition of α-ChT under the exposure of high CdCl 2 concentrations.
Assuntos
Cloreto de Cádmio/química , Quimotripsina/química , Cádmio/química , Domínio Catalítico , HumanosRESUMO
Cadmium exposure is known to increase lung cancer risk, but the underlying molecular mechanisms in cadmium-stimulated progression of malignancy are unclear. Here, we examined the effects of prolonged cadmium exposure on the malignant progression of A549 human lung adenocarcinoma cells and the roles of Notch1, hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor 1 receptor (IGF-1R)/Akt/extracellular signal-regulated kinase (ERK)/p70 S6 kinase 1 (S6K1) signaling pathways. Exposing A549 cells to 10 or 20 µm cadmium chloride (CdCl2) for 9-15 weeks induced a high proliferative potential, the epithelial-mesenchymal transition (EMT), stress fiber formation, high cell motility, and resistance to antitumor drugs. Of note, the CdCl2 exposure increased the levels of the Notch1 intracellular domain and of the downstream Notch1 target genes Snail and Slug. Strikingly, siRNA-mediated Notch1 silencing partially suppressed the CdCl2-induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance. In addition, we found that prolonged CdCl2 exposure induced reduction of E-cadherin in BEAS-2B human bronchial epithelial cells and antitumor drug resistance in H1975 human tumor-derived non-small-cell lung cancer cells depending on Notch1 signaling. Moreover, Notch1, HIF-1α, and IGF-1R/Akt/ERK/S6K1 activated each other to induce EMT in the CdCl2-exposed A549 cells. These results suggest that Notch1, along with HIF-1α and IGF-1R/Akt/ERK/S6K1 signaling pathways, promotes malignant progression stimulated by prolonged cadmium exposure in this lung adenocarcinoma model.
Assuntos
Cloreto de Cádmio/química , Receptor Notch1/metabolismo , Transdução de Sinais , Células A549 , Antígenos CD , Antineoplásicos/química , Brônquios/metabolismo , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
Reviewing the mode of interaction between this kind of active pharmaceutical ingredients and DNA has received much more attention in current years. Anthracycline drugs such as Epirubicin are frequently used in cancer treatment for breast cancer treatment. In the present study, the Epirubicin -calf thymus DNA interaction was investigated by using spectroscopic, fluorimetric and molecular docking methods. Water-soluble quantum dots (QDs) with nanometric particle size fabricated and characterized by transmission electron microscope and photon correlation spectroscopy. The binding constant value and the free energy change for this interaction were obtained to be 3.00×106 M-1 and -42.26 kJ mol-1, using the spectroscopic method and docking investigations, respectively. Additionally, fluorescent thioglycolic acid-capped CdTe QDs were used for investigation of EPI and DNA interaction. Epirubicin as a quencher quenched the fluorescence of CdTe QDs after electrostatic adsorption on the surface of QDs. With the addition of DNA, EPI will be desorbed from the surface of CdTe QDs, inserted into the DNA. Subsequently, fluorescence changes of QDs were used for calculation of binding constant value, which was in good agreement with that obtained by the spectroscopic method. By the comparison of the achieved results, the intercalation mode of interaction between Epirubicin and DNA proved.
Assuntos
Antibióticos Antineoplásicos/química , DNA/química , Epirubicina/química , Simulação de Acoplamento Molecular , Pontos Quânticos/química , Espectrofotometria Ultravioleta , Cloreto de Cádmio/química , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Nanopartículas/química , Tamanho da Partícula , Telúrio/química , Tioglicolatos/químicaRESUMO
Cadmium adversely affects the biological function of the liver. Transferrin might be involved in the detoxification system of cadmium. However, owing to the lack of investigation of the molecular mechanism of cadmium conjugating to transferrin, the role of transferrin in cadmium detoxification in the liver and how transferrin undergoes conformational and functional changes upon cadmium binding are not clear. In this article, we demonstrated the potential role of transferrin in the protection of the mouse primary hepatocytes against cadmium toxicity. After the incubation of hepatocytes with 10 and 100 µM CdCl2, pretreatment with transferrin significantly attenuated the reduction of cell viability in comparison with the samples treated with CdCl2 alone. Furthermore, a detailed molecular mechanism investigation of the interaction of CdCl2 with transferrin was reported using biophysical methods. Multi-spectroscopic measurements showed that CdCl2 formed complexes with transferrin and caused structural and conformational changes of transferrin. Isothermal titration calorimetry measurements revealed that transferrin has two classes of binding sites with different binding constants for CdCl2 binding. Hydrophobic forces and electrostatic forces are the major driving forces of the interaction. Preferred specific binding sites on transferrin were identified by dialysis experiments, molecular docking studies and molecular dynamics simulations. Upon low CdCl2 concentration exposure, no content of iron was released from transferrin because CdCl2 preferentially binds to the surface of transferrin molecules. Upon higher CdCl2 concentration exposure, the release of iron content from transferrin was observed due to the interaction of CdCl2 with the key residues around iron binding sites.
Assuntos
Cloreto de Cádmio/metabolismo , Cloreto de Cádmio/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Transferrina/metabolismo , Animais , Cloreto de Cádmio/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Inativação Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Termodinâmica , Transferrina/químicaRESUMO
An effective biological early warning system for the detection of water contamination should employ undemanding species that rapidly react to the presence of contaminants in their environment. The demonstrated reaction should be comprehensible and unambiguously evidential of the contamination event. This study utilized 96h post fertilization zebrafish larvae and tested their behavioral response to acute exposure to low concentrations of cadmium chloride (CdCl2) (5.0, 2.5, 1.25, 0.625mg/L) and permethrin (0.05, 0.029, 0.017, 0.01µg/L). We hypothesize that the number of larvae that show advanced trajectories in a group corresponds with water contamination, as the latter triggers avoidance behavior in the organisms. The proportion of advanced trajectories in the control and treated groups during the first minute of darkness was designated as a segregation parameter. It was parametrized and a threshold value was set using one CdCl2 trial and then applied to the remaining CdCl2 and permethrin replicates. For all cases, the method allowed distinguishing between the control and treated groups within two cycles of light: dark. The calculated parameter was statistically significantly different between the treated and control groups, except for the lowest CdCl2 concentration (0.625mg/L) in one replicate. This proof-of-concept study shows the potential of the proposed methodology for utilization as part of a multispecies biomonitoring system.
Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Bioensaio/métodos , Cloreto de Cádmio/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/fisiologia , Animais , Biomarcadores , Cloreto de Cádmio/administração & dosagem , Cloreto de Cádmio/química , Relação Dose-Resposta a Droga , Monitoramento Ambiental , Larva/efeitos dos fármacos , Poluentes Químicos da Água/administração & dosagem , Poluentes Químicos da Água/químicaRESUMO
We have engineered a metal-binding site into the novel artificial ß-propeller protein Pizza. This new Pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. The designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. Two copies of the trimer associate in the presence of cadmium chloride in solution, and very high-resolution X-ray crystallographic analysis reveals a nanocrystal of cadmium chloride, sandwiched between two trimers of the protein. This nanocrystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. Our results indicate the feasibility of using rationally designed symmetrical proteins to biomineralize nanocrystals with useful properties.
Assuntos
Cloreto de Cádmio/química , Nanopartículas/química , Proteínas/química , Cristalografia por Raios X , Modelos Moleculares , Engenharia de ProteínasRESUMO
Enzyme activity in biological systems is often governed by control mechanisms in which the catalytic properties are made sensitive or insensitive to differences in enzyme or substrate concentration. Here, we report the first supramolecular system where the catalytic activity is made concentration independent through the use of newly designed inhibitor molecules. The precise concentration dependence of coupled supramolecular equilibriums between free catalyst, inhibited catalyst, active inhibitor, and inactive inhibitor allows to keep the concentration of free catalyst at 1 mM in a broad concentration range, yielding an autoregulated catalytic system.
Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Homeostase/fisiologia , Cloreto de Cádmio/química , Catálise , Desenho de Fármacos , Inibidores Enzimáticos/química , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Estatísticos , Naftiridinas/química , Pirimidinonas/químicaRESUMO
The crystal structure of the protein augmenter of liver regeneration containing a 14-residue hexahistidine purification tag (hsALR) has been determined to 2.4 Å resolution by Cd-SAD using a highly redundant data set collected on a rotating-anode home X-ray source and processed in 1998. The hsALR crystal structure is a tetramer composed of two homodimers bridged by a novel Cd(2)Cl(4)O(6) cluster via binding to the side-chain carboxylate groups of two solvent-exposed aspartic acid residues. A comparison with the native sALR tetramer shows that the cluster dramatically changes the hsALR dimer-dimer interface, which can now better accommodate the extra 14 N-terminal residues associated with the purification tag. The refined 2.4â Å resolution structure is in good agreement with both the X-ray data (R(cryst) of 0.165, R(free) of 0.211) and the expected stereochemistry (r.m.s. deviations from ideality for bond lengths and bond angles of 0.007 Å and 1.15°, respectively).
Assuntos
Ácidos/química , Cloreto de Cádmio/química , Compostos de Cádmio/química , Redutases do Citocromo/química , Proteínas/química , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The use of 1,4-dioxane or pyridine as solvents for the polymerization of isoprene mediated by the acid-functional SG1-based alkoxyamines N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-O-(2-carboxylprop-2-yl)hydroxylamine (BlocBuilder MA) and N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-O-(2-carboxyleth-2-yl)hydroxylamine results in an increase in the rate of consumption of the initiator and narrower molecular weight distributions of the resulting polymer. In pyridine, an improved control of the polymerization was also obtained for a non-acid-functional initiator, and the overall rate of polymerization increased. These effects are likely to be the result of the disruption of intramolecular hydrogen bonding and, in the case of pyridine, stabilization of the polar SG1 free radical.
Assuntos
Butadienos/química , Dioxanos/química , Hemiterpenos/química , Óxidos de Nitrogênio/química , Pentanos/química , Piridinas/química , Solventes/química , Álcoois/química , Aminas/química , Cloreto de Cádmio/química , Catálise , Ligação de Hidrogênio , Peso Molecular , PolimerizaçãoRESUMO
The present work is dedicated to the study of the interactions of CdCl(2) with the synthetic polynucleotides polyAT and polyGC confined in the nanoscopic aqueous compartment of the water-in-oil microemulsion CTAB/pentanol/hexane/water, with the goal to mimic in vitro the situation met by the nucleic acids in vivo. In biological structures, in fact, very long strings of nucleic acids are segregated into very small compartments having a radius exceedingly smaller than the length of the encapsulated macromolecule. For comparison, the behaviour of polyGC was also studied in aqueous solutions of matched composition. The conformational and thermal stabilities of both polynucleotides enclosed in the inner compartment of the microemulsion are scarcely affected by the presence of CdCl(2), whereas in solution immediate and large effects were observed also at room temperature. The lack of effects of CdCl(2) on the properties of the biopolymers entrapped in the aqueous core of the microemulsion has been attributed to the peculiar characteristics of the medium (low dielectric constant, in particular) which cause a total repression of the CdCl(2) dissociation that is not complete even in water. In fact, several of the numerous effects of CdCl(2) observed on the conformational stability of polyGC in aqueous solutions have also been ascribed to the limited dissociation of the cadmium salt.
Assuntos
Cloreto de Cádmio/química , DNA/química , Emulsões/química , Polinucleotídeos/química , Cetrimônio , Compostos de Cetrimônio , Hexanos , Óleos/química , Água/químicaRESUMO
A novel block ionomer complex micelles as drug carrier is developed utilizing self-assemble of poly(ethylene glycol)-block-poly(acrylic acid) (PEG-b-PAA) and cadmium chloride. This micelles are characterized to be have good bio-compatibility, hydrophilicity, passive targeting and sustained slow release property which shows great potential for liver cancer therapy. Block ionomer complex micelles based on PEG-b-PAA and cadmium chloride can self-assemble in distilled water, and Cd(²+) agent is entrapped into the core stabilized by PEG shells. Results showed the block ionomer complex micelles to be spherically shaped. Cadmium was incorporated easily into the ionic core with remarkably high efficiency (34.25% weight (wt)/wt). The cadmium-loaded polymeric micelles exhibited sustained and slow release behavior of cadmium and a potent cytotoxicity against SMMC-7721 in vitro. This novel block ionomer complex micelles with cores of metal antitumor drug indicates to be potential carriers for effective drug delivery.
Assuntos
Acrilatos/farmacologia , Antineoplásicos/farmacologia , Cloreto de Cádmio/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Polietilenoglicóis/farmacologia , Acrilatos/administração & dosagem , Acrilatos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Cloreto de Cádmio/administração & dosagem , Cloreto de Cádmio/química , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Micelas , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Células Tumorais CultivadasRESUMO
Lignin is abundant and contains a large number of aromatic groups. Herein, CdxZn1-xS photocatalyst with tunable band gap energy was successfully synthesized by using 3-mercaptopropionic acid as a structure tuning additive. CdxZn1-xS can depolymerize alkaline lignin to vanillin by the photocatalytic process. Each gram of alkaline lignin can produce 46.5 mg of vanillin. 2-Phenoxy-1-phenylethanol (PP-ol) and other model compounds were used to understand the depolymerizing process of lignin. Fine tuned CdxZn1-xS can effectively cleave the Cß-O-4 bond existed in PP-ol under simulated sunlight. The highest conversion of PP-ol was 89.5% with phenol and acetophenone yields of 66.2% and 33.5%, respectively. The mechanism studies confirm that the Cα-H in PP-ol and lignin is firstly dehydrogenated to form Cα radical intermediates, and then the photogenerated electrons break the adjacent Cß-O bond. This research provides a new strategy to prepare valuable chemicals by virtue of renewable biomass and simulated sunlight.
Assuntos
Benzaldeídos/química , Lignina/química , Sulfetos/química , Ácido 3-Mercaptopropiônico/química , Cloreto de Cádmio/química , Catálise , Estrutura Molecular , Processos Fotoquímicos , Polimerização , Acetato de Zinco/químicaRESUMO
The structural and coordination properties of complexes formed upon the interaction of copper(II) and chromium(II) chlorides with dialkylimidazolium chloride (RMIm(+)Cl(-)) ionic liquids and glucose are studied by a combination of density functional theory (DFT) calculations and X-ray absorption spectroscopy (XAS). In the absence of the carbohydrate substrate, isolated mononuclear four-coordinated MeCl(4)(2-) species (Me = Cu, Cr) dominate in the ionic liquid solution. The organic part of the ionic liquid does not directly interact with the metal centers. The interactions between the RMIm(+) cations and the anionic metal chloride complexes are limited to hydrogen bonding with the basic Cl(-) ligands and the overall electrostatic stabilization of the anionic metal complexes. Exchange of Cl(-) ligands by a hydroxyl group of glucose is only favorable for CrCl(4)(2-). For Cu(2+) complexes, the formation of hydrogen bonded complexes between CuCl(4)(2-) and glucose is preferred. No preference for the coordination of metal chloride species to specific hydroxyl group of the carbohydrate is found. The formation of binuclear metal chloride complexes is also considered. The reactivity and selectivity patterns of the Lewis acid catalyzed reactions of glucose are discussed in the framework of the obtained results.
Assuntos
Cloreto de Cádmio/química , Cobre/química , Glucose/química , Líquidos Iônicos/química , Estrutura Molecular , Teoria Quântica , Espectroscopia por Absorção de Raios XRESUMO
The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl2, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison.
Assuntos
Cádmio/farmacologia , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Dispositivos Lab-On-A-Chip , Cloreto de Cádmio/química , DNA/genética , DNA Polimerase Dirigida por DNA/genética , Células HeLa , Humanos , Oligonucleotídeos/genética , Oxigênio/química , Plasmídeos/metabolismoRESUMO
Genotoxic effects of cadmium on zebra fish Danio rerio have been assessed by random amplified polymorphic DNA and real time PCR, followed by a comparison of the melting temperature patterns between each amplification reaction. Fish were exposed to two concentrations of cadmium chloride dissolved in the medium (1.9+/-0.6 microg Cdl(-1), C(1); 9.6+/-2.9 microg Cdl(-1), C(2)) for 21 days. A discriminative RAPD probe, OPB11, was first selected producing differential band patterns between control and metal-exposed genomic DNAs. RAPD-PCR showed an increase in the relative hybridization efficiency of OPB11 on the genomic DNAs coming from fish exposed to both Cd concentrations as compared to the control condition. In addition, the RAPD-PCR melting temperature patterns showed that with the OPB11 probe, the frequency of PCR products whose fusion temperature belongs to the [86-87 degrees C] interval decreased with Cd contamination, whereas an increase of frequency for the [78-80 degrees C] and [85-86 degrees C] intervals was correlated with Cd exposure.
Assuntos
Cloreto de Cádmio/toxicidade , DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Animais , Cloreto de Cádmio/química , DNA/química , Dano ao DNA , Impressões Digitais de DNA , Monitoramento Ambiental , Marcadores Genéticos , Longevidade/efeitos dos fármacos , Masculino , Testes de Mutagenicidade , Mutagênicos/química , Desnaturação de Ácido Nucleico , Técnica de Amplificação ao Acaso de DNA Polimórfico , Temperatura de Transição , Poluentes Químicos da Água/químicaRESUMO
Evidence of chloride-enhanced cadmium uptake by plants in soil experiments has been reported. However, it is still unclear whether this finding is due to increased rates of Cd2+ diffusion to plant roots or the direct uptake of complexes such as CdC1+. A controlled hydroponic experiment was undertaken to distinguish and quantify the uptake rates of free and inorganic-complexed cadmium and to model the uptake of cadmium by Indian mustard plants with a 'biotic ligand model'. Plants were treated with NaCl (0 to 200 mM) including equivalent Na2SO4 treatments. Cadmium speciation in solution was calculated using the WHAM-VI model. Results of the current trials showed that higher Cl-concentrations in solution generally resulted in greater cadmium accumulation by plants than predicted by the Cd2+ activity. Activities of Cd-chloro complexes showed the best correlations with the cadmium concentrations in the plants compared with the activity of Cd2+. The biotic ligand model showed a reasonable good fit for the plants when assuming competition by Cd2+ and CdCl+ for sorption sites at root level. The relative values of the two reaction constants suggest that root affinity for Cd2+ is 3.4 times greater than for CdCl+. Nevertheless this clearly indicates a substantial role for chloro-complexed cadmium accumulation.
Assuntos
Cádmio/química , Cloretos/química , Ligantes , Mostardeira/metabolismo , Biomassa , Cloreto de Cádmio/química , Cloro/química , Íons , Plantas/metabolismo , Sais/química , Cloreto de Sódio/química , Solo , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água/métodosRESUMO
The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd(2+) ions per Msh2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd(2+) occurs via a nonspecific mechanism. Cd(2+) and Msh2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd(2+):Msh2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the peptide backbone as well. Our study also shows that cadmium inactivates several unrelated enzymes similarly, consistent with a nonspecific mechanism of inhibition. Targeting of a variety of proteins, including Msh2-Msh6, in this generic manner would explain the marked broad-spectrum impact of Cd(2+) on biological processes. We propose that the presence of multiple nonspecific Cd(2+) binding sites on proteins and their propensity to change conformation on interaction with Cd(2+) are critical determinants of the susceptibility of corresponding biological systems to cadmium toxicity.
Assuntos
Pareamento Incorreto de Bases , Cádmio/química , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/fisiologia , Proteína 2 Homóloga a MutS/antagonistas & inibidores , Proteína 2 Homóloga a MutS/fisiologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/fisiologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cádmio/fisiologia , Cloreto de Cádmio/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Hidrólise , Proteína 2 Homóloga a MutS/química , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
IRMPD spectroscopy using the FELIX free electron laser and a Fourier transform ICR mass spectrometer was used to characterize the structures of electrosprayed dimer complexes M(2+)Trp(2) of tryptophan with a series of eight doubly charged metal ions, including alkaline earths Ca, Sr, and Ba, and transition metals Zn, Cd, Mn, Co, and Ni. With the support of DFT thermochemical calculations, at least three different structural motifs were distinguished spectroscopically, depending critically on the nature of the metal ion. The spectral signatures of a ligand in the charge-solvated (CS) configuration, namely peaks near 1730 and 1150 cm(-1), were prominent in all the spectra, and it was clear that all the dimer complexes contain at least one CS ligand. The spectra indicated that the second ligand is zwitterionic (ZW) for all complexes except the Ni case, with the second ligand having an extended binding geometry with smaller metals but showing some admixture of a compact chelated geometry with larger alkaline earths. It was concluded that these dimer complexes have a mixed configuration of ligands, denoted CS/ZW. The Ni(2+)Trp(2) complex is exceptional, with the spectroscopy and the thermochemical calculation both indicating a CS/CS configuration of ligands. This geometry appears to correlate with the exceptionally small size and high binding strength of the Ni(2+) cation. The complex CdClTrp(1+) was also obtained and gave a clear spectrum showing a CS ligand configuration. The presence of a CS ligand in all the dimeric complexes of the 2+ metals is an interesting contrast with the monomer complex Ba(2+)Trp, in which the ligand is ZW.
Assuntos
Cátions Bivalentes/química , Metais/química , Triptofano/química , Bário/química , Cádmio/química , Cloreto de Cádmio/química , Cálcio/química , Conformação Molecular , Estrutura Molecular , Níquel/química , Espectrofotometria Infravermelho , Estrôncio/química , Zinco/químicaRESUMO
Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested.
Assuntos
Receptor alfa de Estrogênio/química , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Cloreto de Cádmio/química , Cloreto de Cádmio/farmacologia , Sistema Livre de Células , Chlorocebus aethiops , Cyprinidae , Dibutilftalato/química , Dibutilftalato/farmacologia , Estradiol/análogos & derivados , Estradiol/química , Estradiol/farmacologia , Etinilestradiol/química , Etinilestradiol/farmacologia , Fulvestranto , Humanos , Ácidos Ftálicos/química , Ácidos Ftálicos/farmacologia , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Temperatura , Zeranol/análogos & derivados , Zeranol/química , Zeranol/farmacologiaRESUMO
Exposure of the brain to cadmium ions (Cd(2+)) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS-supporting cells, are resistant to Cd(2+)-induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl(2) for 24 h at concentrations higher than 20 microM substantially induced astrocytic cytotoxicity, which also resulted from long-term exposure to 5 microM of CdCl(2). Intracellular calcium levels were found to rapidly increase after the addition of CdCl(2) into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd(2+)-treated astrocytes. Adenovirus-mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd(2+)-exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd(2+) exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd(2+)-induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd(2+)-primed astrocytes. This information provides a useful approach for treating Cd(2+)-induced CNS neurological disorders.