Exogenous ONOO Research Articles

Sensitive monitoring mitochondrial peroxynitrite based on a new reaction site and cell imaging by anthracycline-based red emitting fluorescence probe

The change in the level of ONOO− is usually indicative of an abnormality in bodily function. Therefore, there is a need to develop highly reliable ONOO− assays to determine its role in the biological environment. Thus, a long-wavelength anthracycline-based fluorescent probe (LAP) with a new reaction site was presented to detect peroxynitrite (ONOO−) by one step synthesize between 6-hydroxy-1-tetralone and salicylaldehyde. LAP responded to ONOO− about 8-fold fluorescence changes at 628 nm and the detection limit was 3.7 nM. The ONOO− sensing of LAP with the disruption of the conjugated skeleton was highly specific and could identify ONOO− by naked eyes. LAP was specifically localized to the mitochondria and co-localization results of LAP and MitoTrancker Green showed that the merged fluorescent images were observed with high Pearson's co-localization coefficient 0.92 and 0.90 in SMMC-7721 and RAW264.7 cells, respectively. As a specific and sensitive fluorescence probe, LAP with mitochondria-targetable moiety was ability to detect exogenous ONOO− in SMMC-7721 and RAW264.7 cells.

Reasonably constructed NIR fluorescent probes based on dicyanoisophorone skeleton for imaging ONOO− in living cells

Peroxynitrite (ONOO−), a reactive nitrogen species (RNS), engages in various cellular processes including inflammation, apoptosis, and necrosis. Therefore, the development of a powerful tool for imaging endogenous ONOO− is important with regard to revealing its underlying physiological or pathological functions. In this work, we introduced dihalogen (Cl, Br, and I) at the ortho-positions of the hydroxyl group of a dicyanoisophorone skeleton to adjust its pKa to be more suitable for bio-imaging in the physiological environments with a high signal-to-noise ratio. Based on the optimized spectral results and the requirement of biocompatibility, the fluorescent probe NIR-dCl was successfully designed and fabricated for the detection of ONOO−. Upon the addition of ONOO− in solution, an obvious NIR fluorescent signal at 680 nm was observed. Moreover, the probe NIR-dCl exhibited high selectivity for sensing ONOO−. Cell imaging results confirmed that probe NIR-dCl was capable of imaging exogenous and endogenous ONOO− in live cells. Thus, we believe that probe NIR-dCl could be an effective tool for studying biochemical processes and ONOO−-related diseases.

AIE-based fluorescent boronate probe and its application in peroxynitrite imaging

Fluorescent probes have contributed greatly to our understanding of the biological role of peroxynitrite (ONOO–). The ONOO– fluorescence probe characterized by the arlyboronate received a moderate opening fluorescence response, and the borate-masked probe significantly increased the sensitivity of ONOO–. Thus, two simple fluorescent probes (ADB and ANB) with the recognition receptor of phenyl boronate moiety were constructed for the detection of ONOO–. The change of emission spectrum was affected differently by the electron donating (or withdrawing) of the substituents. ANB was shown to have a low sensitivity and quantum yield towards ONOO– in aqueous solution, whereas ADB with aggregation-induced emission (AIE) process exhibited not only good sensitivity for ONOO– with a detection limit of 75 nM, but also ADB could be used to quantitative detecting ONOO– in response to concentrations of ONOO– within 20 s. Importantly, ADB had good performance for the detection of exogenous ONOO– in the RAW 264.7 cells.

A simple dual-response fluorescent probe for imaging of viscosity and ONOO− through different fluorescence signals in living cells and zebrafish

Cellular viscosity is a prominent micro-environmental parameter and peroxynitrite is an essential reactive oxygen special, both of which are involved in various pathological and physiological processes. When the intracellular viscosity is abnormal or the ONOO− concentration is irregular, the normal function of cells will be disturbed. Herein, we rationally designed and synthesized a novel multichannel fluorescent probe (probe 1) for multichannel imaging of viscosity and peroxynitrite. Probe 1 displayed about 108-fold enhancement as the viscosity increased from 1.005 cP to 1090 cP. Moreover, the fluorescence intensity at 540 nm was quickly increased after adding ONOO−. It should be noted that probe 1 has high sensitivity, selectivity and low cytotoxicity, which can be successfully employed for the visualization of exogenous and endogenous ONOO− and imaging viscosity changes in HeLa cells by different fluorescent signals. Furthermore, probe 1 could monitor the change of ONOO− induced by LPS (lipopolysaccharide) and IFN-γ (interferon-γ) in zebrafish. This result reveals that probe 1 may inspire more diagnostic and therapeutic programs for viscosity-peroxynitrite related diseases shortly.

A chromene based fluorescence probe: Accurate detection of peroxynitrite in mitochondria, not elsewhere

• A novel fluorescent probe was synthesized for the detection of ONOO − by conjugating dicyano-vinyl with benzopyran-chromone. • The probe shows specifically selectivity over other biologically relevant ROS/RNS/RSS. • The sensing mechanism is the cleavage of the carbon-carbon double bond by the nucleophilicity and oxidation of peroxynitrite. • The probe has a certain mitochondrial targeting and localization effect. • BC-PN-2 can accurately identify ONOO − in mitochondria by imaging of exogenous ONOO − in HepG2 cells. A novel fluorescent probe (BC-PN-2) for the detection of ONOO − was synthesized by conjugating dicyano-vinyl with benzopyran-chromone. The dicyano-vinyl group can be destructed by ONOO − to generate an aldehyde group resulting in strong fluorescence. Compared with the general fluorescence probes, a novel reactive site to detect ONOO − in mitochondria is used in this probe and the results show short response time, high sensitivity and excellent selectivity toward ONOO − among reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PBS. Subsequent fluorescence imaging shows the high membrane permeability, a certain mitochondrial targeting and localization effect of the probe. In addition, the product BC-aldehyde cannot permeate through the membrane. Meanwhile, BC-PN-2 can accurately identify ONOO − in mitochondria by imaging of exogenous ONOO − in HepG2 cells and prove detected ONOO − are from mitochondria.

Highly Sensitive Near-Infrared Imaging of Peroxynitrite Fluxes in Inflammation Progress.

Inflammation is an important protection reaction in living organisms associated with many diseases. Since peroxynitrite (ONOO-) is engaged in the inflammatory processes, illustrating the key nexus between ONOO- and inflammation is significant. Due to the lack of sensitive ONOO- in vivo detection methods, the research still remains at its infancy. Herein, a highly sensitive NIR fluorescence probe DDAO-PN for in vivo detection of ONOO- in inflammation progress was reported. The probe responded to ONOO- with significant NIR fluorescence enhancement at 657 nm (84-fold) within 30 s in solution. Intracellular imaging of exogenous ONOO- with the probe demonstrated a 68-fold fluorescence increase (F/F0). Impressively, the probe can in vivo detect ONOO- fluxes in LPS-induced rear leg inflammation with a 4.0-fold fluorescence increase and LPS-induced peritonitis with an 8.0-fold fluorescence increase The remarkable fluorescence enhancement and quick response enabled real-time tracking of in vivo ONOO- with a large signal-to-noise (S/N) ratio. These results clearly denoted that DDAO-PN was able to be a NIR fluorescence probe for in vivo detection and high-fidelity imaging of ONOO- with high sensitivity and will boost the research of inflammation-related diseases.

An easily available ratiometric AIE probe for peroxynitrite in vitro and in vivo imaging

Peroxynitrite (ONOO−), a potent oxidative and nitrating reagent that can damage a variety of molecules in cells is reported to be closely related to various diseases. To determine the physiological or pathophysiological concentrations of ONOO− and to deeply understand its pathophysiological processes strongly rely on the development of more powerful detection methods. Hereof, we fabricated the first ratiometric fluorescent probe by using aggregation-induced emission luminogens (AIEgens) with superior photostability for efficient and reliable visualization of ONOO− both in vitro and in vivo. Probe BTCV-PN, obtained via simple two-step process, displayed superior photostability, remarkable sensitivity and outstanding specificity toward ONOO− through a ratiometric response mode. Furthermore, BTCV-PN was successfully applied for imaging of exogenous and endogenous ONOO− in live cells. Notably, in vivo imaging of ONOO− in live mice was also accomplished. The excellent performances plus the superior parameters of BTCV-PN including reliable ratiometric response and superior stability against photobleaching would not only make BTCV-PN a powerful tool-box for facilely investigating the cellular concentrations of ONOO−, but also open up an updated way for straightforward visualization of the pathophysiological process of ONOO− in live cells and animals. Elsevier B.V. All rights reserved.

Nanoliposomal Ratiometric Fluorescent Probe toward ONOO- Flux.

Peroxynitrite (ONOO-), a powerful biological oxidant, is produced in the mitochondria and reacts with many biomolecular targets under various pathological conditions, leading to a range of disease states. In this work, we developed a nanoliposome-encapsulated ratiometrically fluorescent probe (NRF) based on a hemicyanine structure Cy-O obtained by facile synthesis. Upon reaction with ONOO-, the oxidation and hydrolysis of a π-conjugation system within the nanoliposome triggers a ratiometrically fluorescent response and a large-scale emission shift (238 nm), which provides a specific and sensitive means for the ONOO- detection. Moreover, we have performed DFT calculation at the 6-31+G(d,p) level using a suite of Gaussian 09 programs to obtain insights into the chemical structure optical properties of Cy-O. In addition, the practical applications of the nanoprobe to image exogenous and endogenous ONOO- were achieved further in live cells and animals triumphantly.

A novel ultrasensitive peroxynitrite-specific fluorescent probe and its bioimaging applications in living systems

Peroxynitrite, one of the well-known reactive nitrogen species (RNS), which is considered to be an important oxidant. Besides, peroxynitrite plays an important role in the broad spectrum of biological processes, and it is also a vital participant in the emergence of cancer. To this end, the fluorescent probe PN-1 was rationally synthesized, which contains a coumarin-fused coumarin dye and borates as the specific recognition group. Probe PN-1 exhibits several obvious performances, for instance, high specificity, excellent sensitivity, and it could promptly respond toward ONOO−. Using this simple fluorescent probe, we achieved the imaging of endogenous and exogenous ONOO− fluctuations in living cells and zebrafish. Moreover, PN-1 has efficiently differentiated cancerous cells from normal cells owing to image their different native ONOO− levels, these results indicated that PN-1 is expected to be an analytical tool, for researching ONOO− related cancer biology and studying the related effect of ONOO− in food.

Novel near-infrared fluorescence probe with large Stokes shift for monitoring CCl4-induced toxic hepatitis

Toxic hepatitis which is induced by chemical substance is a serious threat to human health. More and more studies have shown that peroxynitrite (ONOO−) is related with the development of toxic hepatitis. So it is important to find a tool to study ONOO− change during the diagnosis and therapy of toxic hepatitis. Herein, a series of novel near-infrared (NIR) fluorescence dyes (DDM-R) with long emission wavelength (740–770 nm) and large Stokes shift (~200 nm) are developed. Among the dyes, DDM-OH with great spectral performance and facilely modified feature is used to construct probe DDM-ONOO-. The probe have the preference of high sensitivity and excellent selectivity for ONOO−. In addition, DDM-ONOO- was applied in detecting exogenous and endogenous ONOO− in cells and further used in detecting ONOO− of CCl4-induced toxic hepatitis in cells by fluorescence imaging, 3D quantification analysis, flow cytometry. More importantly, by visualizing ONOO−, the probe was used to monitor the diagnosis of CCl4-induced toxic hepatitis in mice and evaluate the therapeutic efficacy of hepatoprotective medicines (NAC, SM, DDB). The results show that the probe will provide a powerful tool for the diagnosis and treatment of toxic hepatitis.

A Simple Near-Infrared Fluorescent Probe for the Detection of Peroxynitrite.

Herein, we report the evaluation and synthesis of a reaction based fluorescent probe DCM‐Bpin for the detection of Peroxynitrite (ONOO−). DCM‐Bpin exhibits selective fluorescence off‐on response for ONOO− over other reactive oxygen species, including H2O2. Moreover, DCM‐Bpin is biocompatible and has been used to visualize exogenous ONOO− in HeLa cells.

A highly selective fluorescent probe for monitoring exogenous and endogenous ONOO− fluctuations in HeLa cells

As a typical reactive oxygen species, peroxynitrite (ONOO−) plays vital roles in various biological functions and pharmacological activities. Specific, sensitive and rapid fluorescent probes are helpful tools for monitoring endogenous peroxynitrite. In this work, a highly selective fluorescent probe BTCBE was designed based on benzothiazole-2-acetonitrile and 4-formylphenylboronic acid pinacol cyclic ester. BTCBE showed the merits of rapid response (<1 min) and prominent sensitivity (LOD = 4.7 nM). It showed an excellent selectivity towards ONOO− over NaClO and H2O2. Moreover, BTCBE was successfully used for bioimaging exogenous and endogenous ONOO− in HeLa cells.

H2 O2 -induced microvessel barrier dysfunction: the interplay between reactive oxygen species, nitric oxide, and peroxynitrite.

Elevated H2O2 is implicated in many cardiovascular diseases. We previously demonstrated that H2O2‐induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO‐mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO‐derived peroxynitrite (ONOO−) in H2O2‐induced vascular barrier dysfunction by elucidating the interrelationships between H2O2‐induced NO, superoxide, ONOO−, and changes in endothelial [Ca2+]i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+]i, NO, and O2 − were assessed with fluorescence imaging. Perfusion of vessels with H2O2 (10 µmol/L) induced marked productions of NO and O2 −, resulting in extensive protein tyrosine nitration, a biomarker of ONOO−. The formation of ONOO− was abolished by inhibition of NOS with NG‐Methyl‐L‐arginine. Blocking NO production or scavenging ONOO− by uric acid prevented H2O2‐induced increases in endothelial [Ca2+]i and Lp. Additionally, the application of exogenous ONOO− to microvessels induced delayed and progressive increases in endothelial [Ca2+]i and microvessel Lp, a pattern similar to that observed in H2O2‐perfused vessels. Importantly, ONOO− caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO‐derived ONOO− is essential for H2O2‐induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self‐promoted amplifications of NO‐dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species‐mediated vascular dysfunction in cardiovascular diseases.

A novel hexahydropyridazin-modified rhodamine fluorescent probe for tracing endogenous/exogenous peroxynitrite in live cells and zebrafish

As a reactive nitrogen species (RNS), peroxynitrite (ONOO−) is known for its crucial functions in biological systems. Excessive formation of ONOO− is very closely related to the pathogenesis of a series of diseases, and thus, effective methods are needed for highly sensitive detection of ONOO−. Herein, a simple fluorescent probe RHHP-PN was proposed to detect ONOO−, and manifested obvious color changes from colorless to red in less than 10 s. Besides, it is not only suitable for quantitative detection of ONOO−, but also satisfactory in distinguishing ONOO− from other related species. What is particularly noteworthy is the fluorescence bioimaging for tracing endogenous and exogenous ONOO− in living cells and zebrafish. Therefore, probe RHHP-PN could be used as an imaging tool to visualize intracellular ONOO− in biological systems.

Novel Dimethylhydrazine-Derived Spirolactam Fluorescent Chemodosimeter for Tracing Basal Peroxynitrite in Live Cells and Zebrafish.

The precise cellular function of peroxynitrite (ONOO-) in biosystems remains elusive, primarily owing to being short of ultrasensitive techniques for monitoring its intracellular distribution. In this work, a novel rhodamine B cyclic 1,2-dimethylhydrazine fluorescent chemodosimeter RDMH-PN for highly specific and ultrasensitive monitoring of basal ONOO- in biosystems was rationally designed. The fluorescence titration experiments demonstrated that RDMH-PN was capable of quantitatively detecting 0-100 nM ONOO- (limit of detection = 0.68 nM). In addition, RDMH-PN has outstanding performances of ultrafast measurement, naked-eye detection, and preeminent selectivity toward ONOO- to accurately detect intracellular basal ONOO-. Finally, it has been confirmed that RDMH-PN could not only map the intracellular basal ONOO- level by inhibition tests but also trace the fluctuations of endogenous and exogenous ONOO- levels with diverse stimulations in live cells and zebrafish.

A highly selective and sensitive red-emitting fluorescent probe for visualization of endogenous peroxynitrite in living cells and zebrafish.

Peroxynitrite (ONOO-) has been proven to participate in various physiological and pathological processes, and may also be a contributing factor in many diseases. In view of this, there is a need to develop detection tools for unambiguously tracking a small amount of endogenous ONOO- to reveal its exact mechanisms. In this paper, a colorimetric and red-emitting fluorescent probe Red-PN, based on a rhodamine-type fluorophore and hydrazide reactive site is described. The probe Red-PN possesses the advantages of rapid response (within 5 s), visual color change (from colorless to pink), preeminent sensitivity (detection limit = 4.3 nM) and selectivity. Because of these outstanding performances, it was possible to accurately detect endogenous ONOO-. It was encouraging that the probe Red-PN could be used effectively for tracking the relatively low levels of endogenous and exogenous ONOO- in living cells and zebrafish. Thus, it is envisioned that the probe Red-PN would have promising prospects in applications for imaging ONOO- in a variety of biological settings.

Effect of Peroxynitrite (ONOO&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt;) on the Function of Murine Perivascular Adipose Tissue

Perivascular adipose tissue (PVAT) surrounds the exterior of blood vessels and releases numerous substances such as adiponectin which positively modulate blood vessel tone. In some cardiovascular diseases such as diabetes and high blood pressure, the function of PVAT changes and we speculate that oxidant stress may play a role in this change. PVAT has the ability to generate both superoxide and nitric oxide and these can combine rapidly under physiological conditions to form peroxynitrite (ONOO-). In disease states, the production of ONOO- may be increased and so its effect on the function of PVAT is of great interest. Consequently, we studied the effects of acute addition of the oxidant species ONOO- on vascular tone and production of adiponectin by mouse thoracic aortic PVAT. Murine PVAT was immunostained for nitrotyrosine, indicating that ONOO- is formed in the PVAT. Exogenous ONOO- significantly increased the anticontractile effect of PVAT via increased adiponectin content but had no effect on eNOS expression or phosphorylation. These results suggest that generation of ONOO- within PVAT may be an important regulatory mechanism which influences the activity of PVAT. The effect of chronic exposure to raised levels of ONOO- on PVAT function remains to be determined.

A ratiometric fluorescent probe for the detection of peroxynitrite with simple synthesis and large emission shift and its application in cells image

Peroxynitrite (ONOO−) is an important kind of reactive oxygen species (ROS), played an essential role in physiology and pathology. To date, various fluorescent probes for ONOO− have been constructed successfully, but these probes exhibit turn-on fluorescence signal in single channel. While, ratiometric fluorescent probes, especially those probes with large emission shift, are suitable for bioimage in two channels. Herein, a hemicyanine-based fluorescent probe named Cy-NH2 was designed and synthesized for ratiometric detection of ONOO−. The characteristics of Cy-NH2 are as follows: (1) The probe possesses a simple structure, which can be obtained by easy synthetic steps; (2) The ratiometric fluorescent probe owns large emission shift (248 nm) due to the disruption of π-conjugation system caused by strong oxidizing ability of ONOO−; (3) The probe exhibits high sensitivity to ONOO− with 1728-fold enhancement of fluorescence intensity ratio and superior selectivity for ONOO− over various potentially interfering biological analytes; (4) The ratiometric fluorescent probe is applied for the image detection of exogenous and endogenous ONOO− in living cells successfully.

A ratiometric fluorescent probe based on a coumarin–hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite

In the study described herein, the red emitting probe CHCN, which possesses a linked coumarin–hemicyanine scaffold, was developed for detection of peroxynitrite (ONOO−) under physiological conditions. The studies show that CHCN displays a dual ratiometric and colorimetric response to ONOO− that is caused by an oxidation process. A possible mechanism of this oxidation process was proposed and confirmed by ESI-MS spectra for the first time. CHCN shown highly selective and sensitive towards ONOO− with a low limit of detection LOD (49.7nM). Moreover, CHCN has appreciable cell permeability and, as a result, it is applicable to ratiometric detection of exogenous and endogenous ONOO− in living cells during phagocytic immune response. We anticipate that, owing to their ideal properties, probes of this type will find great use in explorations of the role played by ONOO− in biology.

Peroxynitrite disrupts endothelial caveolae leading to eNOS uncoupling and diminished flow‐mediated dilation in coronary arterioles of diabetic patients (1082.3)

Peroxynitrite (ONOO‐) contributes to coronary microvascular dysfunction in diabetes (DM). We hypothesized that in DM ONOO‐ interferes with the function of coronary endothelial caveolae, which plays an important role in nitric‐oxide (NO)‐dependent vasomotor regulation. Flow‐mediated dilation (FMD) of coronary arterioles was investigated in DM (n=41) and nonDM (n=39) patients undergoing heart surgery. NO‐mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO‐ scavenger, uric‐acid, whereas exogenous ONOO‐ reduced FMD in nonDM subjects. Immuno‐electronmicroscopy demonstrated an increased 3‐nitrotyrosine formation (ONOO‐ specific protein nitration) in endothelial plasma membrane in DM, which co‐localized with caveolin‐1 (Cav‐1), the key structural protein of caveolae. The membrane‐localized Cav‐1 was significantly reduced in DM and also in high glucose‐exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO‐ reduced caveolae number. Correspondingly, pharmacological (methyl‐β‐cyclodextrin) or genetic disruption of caveolae (Cav‐1‐knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM. Thus, we propose that ONOO‐ selectively targets and disrupts endothelial caveolae, which leads to NO synthase uncoupling, hence reduced NO‐mediated coronary vasodilation in DM patients.Grant Funding Source: Supported by NIH HL104126