Mitochondrial Peroxynitrite Research Articles

Naphthylamine-based ratiometric fluorescence probe with a large Stokes shift for monitoring mitochondrial peroxynitrite

The design of ratiometric fluorescence probes with large Stokes shift is greatly significant in addressing the major challenges of detecting and imaging target substances in complex organisms. Such probes with a self-calibration function can minimize the interference of probe concentration and excitation light. In this study, a novel fluorophore of NA-Py with large Stokes shift of 158 nm, was easily obtained in one step. As a proof of concept, based on NA-Py, a fluorescence probe NA-BOH with strong intramolecular charge transfer (ICT) structural characteristics is designed for detecting peroxynitrite (ONOO−). NA-BOH exhibits red emission (627 nm) and a larger Stokes shift of 178 nm. Furthermore, it demonstrats rapid response (within 20 min) and high selectivity towards ONOO−, with ratiometric quantitative response (0–20 μM), and high sensitivity (182 nM). Alongside excellent biocompatibility and remarkable mitochondrial targeting ability, NA-BOH is successfully utilized for the proportional imaging of ONOO− in living cells and zebrafish.

Formation and Decomposition of the Mn(II)P-NO Complex In Vitro: Potential Role in Mitochondrial Peroxynitrite Formation

Formation and Decomposition of the Mn(II)P-NO Complex In Vitro: Potential Role in Mitochondrial Peroxynitrite Formation

Coumarin-cyanine hybrid: A ratiometric fluorescent probe for accurate detection of peroxynitrite in mitochondria

There is an urgent need to develop highly sensitive and selective fluorescence probes for ONOO− in mitochondria. Herein, we reported a ratiometric fluorescent probe COUS with coumarin-cyanine hybrid as fluorophore and C = C bonds as reaction sites of ONOO−. The probe COUS was sensitive and selective to ONOO−, and had a large fluorescence emission shift (239 nm) as well as a low detection limit (41.88 nM). Moreover, COUS showed the mitochondrial targeting ability, and the targeting moiety could dissociate from the probe when reacting with ONOO−, which enabled COUS to accurately detect ONOO− in mitochondria.

A large Stokes shift NIR fluorescent probe for visual monitoring of mitochondrial peroxynitrite during inflammation and ferroptosis and in an Alzheimer's disease model.

The excessive formation of peroxynitrite (ONOO-) in mitochondria has been implicated in various pathophysiological processes and diseases. However, owing to short emission wavelengths and small Stokes shifts, previously reported fluorescent probes pose significant challenges for mitochondrial ONOO- imaging in biological systems. In this study, a near-infrared (NIR) fluorescent probe, denoted as DCO-POT, is designed for the visual monitoring of mitochondrial ONOO-, displaying a remarkable Stokes shift of 170 nm. The NIR fluorophore of DCO-CHO is released by DCO-POT upon the addition of ONOO-, resulting in off-on NIR fluorescence at 670 nm. This phenomenon facilitates the high-resolution confocal laser scanning imaging of ONOO- generated in biological systems. The practical applications of DCO-POT as an efficient fluorescence imaging tool are verified in this study. DCO-POT enables the fluorometric visualization of ONOO- in organelles, cells, and organisms. In particular, ONOO- generation is analyzed during cellular and organism-level (zebrafish) inflammation during ferroptosis and in an Alzheimer's disease mouse model. The excellent visual monitoring performance of DCO-POT in vivo makes it a promising tool for exploring the pathophysiological effects of ONOO-.

Imaging of peroxynitrite in mitochondria by a near-infrared fluorescent probe with a large Stokes shift

As a result of large imaging depth, low background biofluorescence, facile operation, and high tempo-spatial resolution, the small-molecule near-infrared (NIR) fluorescence imaging technique has been one of the most popular tools for in situ monitoring of cellular oxidative stress (OS) processes. Nevertheless, the recent near infrared probes have low signal-to-noise ratio due to the small stokes shift. To this end, this work quinone-cyanine-7 (QCy7, λmaxabs = 582 nm, ε = 9329 M−1 cm−1 and λmaxem = 719 nm, Ф = 0.12) with large Stokes shift (137 nm) was used as a fluorophore, and 1-methylindoline-2,3-dione was used as the “Turn-ON” cyanine NIR Peroxynitrite (ONOO−) fluorescent probe HJ-ONOO-P3 (with 41-fold fluorescence enhancement with ONOO− concentration in the range of 0–35 μM). The indole quaternary ammonium heterocycle with a positive charge itself could be used as the targeted group for mitochondria. This probe exhibited a rapid response (5 min complete response) and high selectivity and sensitivity to ONOO− (detection limit as low as 25.4 nM). Meanwhile, this probe could achieve the task of in-situ imaging of OS during cellular oxygen-glucose deprivation/reperfusion (OGD/R) and middle cerebral artery occlusion (MCAO) in mice. The results demonstrated that the mitochondria-targeted NIR probe with a large Stokes shift possess great potential in studies of stroke and the induced cellular OS.

Photocontrollable Fluorescence Imaging of Mitochondrial Peroxynitrite during Ferroptosis with High Fidelity.

Ferroptosis, a new regulatory cell death modality, underlies the pathogenesis of a broad range of disorders. Although much efforts have been made to uncover the molecular mechanisms, some mechanistic details of ferroptosis still remain poorly understood. Particularly, the functional relevance of mitochondrial reactive oxygen species (ROS) in ferroptosis is still highly controversial, which is partially due to the fact that it still remains puzzled how the mitochondrial ROS level varies during ferroptosis. The conventional mitochondria-targeted probes may react with cytosolic ROS and show fluorescence variation before entering mitochondria, thus probably giving a false result on the mitochondrial ROS level and leading to the misjudgment on its biofunction. To circumvent this issue, we rationally designed a photocontrollable and mitochondria-targeted fluorescent probe to in situ visualize the mitochondrial peroxynitrite (ONOO-), which is the ROS member and mediator of ferroptosis. The photoactivated probe was endowed with a highly specific and sensitive fluorescence response to ONOO-. Notably, the response activity could be artificially regulated with light irradiation, which ensured that all the probe molecules passed through the cytosol in the locked status and were then photoactivated after reaching mitochondria. This photocontrolled fluorescence imaging strategy eliminated the interference of ONOO- outside the mitochondria, thus potentially afforded improved fidelity for mitochondrial ONOO- bioimaging in live cells and animal models. With this probe, for the first time, we revealed the mitochondrial ONOO- flux and its probable biological source during erastin-induced ferroptosis. These results suggest a tight correlation between mitochondrial ONOO-/ROS and ferroptotic progression, which will further facilitate the comprehensive exploration and manipulation of ferroptosis.

A deep red ratiometric fluorescent probe for accurate detection of peroxynitrite in mitochondria

Peroxynitrite (ONOO−) is widespread within living organisms and has been implicated in many physiological and pathological processes. Since ONOO− is mainly produced in mitochondria, accurate detection of ONOO− in mitochondria can help us understand its specific mechanism of action in the organism. Rather than single-wavelength emissive mitochondrial probes, ratiometric fluorescent probes with longer emission wavelength, large emission shift, and specific mitochondrial targeting properties are more likely to obtain a more accurate ONOO− content in mitochondria. To further avoid the interference by cytoplasmic ONOO−, we constructed a fluorescent probe MXMP with deep red emission and ratio properties, and it will be forbidden to enter the mitochondria after oxidation. In addition to its excellent selectivity and sensitivity, it shifted its fluorescence emission by up to 130 nm, with a detection limit of 84 nM.

Phosphinate–benzoindocyanin fluorescent probe for endogenous mitochondrial peroxynitrite detection in living cells and gallbladder access in inflammatory zebrafish animal models

Potent oxidants such as peroxynitrite (ONOO−) play important roles in the regulation of different physiopathological processes; their overproduction is thought to potentially cause several diseases in living organisms. Hence, the precise and selective monitoring of ONOO− is imperative for elucidating its interplay and roles in pathological and physiological processes. Herein, we present a novel diphenyl phosphinate-masked benzoindocyanin “turn-on” fluorogenic probe to help detect mitochondrial ONOO− in living cells and zebrafish models. A pale yellow color solution of BICBzDP turns rose-red upon the addition of ONOO−, selectively, contrary to that of other competitive bioactive molecules. BICBzDP displays an ultra-sensitivity detection limit (47.8 nM) with outstanding selectivity and sensitivity towards mitochondrial ONOO− and possesses a notable 68-fold fluorescence enhancement involving a large redshift of 91 nm. Importantly, further biological experimental investigations with BICBzDP indicate specific sensitivity and reliability of the probe to track the ONOO− level, not only in live cells, but also demonstrates dynamic fluctuations in the inflammatory zebrafish animal models. Thus, BICBzDP could be employed as a future potential biological tool for exploiting the role of ONOO− in a variety of different physiological systems.

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.

Rational construction of a novel ratiometric far-red fluorescent probe with excellent water solubility for sensing mitochondrial peroxynitrite

Peroxynitrite (ONOO−) is closely associated with various pathophysiological processes and mainly produced within the mitochondria. Therefore, developing an efficient method for mitochondrial ONOO− detection is crucial for better understanding of its functions. In view of the advantages of ratiometric fluorescent probes and far-red/NIR fluorescent probes, a kind of novel mitochondrion-targeting ratiometric far-red fluorescent probe (ANI-DP) was reasonably constructed by employing isophorone derivative as fluorescence group, diphenyl phosphinate as response group, and pyridinium cation as mitochondria-targetable group. Probe ANI-DP displayed a distinctive ratiometric fluorescence response toward ONOO− with a red-shifted emision in far-red to NIR region owing to the cleavage of a phosphinate group and change of intramolecular charge transfer (ICT) efficiency induced by ONOO−. In 100 % aqueous solution, probe ANI-DP was capable of quantitatively detecting ONOO− levels ranging from 0 to 10 μM with a low detection limit of 13.3 nM, and showed high specificity for ONOO− over other biological relevant substances. Besides, it featured large Stokes shift (202 nm), high photostability, low cytotoxicity as well as good mitochondria targetable ability. Encouraged by the outstanding properties described above, it was successfully applicated for ratiometric fluorescence imaging of ONOO− in mitochondria of living cells as well as visualization of ONOO− in zebrafish, which indicated that it holds great potential for revealing the functions of ONOO− in living biosystems.

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.

A water-soluble boronate masked benzoindocyanin fluorescent probe for the detection of endogenous mitochondrial peroxynitrite in live cells and zebrafish as inflammation models

Endogenous peroxynitrite a highly reactive oxidant plays significant roles in various physiopathological processes in living organisms. Therefore, sensing and monitoring of peroxynitrite with accurate detection specifically, is an immense challenge. To address this issue, we synthesized a water soluble, mitochondria-targeting “turn-on” fluorescent probe (BICBzBF) for the detection of mitochondrial peroxynitrite. BICBzBF (pale yellow) demonstrated high sensitivity and selectivity in fluorometric tracing of peroxynitrite over other ROS/RNS tested. This arylboronate-masked probe responds to peroxynitrite, resulting in the corresponding phenol derivative; a strong fluorescence accompanied a change to red rose as observed by the naked eye. The probe responds to peroxynitrite within <120 s, shows a lower nanomolar detection limit (60.5 nM), exhibits a remarkable ca. 32-fold intense increase. Taking these features fully into account, BICBzBF was evaluated in how it monitors peroxynitrite exogenously and endogenously. Moreover, BICBzBF successfully traced ONOO− in inflammatory living zebrafish animal model which are chemically induced for inflammation. The results obtained in both exogenous and endogenous studies demonstrate that BICBzBF actively detects ONOO−; major accumulation in retina and intestine indicate BICBzBF could serve as a potential molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts.

The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

Activity-Based Fluorescent Molecular Logic Gate Probe for Dynamic Tracking of Mitophagy Induced by Oxidative Stress.

Visualizing and modulating the mitophagy process is essential for understanding the role of mitophagy in cellular homeostasis, physiology, and pathology. To overcome the sensing limitation of available mitophagy probes to only lysosome fusion or degradation, a molecular logic gate probe showing multiple fluorescence responses to different mitophagy stages was proposed in this study to sense the oxidative stress-induced mitophagy via a dual-channel mode. This new fluorescent molecular logic gate probe, Mito-PN, was composed by integrating a peroxynitrite-responsive 1,8-naphthalimide with an acidity-activatable rhodamine spirolactam and possesses the mitochondria-targeting capability due to its triphenylphosphonium group. This probe is able to sense both the mitophagy initiation triggered by peroxynitrite and lysosome fusion at different fluorescence wavelengths. It can be rapidly activated by mitochondrial peroxynitrite to turn on the green fluorescence of naphthalimide, and subsequent lysosome/mitophagosome fusion activates the probe with protons to generate red fluorescence. Moreover, our preliminary results demonstrate that the fluorescence response of Mito-PN to peroxynitrite-induced mitophagy can be discriminated from the mitophagy stimulated by carbonyl cyanide m-chlorophenyl hydrazone, which further proves the specific mitophagy tracking ability of Mito-PN. Overall, this research offers a potentially powerful tool for studying the role played by peroxynitrite in mitophagy and provides a versatile strategy for monitoring oxidative stress-related pathological processes.

ICT-based fluorescent ratiometric probe for monitoring mitochondrial peroxynitrite in living cells

Mitochondria-targeted near-infrared fluorescent probe for the detection of peroxynitrite and the bioimaging of peroxynitrite in cells.

An ICT-based fluorescent probe for ratiometric monitoring the fluctuations of peroxynitrite in mitochondria

Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) which closely involves in various physiological and pathological processes. It is reported that mitochondria are the major site of ONOO− production. Normal level of ONOO− benefits mitochondria biological functions, while its overproduction will induce oxidative stress, resulting in an imbalance of mitochondria homeostasis. Therefore, accurate monitoring of ONOO− in mitochondria will be beneficial for elucidating the physiological metabolism and fluctuations of ONOO−. Herein, a novel mitochondria-targeted ratiometric fluorescent probe Mito-NA for ONOO− was designed rationally by regulating its intramolecular charge transfer (ICT) effect. Once reaction with ONOO−, Mito-NA manifested remarkable ratiometric fluorescence changes (18-fold) with a large red-shift (∼104 nm) emission, which was ascribed to the enhanced ICT process caused by the elimination of the electron-withdrawing recognition group 3-(trifluoromethyl) cinnamic acid and the exposure of electron-donating hydroxyl on Mito-NA structure. Furthermore, Mito-NA exhibited satisfactory performances for ONOO− such as good sensitivity (LOD =0.12 μM), high selectivity to ONOO− over other ROS, fast response and good mitochondrial targeting. More importantly, Mito-NA was successfully applied for ratiometric imaging and monitoring the fluctuations of mitochondrial ONOO− in living cells.

A novel pyridinium-based fluorescent probe for ratiometric detection of peroxynitrite in mitochondria.

Peroxynitrite (ONOO-) is a primary kind of reactive oxygen species. Excessive ONOO- can induce oxidative damage to biomolecules and further results in various diseases. So, quantitative monitoring ONOO- with excellent selectivity and sensitivity is imperative for elucidating its role in biological processes. In this study, a novel pyridinium fluorescent ONOO- probe (CPC) has been constructed base on ICT-modulated by combining coumarin fluorophore and diphenylphosphinate recognition group. The fluorescence response of CPC for ONOO- is realized via the removal of diphenylphosphinate group. The probe CPC shows prominent features for detection of ONOO- including fast response rate (within 3 min), excellent selectivity and sensitivity, distinct colorimetric (red to green), and a large emission wavelength shift (105 nm). The emission intensity ration (I538/I643) exhibits 153-fold enhancement along with the increasing ONOO- and the detection limit is as low as 1.60 × 10-8 M. These good response properties make CPC possible to quantitative detection of ONOO- concentration. By using the strategy, the ratiometric CPC has been employed to detection of mitochondrial ONOO- in live cell successfully.

Rapid peroxynitrite reduction by human peroxiredoxin 3: Implications for the fate of oxidants in mitochondria

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Mitochondrial Peroxynitrite Mediation of Anthracycline-Induced Cardiotoxicity as Visualized by a Two-Photon Near-Infrared Fluorescent Probe.

Anthracyclines rank among the most efficacious anticancer medications. However, their clinical utility and oncologic efficacy are severely compromised by the cardiotoxicity risk facing the early-diagnosis difficulty and their unclear molecular mechanism. Herein, a two-photon-excitable and near-infrared-emissive fluorescent probe, TPNIR-FP, was fabricated and endowed with extraordinary specificity and sensitivity and a rapid response toward peroxynitrite (ONOO-), as well as mitochondria-targeting ability. With the aid of TPNIR-FP, we demonstrate that mitochondrial ONOO- is upregulated in the early stage and contributes to the onset and progression of anthracycline cardiotoxicity in cardiomyocyte and mouse models; therefore, it represents an early biomarker to predict subclinical cardiotoxicity induced by drug challenge. Furthermore, TPNIR-FP is proved to be a robust imaging tool to provide critical insights into drug-induced cardiotoxicity and other ONOO--related pathophysiological processes.

Imaging viscosity and peroxynitrite by a mitochondria-targeting two-photon ratiometric fluorescent probe

The irregular viscosity and peroxynitrite (ONOO–) concentration in mitochondria would result in mitochondria disorders. However, fluorescent probes that can detect both of viscosity and peroxynitrite have not been explored. For this reason, a two-photon fluorescent molecule TP-1Bz was designed and synthesized in this work for the dual-ratiometric-imaging of viscosity and exogenous and endogenous ONOO– in mitochondria. Results indicated that TP-1Bz has two emissions at 407 nm and 650 nm respectively, their ratio (I650/I407) showed linear relationship to the viscosity of the media in the logarithmic plot, and its two-photon action cross-section (Φσ) at 880 nm was increased from 1.2 GM in aqueous solution to be 86.3 GM (72-fold) in 99% glycerol. With a good targeting capability to mitochondria, TP-1Bz allowed us to measure the average viscosity in the mitochidria of HepG2 cells (73.3 cp) and the mitochondria viscosity change upon the addition of ionpores. TP-1Bz also showed the high sensitivity to ONOO– (LOD close to 12.1 nM) in water/glycerol (2/3) solvent as well as the high selectivity over other ROS. With the advantages of its lower cytotoxicity, pH- and photo- stabilities, TP-1Bz was successfully employed for imaging ONOO– in living cells and even in tissue samples of rat liver through the monitoring the emission intensities of the blue and red channels. As the first example of two-photon ratiometric fluorescent probe for dual-imaging viscosity and peroxynitrite in mitochondria, TP-1Bz has great potential to be developed for clinical applications.