Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses.
ABSTRACT: Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.
Project description:Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), 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 O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ 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.
Project description:The current lack of suitable probes has limited the in vivo imaging of reactive oxygen/nitrogen species (ROS/RNS). ROS/RNS are often generated by ischemia-induced inflammation; defining the extent of tissue involvement or ROS/RNS-related damage would have a significant clinical impact. We present the preparation and demonstration of a fluorogenic sensor for monitoring peroxynitrite (ONOO(-)) and myeloperoxidase (MPO) mediated hypochlorous acid (HOCl/OCl(-)) production. The sensor consists of a long circulating biocompatible nanoparticle that targets phagocytic cells in vivo and is coated with approximately 400 quenched oxazine fluorophores that are released by reaction with HOCl or ONOO(-) but are stable toward oxidants such as hydroxyl radical, hydrogen peroxide, and superoxide. MPO-dependent probe activation is chloride ion dependent and is negated in flow cytometry studies of MPO inhibitor treated neutrophils. Fluorescence reflectance imaging and microscopic fluorescence imaging in mouse hearts after myocardial infarction showed probe release into neutrophil-rich ischemic areas, making this ROS/RNS sensor a novel prognostic indicator.
Project description:Two novel drug-conjugates based on a "coumarin linker" have been designed for the synergic release of a therapeutic agent and fluorescent probe for the potential application of theranostics. The drug conjugates; CC-RNS and CI-RNS were designed to be activated by reactive oxygen species or reactive nitrogen species (ROS/RNS). The fluorescence OFF-ON response was triggered by the peroxynitrite-mediated transformation of a boronic acid pinacol ester to a phenol moiety with simultaneous release of the therapeutic agents (Confirmed by HRMS). The limit of detection for peroxynitrite using CC-RNS and CI-RNS was 0.29 and 37.2 ?M, respectively. Both CC-RNS and CI-RNS demonstrated the ability to visualize peroxynitrite production thus demonstrating the effectiveness of these probes for use as tools to monitor peroxynitrite-mediated drug release in cancer cell lines.
Project description:Developmental transitions and stress reactions in both eukaryotes and prokaryotes are tightly linked with fast and localized modifications in concentrations of reactive oxygen and nitrogen species (ROS and RNS). Fluorescent microscopic analyses are widely applied to detect localized production of ROS and RNS in vivo. In this mini-review we discuss the biological characteristics of studied material (cell wall, extracellular matrix, and tissue complexity) and its handling (concentration of probes, effect of pressure, and higher temperature) which influence results of histochemical staining with "classical" fluorochromes. Future perspectives of ROS and RNS imaging with newly designed probes are briefly outlined.
Project description:Photoacoustic imaging is attracting a great deal of interest owing to its distinct advantages over other imaging techniques such as fluorescence or magnetic resonance image. The availability of photoacoustic probes for reactive oxygen and nitrogen species (ROS/RNS) could shed light on a plethora of biological processes mediated by these key intermediates. Tetramethylbenzidine (TMB) is a non-toxic and non-mutagenic colorless dye that develops a distinctive blue color upon oxidation. In this work, we have investigated the potential of TMB as an acoustogenic photoacoustic probe for ROS/RNS. Our results indicate that TMB reacts with hypochlorite, hydrogen peroxide, singlet oxygen, and nitrogen dioxide to produce the blue oxidation product, while ROS, such as the superoxide radical anion, sodium peroxide, hydroxyl radical, or peroxynitrite, yield a colorless oxidation product. TMB does not penetrate the Escherichia coli cytoplasm but is capable of detecting singlet oxygen generated in its outer membrane.
Project description:<i>In situ</i> fluorescence imaging of nitric oxide (NO) is a powerful tool for studying the critical roles of NO in biological events. However, the selective imaging of NO is still a challenge because most currently available fluorescent probes rely on the <i>o</i>-phenylenediamine (OPD) recognition site, which reacts with both NO and some abundant reactive carbonyl species (RCS) (such as dehydroascorbic acid and methylglyoxal) and some reactive oxygen/nitrogen species (ROS/RNS). To address this problem, a new fluorescent probe, <b>NCNO</b>, based on the <i>N</i>-nitrosation of aromatic secondary amine was designed to bypass the RCS, ROS, and RNS interference. As was expected, the probe <b>NCNO</b> could recognize NO with pronounced selectivity and sensitivity among ROS, RNS, and RCS. The probe was validated by detecting NO in live cells and deep tissues owing to its two-photon excitation and red-light emission. It was, hence, applied to monitor NO in ischemia reperfusion injury (IRI) in mice kidneys by two-photon microscopy for the first time, and the results vividly revealed the profile of NO generation <i>in situ</i> during the renal IRI process.
Project description:Reactive oxygen species (ROS) production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state, a requirement for functional spermatozoa. However, an increase in oxidative stress, due to an abnormal production of ROS, has been shown to be related to loss of sperm function, highlighting the importance of an accurate detection of sperm ROS, given the specific nature of this cell. In this work, we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species (RNS) in human sperm, to define their specificity. Using both flow cytometry (FC) and fluorescence microscopy (FM), we confirmed that MitoSOX™ Red and dihydroethidium (DHE) detect superoxide anion (as determined using antimycin A as a positive control), while DAF-2A detects reactive nitrogen species (namely, nitric oxide). For the first time, we also report that RedoxSensor™ Red CC-1, CellROX® Orange Reagent, and MitoPY1 seem to be mostly sensitive to hydrogen peroxide, but not superoxide. Furthermore, mean fluorescence intensity (and not percentage of labeled cells) is the main parameter that can be reproducibly monitored using this type of methodology.
Project description:Here we report that ferricytochrome c (cyt c(3+)) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c(3+)-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E(+)-E(+), Mito-HE-Mito-HE, and Mito-E(+)-Mito-E(+)) and heterodimers (HE-E(+) and Mito-HE-Mito-E(+)), as well as the monomeric ethidium (E(+)) and mito-ethidium (Mito-E(+)). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c(3+) were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E(+) or Mito-E(+), the dimeric analogs (E(+)-E(+) and Mito-E(+)-Mito-E(+)) were not fluorescent. Superoxide (O(2)(*-)) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E(+)) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c(3+)-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.
Project description:Current drug-safety assays for hepatotoxicity rely on biomarkers with low predictive power. The production of radical species, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), has been proposed as an early unifying event linking the bioactivation of drugs to hepatotoxicity and as a more direct and mechanistic indicator of hepatotoxic potential. Here we present a nanosensor for rapid, real-time in vivo imaging of drug-induced ROS and RNS for direct evaluation of acute hepatotoxicity. By combining fluorescence resonance energy transfer (FRET) and chemiluminescence resonance energy transfer (CRET), our semiconducting polymer-based nanosensor simultaneously and differentially detects RNS and ROS using two optically independent channels. We imaged drug-induced hepatotoxicity and its remediation longitudinally in mice after systemic challenge with acetaminophen or isoniazid. We detected dose-dependent ROS and RNS activity in the liver within minutes of drug challenge, which preceded histological changes, protein nitration and DNA double-strand-break induction.
Project description:Chronic elevations in cellular redox state are known to result in the onset of various pathological conditions, but transient increases in reactive oxygen species (ROS)/reactive nitrogen species (RNS) are necessary for signal transduction and various physiological functions. There is a distinct lack of reversible fluorescent tools that can aid in studying and unraveling the roles of ROS/RNS in physiology and pathology by monitoring the variations in cellular ROS levels over time. In this work, we report the development of ratiometric fluorescent sensors that reversibly respond to changes in mitochondrial redox state.Photophysical studies of the developed flavin-rhodamine redox sensors, flavin-rhodamine redox sensor 1 (FRR1) and flavin-rhodamine redox sensor 2 (FRR2), confirmed the reversible response of the probes upon reduction and re-oxidation over more than five cycles. The ratiometric output of FRR1 and FRR2 remained unaltered in the presence of other possible cellular interferants (metals and pH). Microscopy studies indicated clear mitochondrial localization of both probes, and FRR2 was shown to report the time-dependent increase of mitochondrial ROS levels after lipopolysaccharide stimulation in macrophages. Moreover, it was used to study the variations in mitochondrial redox state in mouse hematopoietic cells at different stages of embryonic development and maturation.This study provides the first ratiometric and reversible probes for ROS, targeted to the mitochondria, which reveal variations in mitochondrial ROS levels at different stages of embryonic and adult blood cell production.Our results suggest that with their ratiometric and reversible outputs, FRR1 and FRR2 are valuable tools for the future study of oxidative stress and its implications in physiology and pathology. Antioxid. Redox Signal. 24, 667-679.