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Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18-labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.
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Ácidos Borônicos/química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Oxidantes/química , Oxidantes/metabolismo , Isótopos de Oxigênio/química , Encéfalo/metabolismo , Marcação por IsótopoRESUMO
The mediated oxidation of acetate and octanoate ions in acetonitrile was used to covalently modify carbon surfaces with films bearing saturated aliphatic chains of different length. Film thickness increases proportionally with the length of the aliphatic chain within the carboxylate precursor. The thickest film was obtained from octanoate oxidation and rectification occurs when ferrocene is used as redox probe in acetonitrile solution. This effect increases with the bulky and hydrophobic nature of the supporting electrolyte cations; n-Hx4 N+ >n-Bu4 N+ >Me4 N+ . The combination of the bulky and hydrophobic properties of the supporting electrolyte ions as well as the hydrophobic properties of the electrografted films is the basis of rectification of ferrocene in cyclic voltammetry experiments. This phenomenon was simulated through a CEC mechanism in solution, where the mass transport inside the film channels was emulated through single chemical equilibria.
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Nanoneedles, defined as high aspect ratio structures with tip diameters of 5 to approximately 500 nm, are uniquely able to interface with the interior of living cells. Their nanoscale dimensions mean that they are able to penetrate the plasma membrane with minimal disruption of normal cellular functions, allowing researchers to probe the intracellular space and deliver or extract material from individual cells. In the last decade, a variety of strategies have been developed using nanoneedles, either singly or as arrays, to investigate the biology of cancer cells in vitro and in vivo. These include hollow nanoneedles for soluble probe delivery, nanocapillaries for single-cell biopsy, nano-AFM for direct physical measurements of cytosolic proteins, and a wide range of fluorescent and electrochemical nanosensors for analyte detection. Nanofabrication has improved to the point that nanobiosensors can detect individual vesicles inside the cytoplasm, delineate tumor margins based on intracellular enzyme activity, and measure changes in cell metabolism almost in real time. While most of these applications are currently in the proof-of-concept stage, nanoneedle technology is poised to offer cancer biologists a powerful new set of tools for probing cells with unprecedented spatial and temporal resolution.
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Fenômenos Fisiológicos Celulares , Membrana Celular , Citosol , Espaço IntracelularRESUMO
This study describes the theoretical basis and the methods for the facile synthesis and characterization of four fluorogenic probes, N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG), N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2) and N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2). In addition, the study describes the methodology for the application of these reagents for measuring and imaging of free thiols on cell surfaces as well as their use as pseudo substrates for the thiol reductase and S-nitrosothioldenitrosylase activities of protein disulfide isomerase (PDI) and S-nitrosothiol reductase activity of S-nitrosoglutathione reductase (GSNOR) in vitro and on live cells in culture.
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Dissulfetos/metabolismo , Corantes Fluorescentes/química , Oxirredutases/metabolismo , S-Nitrosoglutationa/química , Membrana Celular/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/metabolismo , Epitélio Pigmentado da Retina/citologia , Compostos de SulfidrilaRESUMO
Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2ÌÌ), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2ÌÌ) and forms a stable fluorescent product or reacts with O2ÌÌ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.
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Mitocôndrias/metabolismo , Técnicas de Sonda Molecular , Espécies Reativas de Oxigênio/metabolismo , Aconitato Hidratase/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
Redox signalling plays an important role in many aspects of physiology, including that of the cardiovascular system. Perturbed redox regulation has been associated with numerous pathological conditions; nevertheless, the causal relationships between redox changes and pathology often remain unclear. Redox signalling involves the production of specific redox species at specific times in specific locations. However, until recently, the study of these processes has been impeded by a lack of appropriate tools and methodologies that afford the necessary redox species specificity and spatiotemporal resolution. Recently developed genetically encoded fluorescent redox probes now allow dynamic real-time measurements, of defined redox species, with subcellular compartment resolution, in intact living cells. Here we discuss the available genetically encoded redox probes in terms of their sensitivity and specificity and highlight where uncertainties or controversies currently exist. Furthermore, we outline major goals for future probe development and describe how progress in imaging methodologies will improve our ability to employ genetically encoded redox probes in a wide range of situations. This article is part of a special issue entitled "Redox Signalling in the Cardiovascular System."
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Oxirredução , Animais , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Proteínas Luminescentes/metabolismoRESUMO
Diamond electrochemistry is primarily influenced by quantities of sp3-carbon, surface terminations, and crystalline structure. In this work, a new dimension is introduced by investigating the effect of using substrate-interlayers for diamond growth. Boron and nitrogen co-doped nanocrystalline diamond (BNDD) films are grown on Si substrate without and with Ti and Ta as interlayers, named BNDD/Si, BNDD/Ti/Si, and BNDD/Ta/Ti/Si, respectively. After detailed characterization using microscopies, spectroscopies, electrochemical techniques, and density functional theory simulations, the relationship of composition, interfacial structure, charge transport, and electrochemical properties of the interface between diamond and metal is investigated. The BNDD/Ta/Ti/Si electrodes exhibit faster electron transfer processes than the other two diamond electrodes. The interlayer thus determines the intrinsic activity and reaction kinetics. The reduction in their barrier widths can be attributed to the formation of TaC, which facilitates carrier tunneling, and simultaneously increases the concentration of electrically active defects. As a case study, the BNDD/Ta/Ti/Si electrode is further employed to assemble a redox-electrolyte-based supercapacitor device with enhanced performance. In summary, the study not only sheds light on the intricate relationship between interlayer composition, charge transfer, and electrochemical performance but also demonstrates the potential of tailored interlayer design to unlock new capabilities in diamond-based electrochemical devices.
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The most well-known criterion for POC devices is ASSURED, and affordability, i.e., using low-cost instrumentation, is the most challenging one. This manuscript provides a pathway for transitioning ESSENCE, an impedance-based biosensor platform, from using an expensive benchtop analyzer-KeySight 4294A (~$50k)-to using a significantly portable and cheaper USB oscilloscope-Analog Discovery 2 (~$200) -with similar sensitivity (around 100 times price difference). To achieve this, we carried out a fundamental study of the interplay between an electrolyte like potassium chloride (KCl), and an electrolyte buffer like phosphate buffered saline (PBS) in the presence and absence of a redox buffer like ferro/ferricyanide system and ([Ru(bpy)3]2+). Redox molecules in the electrolyte caused a significant change in the Nyquist curve of the impedance depending on the redox molecule type. The redox species and the background electrolyte have their own RC semicircles in the Nyquist curve, whose overlap depends on the redox concentration and electrolyte ionic strength. We found that by increasing the electrolyte ionic strength or the redox concentration, the RC semicircle moves to higher frequencies and vice versa. Importantly, the use of the buffer electrolyte, instead of KCl, led to a lower standard deviation and overall signal (lesser sensitivity). However, to achieve the best results from the biorecognition signal, we chose a buffered electrolyte like PBS with high ionic strength and lowered the redox probe concentrations to minimize the standard deviation and reduce any noise from migrating to the low-cost analyzer. Comparing the two analyzers shows similar results, with a lowered detection limit from the low-cost analyzer.
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Técnicas Biossensoriais , Eletrólitos , Impedância Elétrica , OxirreduçãoRESUMO
Organophosphorus pesticides (OPs) are one of the most frequently used pesticides in agriculture, and their residues in environment have caused serious human health and environmental concerns. In this work, we reported a dual-ratiometric electrochemical aptasensor based on carbon nanohorns/anthraquinone-2-carboxylic acid/Au nanoparticles (CNHs/AQ/AuNPs) for simultaneous detection of malathion (MAL) and omethoate (OMT). Here, CNHs/AQ/AuNPs composites were synthesized by a simple room temperature method, and used as a substrate to generate a reference signal (IAQ) and enlarge response signals. Hairpin DNA was then immobilized, offering independent and specific binding sites to further adsorb MB-labelled MAL aptamer (MB-Apt1) and Fc-labelled OMT aptamer (Fc-Apt2). Upon the addition of MAL or OMT, the formation of aptamer-target complex caused the release of MB-Apt1 or Fc-Apt2 from the electrode, resulting in a decrease in IMB or IFc, while IAQ kept unchanged. Based on this principle, the ratiometric signals of IMB/IAQ and IFc/IAQ were used to simultaneously detect MAL and OMT, offering a linear range of 3 pg mL-1 to 3 ng mL-1 for MAL and 10 pg mL-1 to 10 ng mL-1 for OMT, and no significant cross-reactivity existed. By taking advantage of the excellent conductivity and large specific area of CNHs/AQ/AuNPs and the stable two-dimensional structure of hairpin DNA, the aptasensor exhibited high sensitivity, selectivity and reliability. Our work has offered a novel way for simultaneous detection of multiple OPs.
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Nanopartículas Metálicas , Praguicidas , Humanos , Malation , Ouro , Compostos Organofosforados , Reprodutibilidade dos Testes , DNA , CarbonoRESUMO
Currently, electrochemical sensors are regarded as an efficient tool for the biological and environmental sensing. Electrochemical sensors, such as voltammetric, amperometric, and impedimetric sensors, have gained great attention due to their simplicity, sensitivity, and selectivity. The performance of these electrochemical sensors could be enhanced by surface engineered nano/micro structured materials with conducting dyes/redox species. In this review, a great focus has been put on the redox-active dyes because of their electronic, optical, electrochromic, and conductivity properties. The mechanisms of oxidation and subsequent polymerization of different redox-active dyes at the surface of electrodes have been studied. Additionally, their role in catalyzing the oxidation or reduction of the target analytes at the surfaces of electrodes has also been highlighted. The redox-active dyes were used as electrochemical probes for detecting various analytes in biological and environmental samples. Overall, redox-active dyes are considered promising conducting polymers for the assessment of many analytes such as drugs, pesticides, surfactants, and heavy metal ions.
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Cytokines are small proteins and form complicated cytokine networks to report the status of our health. Thus, accurate profiling and sensitive quantification of multiple cytokines is essential to have a comprehensive and accurate understanding of the complex physiological and pathological conditions in the body. In this study, we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1ß, and TNF-α. First, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1ß, and TNF-α, respectively, to obtain Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, the capture monoclonal antibody for IL-6, IL-1ß, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1ß, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity, and stability, and recovery of 93.6%-105.5% was achieved for determining cytokines spiked in the whole mouse serum.
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Técnicas Biossensoriais/métodos , Citocinas/sangue , Técnicas Eletroquímicas , Grafite/química , Imunoensaio , Animais , Anticorpos/imunologia , Benzenossulfonatos/química , Citocinas/imunologia , Eletrodos , Compostos Ferrosos/química , Interleucina-6/sangue , Interleucina-6/imunologia , Limite de Detecção , Metalocenos/química , Camundongos , Oxazinas/química , Oxirredução , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Redox reactions play a key role in maintaining essential biological processes. Deviations in redox pathways result in the development of various pathologies at cellular and organismal levels. Until recently, studies on transformations in the intracellular redox state have been significantly hampered in living systems. The genetically encoded indicators, based on fluorescent proteins, have provided new opportunities in biomedical research. The existing indicators already enable monitoring of cellular redox parameters in different processes including embryogenesis, aging, inflammation, tissue regeneration, and pathogenesis of various diseases. In this review, we summarize information about all genetically encoded redox indicators developed to date. We provide the description of each indicator and discuss its advantages and limitations, as well as points that need to be considered when choosing an indicator for a particular experiment. One chapter is devoted to the important discoveries that have been made by using genetically encoded redox indicators.
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Técnicas Biossensoriais , Imagem Molecular/métodos , Sondas Moleculares/biossíntese , Espécies Reativas de Oxigênio/análise , Proteínas Recombinantes de Fusão/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desenvolvimento Embrionário/genética , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Inflamação , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Oxirredução , Estrutura Secundária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Mitochondrially generated oxidants are believed to play important roles in both physiology and pathophysiology. Therefore, it is of significant interest to better understand the metabolic conditions leading to enhanced mitochondrial oxidant generation. Here, we investigate the influence of oxygen and glucose availability on the redox state of peroxiredoxin-based redox probes, expressed in the cytosol and mitochondrial matrix of yeast cells. We observe that the redox state of peroxiredoxin probes reflects the balance between dioxygen-dependent peroxide generation and glucose-dependent generation of reducing equivalents. The oxidative pentose phosphate pathway appears to be the dominant source of NADPH in the system under study.
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Hydrogen sulfide (H2S) was discovered as a third gasotransmitter in biological systems and recent years have seen a growing interest to understand its physiological and pathological functions. However, one major limiting factor is the lack of robust sensors to quantitatively track its production in real-time. We described a facile electrochemical assay based on latent redox probe approach for highly specific and sensitive quantification in living cells. Two chemical probes, Azido Benzyl ferrocene carbamate (ABFC) and N-alkyl Azido Benzyl ferrocene carbamate (NABFC) composed of azide trigger group were designed. H2S molecules specifically triggered the release of reporters from probes and the current response was monitored using graphene oxide film modified electrode as transducer. The detection limits are 0.32µM (ABFC) and 0.076µM (NABFC) which are comparable to those of current sensitive methods. The probes are successful in the determination of H2S spiked in whole human blood, fetal bovine serum, and E. coli. The continuous monitoring and quantification of endogenous H2S production in E. coli were successfully accomplished. This work lays first step stone towards real-time electrochemical quantification of endogenous H2S in living cells, thus hold great promise in the analytical aspects of H2S.
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Azidas/química , Técnicas Biossensoriais/métodos , Escherichia coli/química , Compostos Ferrosos/química , Sulfeto de Hidrogênio/análise , Sulfeto de Hidrogênio/sangue , Animais , Bovinos , Técnicas Eletroquímicas/métodos , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Sulfeto de Hidrogênio/metabolismo , Metalocenos , OxirreduçãoRESUMO
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.
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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
This article contains representative fluorescence excitation spectra of roGFP2-based probes used for ratiometric analysis of redox changes as presented in the article "Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species" [1]. The recombinant probes roGFP2, roGFP2-Orp1, and Grx1-roGFP2 were exposed to various oxidative and nitrosative species, including hydrogen peroxide (H2O2), aldrithiol-2 (AT-2), glutathione disulfide (GSSG), hypochlorous acid (HOCl), S-nitrosoglutathione (GSNO), peroxynitrite (ONOO-), potassium polysulfide (K2Sx), spermine NONOate (SperNO), and diethyl amino NONOate (DeaNO) at different molar ratios. Fluorescence excitation spectra of the probes were recorded in the excitation wavelength range between 350 and 500 nm and for a total of 60 min. Analysis and interpretation of the data is presented in an associated article [1].
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The density of electronic states (DOS) is an intrinsic electronic property that works conclusively in the electrochemistry of carbon materials. However, seldom has it been reported how the DOS at the Fermi level influences the electrochemical activity. In this work, we synthesized partially and fully unzipped carbon nanotubes by longitudinally unzipping pristine carbon nanotubes (CNTs). We then studied the electrochemical activity and biosensitivity of carbon materials by means of the CNTs and their derivatives to elucidate the effect of the DOS on their electrochemical performances. Tailoring of the DOS for the CNT derivatives could be conveniently realized by varying the sp(2)/sp(3) ratio (i.e., graphite concentration) through manipulating the oxidative unzipping degree. Despite the diverse electron transfer mechanisms and influence factors of the four investigated redox probes (IrCl6(2-), [Fe(CN)6](3-), Fe(3+), and ascorbic acid), the CNT derivatives exhibited consistent kinetic behaviors, wherein CNTs with a high DOS showed superior electrochemical response compared with partially and fully unzipped carbon nanotubes. For biological detection, the CNTs could simultaneously distinguish ascorbic acid, dopamine, and uric acid, while the three CNT derivatives could all differentiate phenethylamine and epinephrine existed in the newborn calf serum. Moreover, the three CNT derivatives all presented wide linear detection ranges with high sensitivities for dopamine, phenethylamine, and epinephrine.
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Técnicas Eletroquímicas/métodos , Elétrons , Nanotubos de Carbono/química , Animais , Animais Recém-Nascidos , Bovinos , Espectroscopia Dielétrica , Dopamina/análise , Eletrodos , Epinefrina/análise , Nanotubos de Carbono/ultraestrutura , Fenetilaminas/análise , Espectroscopia Fotoeletrônica , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H2O2 and Zn(2+) have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn(2+) to cause cellular H2O2 production. To determine the role of Zn(2+)-induced H2O2 production in the human airway epithelial cell response to Zn(2+) exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn(2+) for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn(2+) exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn(2+)-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn(2+)-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn(2+) leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.
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Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Mucosa Respiratória/metabolismo , Zinco/metabolismo , Adaptação Biológica/genética , Catalase/genética , Catalase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Mucosa Respiratória/efeitos dos fármacos , Zinco/farmacologiaRESUMO
Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H2O2 reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H2O2 treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H2O2 was still present. Although HyPer should be H2O2 specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl(-) exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H2O2-albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H2O2 increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H2O2 decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.