Characterization of Fluorescein-Based Monoboronate Probe and Its

Apr 15, 2016 - Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Tre...
5 downloads 9 Views 2MB Size
Download PDF
Article pubs.acs.org/crt

Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin Karolina Dębowska,† Dawid Dębski,† Bartosz Michałowski,† Agnieszka Dybala-Defratyka,† Tomasz Wójcik,‡ Radosław Michalski,† Małgorzata Jakubowska,† Anna Selmi,‡ Renata Smulik,† Łukasz Piotrowski,† Jan Adamus,† Andrzej Marcinek,† Stefan Chlopicki,‡,§ and Adam Sikora*,† †

Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Kraków, Poland § Chair of Pharmacology, Jagiellonian University Medical College, Kraków, Poland ‡

S Supporting Information *

ABSTRACT: Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 105 M−1 s−1, (8.6 ± 0.5) × 103 M−1 s−1, and (0.96 ± 0.03) M−1 s−1, respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.

■

(O2•−), has been considered as a key cellular oxidant formed in various pathophysiological states.2,8,9 In the past decade, a large number of molecular probes have been designed and tested for the detection of peroxynitrite. The mechanisms of action of these probes are based on various peroxynitrite-triggered reactions including nitration of the aromatic moiety,10,11 formation of dioxirane with activated ketones,12−14 oxidation of organoselenium compounds,15,16 oxidative conversion of boronates,4,17−22 and other reactions.23−26 Initially, boronatebased fluorogenic probes were proposed as a tool for the selective detection of hydrogen peroxide.27,28 Recently, it has been shown that boronic acids and their esters react directly with peroxynitrite a million times faster than with hydrogen peroxide, and the corresponding phenols are formed as the major products.21,29 The oxidation mechanism of boronic acids by peroxynitrite has been already described in details.22,30 The

INTRODUCTION

During the last 40 years, tremendous progress in the understanding of the biological chemistry of reactive oxygen and nitrogen species (ROS and RNS) has been made.1,2 In order to understand how these reactive species affect cell functions, it is important to determine within living cells, and in real time, what kind of species are produced, and in what quantities.3−7 As the direct detection of intracellular reactive species is impossible because of their short lifetime and rapid intracellular scavenging, various probes that can react with them, producing easily detectable, relatively stable products, have been developed. Because of the high sensitivity of fluorescent methods, fluorogenic probes for reactive oxygen species became important tools in the studies on oxidative stress. However, the rational use of those probes requires a deep understanding of their mechanism of action.3,5−7 Peroxynitrite (ONOO−/ONOOH), a powerful oxidizing and nitrating agent formed in the diffusion-controlled reaction between nitric oxide (•NO) and superoxide radical anion © 2016 American Chemical Society

Received: October 15, 2015 Published: April 15, 2016 735

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology fluorogenic probes with boronate groups may be used for the detection of peroxynitrite, and some of those probes have been successfully used for that purpose in enzymatic and cellular systems.4,18,21,31,32 It has been also shown that boronate probes can be oxidized by aliphatic and aromatic hydroperoxides.30,33 A number of fluorogenic boronate probes for the detection of hydrogen peroxide and peroxynitrite have been developed.34 Although a number of boronate-based probes have been described in the literature, only few are commercially available or are readily accessible via simple synthesis and have been fully characterized in terms of their reactivity toward both peroxynitrite and hydrogen peroxide.18,21 Recently, a simple synthetic protocol has been proposed for the synthesis of boronate fluorogenic probes, based on the reaction of boronobenzylation of fluorescent dyes.18,35−38 Here, we show how this approach can be used for the synthesis of a fluorescein-based monoboronate probe. We also present the study on the reactivity of this novel probe, 4-(pinacol boronate)benzyl-derivative of fluorescein methyl ester (FBBE), toward peroxynitrite, hypochlorous acid, hydrogen peroxide, and tyrosyl hydroperoxide39,40 (TyrOOH). The effect of glutathione on the FBBE reaction with peroxynitrite, H2O2, HOCl, and TyrOOH was also studied. The oxidative conversion of the FBBE probe to the fluorescent product has been also studied in the presence and absence of glutathione in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide. Additionally, we present the results of the DFT quantum mechanical calculations of the oxidation of phenylboronate ester by the deprotonated hydrogen peroxide (HO2−) and peroxynitrite. Finally, the FBBE probe was used to study the production of ROS and RNS in Ea.hy926 endothelial cells incubated with doxorubicin (DOX), a quinone anthracycline antibiotic. DOX is widely used as an anticancer drug, but its clinical use is limited by its cardiac and vascular toxicities. Although DOX has been used for nearly half a century in the treatment of various malignancies, the mechanisms of its side effects are still not entirely clear.41 One of the proposed mechanisms of DOX cardiotoxicity involves an increased formation of reactive oxygen species, particularly the superoxide radical anion,42−46 due to the one-electron enzymatic reduction of DOX yielding the DOX semiquinone radical, and the further reoxidation of this radical by molecular oxygen. More recently, it has been found that DOX can uncouple NOS and transform the NO synthase into a NADPH oxidase with the concomitant formation of peroxynitrite.47 In many studies, the doxorubicin-induced peroxynitrite formation was deduced from the observation of increased 3-nitrotyrosine formation.48−51 It has been shown that extensive protein nitration of cardiac myofibrils correlated with DOX-induced cardiac dysfunction.48 It has been also postulated that DOX-induced inactivation of cardiac mitochondrial creatine kinase, resulting in lower phosphocreatine-ATP, is related to the formation of peroxynitrite.52,53 Further studies have shown that DOX induces increased myocardial iNOS, ROS generation, and dosedependent increase in cellular 3-nitrotyrosine.54 Altogether, there is a large body of evidence suggesting that doxorubicininduced cardiac toxicity is related to oxidative stress and peroxynitrite formation.55,60,59 However, the formation of peroxynitrite in DOX-induced endothelial cells injury is not that well documented. We have recently proposed that ROS generation was a consequence of the DNA-damage-induced endothelial stress response41 caused by anthracyclines accumu-

lation in the nucleus.55 However, it was suggested that the toxicity of DOX may be rather related to TopIIβ inhibition or activation of the CREB3L1 transcription factor.56 Here, we tested whether DOX toxicity toward endothelium cells was associated with peroxynitrite generation.

■

MATERIALS AND MEASUREMENTS

Chemicals. Synthesis of bis[(4-pinacol boronate)benzyl)]fluorescein (FbisBBE) and the 4-(pinacol boronate)benzyl-derivative of fluorescein methyl ester (FBBE) is described in Supporting Information. The monoboronate resorufin derivative PC1 (3-oxo3H-phenoxazin-7-yl pinacolatoboron) was synthesized according to the literature.57 All other chemicals (of the highest purity available) were from Sigma-Aldrich Corp. All solutions were prepared using deionized water (Millipore MilliQ system). Because of the poor solubility of the FBBE probe in water, CH3CN was used as a cosolvent (up to 10% v/v). We have tested the effect of CH3CN on the kinetics and yield of boronate oxidation by peroxynitrite, hypochlorite, and hydrogen peroxide using coumarin boronic acid (CBA) that is well soluble in water. The addition of CH3CN (10% v/v) does not affect the yield of oxidation and only slightly influences the reaction kinetics (the rate constants determined in water and in 10% CH3CN differ by less than 20%). Peroxynitrite was prepared by reacting nitrite with H2O2, according to the published procedure.58 The concentration of ONOO− in alkaline aqueous solutions (pH > 12) was determined by measuring the absorbance at 302 nm (ε = 1.7 × 103 M−1 cm−1).59 UV−Vis Absorption and Fluorescence Measurements. The UV−vis absorption spectra were collected using an Agilent 8453 spectrophotometer equipped with a diode array detector and thermostated cell holder. Fluorescence spectra were collected at room temperature using a Varian Cary Eclipse fluorescence spectrophotometer. Kinetic Studies. The competition kinetic approach was used to determine the rate constant of the FBBE reaction with peroxynitrite and hypochlorite. The resorufin boronate derivative PC1 was used as a competitor in kinetic experiments (for details, see Supporting Information). Stopped-Flow Measurements. Stopped-flow experiments were performed on an Applied Photophysics SX20 stopped-flow spectrophotometer. The thermostated cell (25 °C) with a 10 mm optical pathway was used for kinetic measurements. Determination of O2•− and •NO Fluxes. Nitric oxide fluxes were determined from the measured rate of the decomposition of PAPANONOate by following the decrease in its characteristic absorbance at 250 nm (ε = 8 × 103 M−1cm−1).60 This rate was multiplied by a factor of 2 to get the rate of •NO production (assuming that two molecules of •NO are released from one molecule of PAPA-NONOate). The flux of O2•− was determined by monitoring the cytochrome c reduction following the increase in absorbance at 550 nm (using molar absorption coefficient of 2.1 × 104 M−1 s−1).61 Generation of Singlet Oxygen. The reactivity toward singlet oxygen was studied as follows: the solution of FBBE probe (10 μM) and rose bengal (10 μM) in phosphate buffer (50 mM, pH 7.4) was placed in a fluorometer quartz cell and bubbled with oxygen. Then, the sample was illuminated with visible light using a 200-W xenon lamp at room temperature with continuous oxygenation. The illuminating light was passed through a water filter and a 400 nm cutoff filter. Next, the fluorescence spectra of the reaction mixture were collected at different times of photolysis. The influence of GSH on the reaction of the FBBE probe with singlet oxygen was investigated similarly, but in the presence of various concentrations of GSH. Generation of Tyrosyl Hydroperoxide. It is well known that tyrosyl hydroperoxide is formed with high yield during the reaction of tyrosine with singlet oxygen.33,62 Thus, for the purpose of the experiment, tyrosyl hydroperoxide was generated in the system containing a singlet oxygen generator, rose bengal. The solution of tyrosine (1 mM), phosphate buffer (50 mM, pH 7.4), and rose bengal (10 μM) was placed in a fluorometer quartz cell and was oxygenated. 736

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology Scheme 1. Reaction Scheme of FbisBBE and FBBE Oxidation by ONOO− and H2O2

Then, the sample was illuminated for 30 min with visible light using a 200-W xenon lamp at room temperature with continuous oxygenation. The illuminating light was passed through a water filter and a 400 nm cutoff filter. After photolysis, the sample was incubated with catalase (100 U/mL) in order to remove any hydrogen peroxide formed during the photolysis. Such prepared sample of tyrosyl hydroperoxide was further diluted with FBBE/GSH solution, and the fluorescence signal was measured. Theoretical Studies. All calculations were performed with the use of the Gaussian 09, rev.A.02 (G09), package.63 Live Cell Fluorescence Imaging. EA.hy926 human endothelial cells were passaged in a 96 well tissue culture treated plate for fluorescence imaging (BD Falcon), plated at a density of 10 000 cells per well, and incubated at 37 °C, 5% CO2, overnight. Cells were then treated with doxorubicin hydrochloride (Sigma) at a concentration of 0.1, 0.5, and 1 μM for 24 h. Appropriate positive control experiments were done as follows: superoxide dismutase-polyethylene glycol from bovine erythrocytes (PEG-SOD, Sigma) and/or catalase-polyethylene glycol from bovine liver (PEG-CAT, Sigma) at a concentration of 100 U/mL were added simultaneously with doxorubicin, while 1 mM L-

NAME (Sigma) was added 18 h after the addition of doxorubicin. After the incubation with doxorubicin, cells were washed twice with calcium and magnesium free Dulbecco’s phosphate-buffered saline (DPBS, Life technologies), and FBBE (5 μM in serum-free medium, DPBS) was added for 1 h of incubation at 37 °C. At the same time, cells were counterstained with Hoechst 33342 (Life technologies) in DPBS. After incubation with FBBE, cells were washed again with DPBS. Two hundred microliters of DPBS was then added per well, and the plate was placed in an Olympus ScanR incubation chamber (37 °C, 5% CO2) for live cell imaging. Fluorescence images were collected using an Olympus ScanR microscope, equipped with a ×40 objective, in a 495/519 nm FITC channel (exposure time: 80 ms) for FBBE oxidation product imaging and a 358/461 nm DAPI channel (exposure time: 5 ms) for Hoechst 33342 imaging of nuclei of endothelial cells. For quantitative analysis of FBBE oxidation derived fluorescence, images were imported into a Columbus (PerkinElmer) server for image analysis using the associated Columbus analysis building blocks. The mean intensity and standard deviation per single cell were quantitated in duplicate. The differences in the measured 737

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology fluorescence intensity were statistically analyzed with the use of Student’s t-test. Caspases-3 and -7 Activity Assay. For the caspases-3 and -7 activities assay, EA.hy926 cells were passaged on white-walled 96-well plates for chemiluminescence measurements (PerkinElmer, Waltham, MA, USA) and treated with doxorubicin in concentrations of 0.1, 0.5, 1, and 10 μM. Twenty-four hours post-treatment, cells were washed twice with DPBS, and 100 μL of medium was added to each well. One hundred microliters of Caspase Glo 3/7 reagent (Promega, Madison, WI, USA) was added to each well, and the plates were incubated for 3 hours in the dark. Luminescence was measured with a Synergy 4 plate reader (Bioteck, Winooski, VT, USA). The results obtained were normalized by protein concentrations in samples, calculated based on the BCA method.

■

RESULTS AND DISCUSSION

It is known from the literature, that the reaction of fluorescein with benzyl chloride leads both to the benzylation of the hydroxyl and carboxyl groups of the dye.64,65 Similarly, the direct benzylation of fluorescein with the use of 4(iodomethyl)phenylboronic acid pinacol ester results in the dibenzylated derivative of the dye (Scheme S1A), as it has been recently shown for the reaction with 4-(bromomethyl)phenylboronic acid pinacol ester.37 As a consequence, the synthesized probe is characterized by a reduced sensitivity toward oxidants, as there are two distinct reaction pathways of the oxidative deboronation, and only one leads to the formation of the fluorescent compound (Scheme 1A). To overcome this limitation, we synthesized the 4-boronobenzylated derivative of fluorescein methyl ester (Scheme S1B) that in the reaction with oxidants (ONOO−, H2O2, HOCl, and ROOH) is converted into the fluorescent fluorescein methyl ester (FlOMe, Scheme 1B). Oxidation of FBBE by Peroxynitrite, Hydrogen Peroxide, and Hypochlorite. First, we investigated the spectral changes accompanying the peroxynitrite derived oxidation of the FBBE probe. The UV−vis absorption spectra of the reaction mixtures (Figure 1A) containing the FBBE probe (20 μM) and the increasing concentrations of peroxynitrite (0−20 μM) clearly indicated the formation of the dye with the narrow and strong absorption band with the maximum at 494 nm. A single isosbestic point was observed at 463 nm for this oxidative conversion. Increase of the measured absorbance at 494 nm is linear up to approximately 1:1 boronate−oxidant ratio (Figure 1B), while further addition of peroxynitrite does not cause additional increase of the product absorption or its decay. Therefore, the reaction of the FBBE probe with peroxynitrite occurs in a stoichiometric manner that is consistent with data obtained previously for other boronates.21,22,29 Reaction of FBBE with oxidant (peroxynitrite, hypochlorite, etc.) leads to the formation of a fluorescent product (Figures S1 and S2). The product formed upon oxidation has intensive fluorescence with a single emission peak at 518 nm, which is similar to the emission spectrum of fluorescein. For low peroxynitrite concentrations (up to 3 μM), increase of both excitation and emission bands is a linear function of oxidant concentration (data not shown). Similar changes in absorption and fluorescence spectra, corresponding to the oxidative conversion of the probe, are observed in the aerated FBBE solutions containing peroxynitrite generator SIN1 or HNO donors, as has been observed before for other boronates66 (Figures S10 and S11). Kinetics analysis of the oxidative conversion of FBBE has shown that FBBE oxidation is a two-step reaction. The reaction

Figure 1. (A) Absorption spectra of an aqueous solution of 20 μM FBBE, 50 mM PB (pH 7.4), 100 μM dtpa, and 10% CH3CN obtained after the addition of 0−20 μM ONOO−. (B) Absorption measured at 494 nm for the solution of 20 μM FBBE, 50 mM PB (pH 7.4), 100 μM dtpa, and 10% CH3CN after the addition of 0−26 μM ONOO−. Spectra were measured 15 min after bolus addition of ONOO−.

of oxidant (peroxynitrite, hypochlorite, hydrogen peroxide, etc.) with boronate group leading to the corresponding phenol is the first step of this process. The second step can be ascribed to slow quinone methide elimination leading to the formation of a FlOMe. The observed changes in absorption and fluorescence spectra correspond to the formation of FlOMe. FBBE reacts with biological oxidants (ONOO−, H2O2, and HOCl) with the rate constants typical for phenylboronic acids (Table 1 and Figures S7, S8, S9, and S13; for methodological Table 1. Second-Order Rate Constants of the Reaction of FBBE with Peroxynitrite, Hypochlorite, and Hydrogen Peroxide at pH 7.4 oxidant peroxynitrite hypochlorite hydrogen peroxide

k (M−1s−1)

2

(2.8 ± 0.2) × 105 (8.6 ± 0.5) × 103 0.96 ± 0.03

details of kinetics measurements, see Supporting Information).18,21,22,29 The Gibbs free energies of activation predicted by performed theoretical calculations (see Supporting Information) are equal to 79.1, 54.4, and 52.3 kJ/mol for the reaction of phenylboronic esther with HO2− cis- and transperoxynitrite, respectively. Although the rate constant for the reaction of FBBE with ONOO− is high (>105 M−1 s−1), the buildup of the product with the absorption at 494 nm is relatively slow (k = 0.09 s−1, Figure 2A) and occurs on a seconds time scale, due to the sluggish quinine methide elimination. According to the acid− base equilibrium of the FBBE probe and the product of its 738

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology

H2O2-derived oxidation of the probe was significant (Figure 3A). The presence of 0.5−5 mM GSH during the oxidation of

Figure 2. (A) Absorption spectra of an aqueous solution of 10 μM FBBE, 50 mM PB (pH 7.4), 100 μM dtpa, and 4% CH3CN obtained 0−40 s after the addition of 5 μM ONOO−. Inset: kinetic trace of the growth of absorption at 494 nm with a nonlinear fit to the first-order reaction equation. (B) Dependence of the observed rate constant of methide quinone elimination on the pH of the solution. Solutions consisted of 10 μM FBBE, 100 mM PB (pH 5.96−9.72), 3% CH3CN, and 10 μM ONOO−. Nonlinear fitting was performed in a pH range of 6.9−8.8.

Figure 3. (A) Concentration of FlOMe formed in the reaction of 20 μM FBBE, 50 mM PB (pH 7.4), 100 μM dtpa, 10% CH3CN (ONOO−), or 20% CH3CN (H2O2) and 0−6 mM GSH with 100 μM ONOO− or H2O2, respectively. (B) The concentration of FlOMe formed in the reaction of 25 μM FBBE, 50 mM PB (pH 7.4), 100 μM dtpa, 10% CH3CN, and 0−6 mM GSH with 10 μM ONOO−. Concentration of the FlOMe was determined based on the absorbance at 494 nm with the use of Δε494 nm = 62000 M−1 cm−1.

oxidation, the pH-dependent rate constant of FlOMe formation in the reaction with peroxynitrite has a complex profile (Figure 2B). At pH below 8.8, the observed rate constant of FlOMe formation can be described by eq 1: ⎞ ⎛ 10−pKa k pHdep = k pHindep·⎜ −pH ⎟ ⎝ 10 + 10−pKa ⎠

25 μM FBBE by 100 μM peroxynitrite led to a small increase in the product formation yield, whereas the addition of GSH (0.5−5 mM) prior to oxidation by 100 μM H2O2 led to the inhibition of FlOMe formation. H2O2 may be efficiently scavenged by GSH, especially in biological systems, as rates of FBBE and GSH oxidation by H2O2 are within the same order of magnitude.68 Moreover, H2O2 scavenging by GSH in cells may be catalyzed by the presence of glutathione peroxidases limiting the probe response to oxidation by H2O2. For lower peroxynitrite concentrations, the scavenging of oxidant is also visible (Figure 3B). Nevertheless, only partial reduction of the fluorescent product formation is observed, which can be explained by the competition between the probe and GSH for peroxynitrite. In fact, the competition kinetics approach can be used to analyze the observed yield of FlOMe formation (Figure 3B), for the evaluation of the rate constant of the reaction between ONOO− and the FBBE probe [kGSH = 1.35 × 103 M−1 s−1,69 and kGSH/kFBBE = (4.6 ± 0.2) × 10−3 (see Figure S8) gives kperoxynitrite = (2.9 ± 0.1) × 105 M−1 s−1, in excellent agreement with the value (2.8 ± 0.2) × 105 M−1 s−1 reported above].

(1)

At pH higher than 9, the probe undergoes complexation with HO− (Scheme S2) giving a product unreactive toward ONOO−, which strongly affects the rate constant of FlOMe formation. We have determined the pH-independent component of the first order rate constant [kpH indep = (8 ± 2) s−1] which allows for the prediction of the methide quinone elimination rate in a pH range between 6.9 and 8.8. Effect of Glutathione on FBBE Oxidation by Peroxynitrite, H2O2, and HOCl. Glutathione (GSH), as a ubiquitous thiol present in millimolar concentrations in living cells, should be always considered as a compound that may interfere with the molecular probes used for the ROS and RNS detection. Thus, the influence of glutathione on FBBE oxidation by peroxynitrite, hydrogen peroxide, and hypochlorous acid was investigated. Glutathione affected the oxidation of the probe by all three studied oxidants. The hypochloritemediated oxidation of FBBE was completely blocked in the presence of GSH, due to very fast scavenging of oxidant by the −SH group67 (data not shown). The influence of GSH on 739

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology Oxidation of FBBE by Hydroperoxides and the Effect of GSH. Recently, the reactivity of boronates toward amino acid, peptide, and protein hydroperoxides has been studied.33 It has been shown that boronates are oxidized by these species to appropriate phenols and that has been also observed for the reaction of FBBE with tyrosyl hydroperoxide (Figure 4).

reaction of tyrosyl hydroperoxides with the studied probe led to the inhibition of the fluorescence signal. Oxidative Conversion of the FBBE Probe in the System Generating 1O2. Several nonphotochemical reactions lead to the formation of singlet oxygen in biological systems.70 It has been shown that singlet oxygen is formed by a peroxidase enzymes and during lipoxygenase-catalyzed reactions. The bimolecular termination reaction of peroxyl radicals or reaction of H2O2 with HOCl or peroxynitrite are other sources of singlet oxygen. Furthermore, evidence that singlet oxygen is produced by stimulated macrophages, eosinophils, and neutrophils has been presented. Besides, 1O2 is also produced in biological systems during their exposure to UV or visible light in the presence of endogenous or exogenous photosensitizers.70 Singlet oxygen, an oxidizing species, undergoes a reaction with a broad range of biological targets including proteins, lipids, DNA, and RNA.70 Glutathione is an efficient scavenger of singlet oxygen (2k = 2.4 × 106 M−1 s−1, cellular levels up to 10 mM) that provides natural protection of cellular components against this ROS.71 In order to fully characterize the FBBE probe, we have decided to test whether singlet oxygen reacts with the probe, and find out what the influence of glutathione on this reaction is. Recently, we have shown that another boronate probe, coumarin boronic acid (CBA), does not form a fluorescent product in the reaction with singlet oxygen but the presence of both, glutathione and singlet oxygen, is necessary to oxidize the CBA probe to the fluorescent 7-hydoxycoumarin.33 Considering the structure of FBBE and its electron-rich character, the reaction of 1O2 with this probe cannot be excluded.72 Nevertheless, in the mixture containing the FBBE probe and photosensitizer (10 μM rose bengal) during the illumination with visible light only a slight oxidation of the probe was observed (Figure 5A). It is unlikely that the reaction of FBBE with singlet oxygen leads directly to the fluorescent product. The fluorescent product is probably formed in the reaction of the FBBE probe with its oxidation products, but this should be a minor process in the cellular system with naturally occurring singlet oxygen scavengers. The reaction mixtures have contained also catalase (100 U/mL) to exclude the formation of H2O2, that can be produced during illumination according to a type I mechanism. Continuing the experiment, we have used various concentrations of GSH (0.5−5 mM) to observe whether glutathione

Figure 4. Fluorescence spectra of an aqueous solution of 10 μM FBBE, 50 mM PB (pH 7.4), 10% CH3CN, and 100 U/mL catalase containing tyrosyl hydroperoxide. Ex. slit: 2.5 nm, em. slit: 5 nm, λex = 494 nm, and PMT voltage = 750 V. Inset: kinetic traces collected for the reaction mixtures containing 10 μM FBBE, tyrosyl hydroperoxide, and various concentrations of GSH (0−10 mM), λex = 494 nm, and λem = 518 nm.

Although we were unable to measure the rate constant of the reaction of FBBE with tyrosyl hydroperoxide because of the lack of its standard and low solubility of the probe, it is reasonable to assume that this rate constant is similar to other boronates. Keeping in mind that various amino acid hydroperoxides react with boronate probes with similar rates, and peptide or protein hydroperoxides react even slower,33 this kind of species should not interfere with peroxynitrite detection by the FBBE probe. Importantly, the influence of GSH on tyrosyl hydroperoxidederived oxidation of the FBBE probe was also significant (Figure 4, inset). The presence of 0.3−10 mM GSH during the

Figure 5. Oxidation of FBBE in the presence of 1O2 and GSH. (A) Aqueous solution of 10 μM FBBE, 50 mM PB (pH 7.4), 10% CH3CN, and 100 U/mL catalase illuminated with visible light (λ > 400 nm) in the presence of 10 μM rose bengal. Time of photolysis is given in the figure legend. Inset: dependence on GSH concentration (0−5 mM). (B) The same as (A), but the mixture contains GSH (5 mM). Conditions: λex = 494 nm, ex. slit = 2.5 nm, em. slit = 1.5 nm, and PMT voltage = 800 V. 740

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology

Figure 6. Changes of fluorescence intensity of an aqueous solution of 20 μM FBBE during the generation of NO and/or O2•− in the presence or absence of GSH and/or SOD. (A) Incubation mixtures containing 2 mM xanthine, xanthine oxidase (generating of a flux of O2•− of 0.5 μM/min), 5 mM GSH, and/or 2 μM SOD. (B) PAPA-NONOate (generating a flux of NO of 0.5 μM/min) and/or 2 μM SOD. (C) 2 mM xanthine, xanthine oxidase (generating a flux of O2•− of 0.5 μM/min), PAPA-NONOate (generating a flux of NO of 0.5 μM/min), 5 mM GSH, and 2 μM SOD. All solution containing 10% CH3CN, 50 mM phosphate buffer, pH 7.4, and 100 μM dtpa. Parameters of measurements: λex = 494 nm, λem = 518 nm, ex. slit = 5 nm, em. slit = 2.5 nm, and PMT voltage = 600 V.

of peroxynitrite (Scheme 2).74 The addition of PAPANONOate to the solutions containing FBBE and X/XO

and singlet oxygen, together, increase the amount of observed fluorescent product (Figure 5A, inset). According to the literature data, GSH reacts with singlet oxygen producing disulfide, sulfoxide, sulfonate, and sulphinate.71 The intermediate formed during the reaction of 1O2 with R-SH can have a structure of zwitterion (RS(H)+-OO−), thiol hydroperoxide (RSOOH), or its deprotonated form RSOO−. The observed rate constants of thiols with 1O2 strongly depend on the pH indicating that the unprotonated form of the thiol reacts with 1O271,73 and thus the deprotonated form of thiol hydroperoxide (RSOO−) should be the intermediate formed.73 This intermediate should be a strong nucleophile able to oxidize the FBBE probe to the appropriate fluorescent product, and indeed, we observed instant oxidation of FBBE when both, singlet oxygen and glutathione, were present in the mixture (Figure 5B). This reaction was independent of the GSH concentration in the tested range (Figure 5A, inset). Oxidation of FBBE in X/XO and the PAPA-NONOate System. The oxidative conversion of the FBBE probe was also investigated in the systems generating superoxide radical anion and nitric oxide. FBBE (20 μM) was incubated with xanthine (X, 2 mM) and XO (generating a flux of superoxide of 0.5 μM/ min) in phosphate buffer (50 mM, pH 7.4) containing dtpa (100 μM). Similar incubations were performed in the presence of a nitric oxide donor, PAPA-NONOate (generating a flux of • NO of 0.5 μM/min). In the X/XO system, the modest formation of fluorescent product in the reaction between FBBE and H2O2, generated by XO and by dismutation of O2•−, was almost completely mitigated in the presence of 5 mM GSH (Figure 6A). In the aqueous solution of nitric oxide donor, PAPA-NONOate, no oxidation of FBBE probe was observed in the absence of glutathione, but in the presence of the thiol, there was an intense conversion of FBBE to FlOMe that was partially inhibited by SOD (Figure 6B). The nitric oxide/glutathione/ oxygen system has been extensively studied in the past, due to the formation of S-nitrosoglutathione (GSNO).74 In the chemical description of such system, three different pathways can be proposed for the formation of S-nitrosothiol, and two of them can be also a source of superoxide radical anion. Subsequent reaction of O2•− with •NO results in the formation

Scheme 2. Proposed Pathways of Peroxynitrite Formation in the X/XO/PAPA-NONOate/GSH System

significantly enhanced the formation of FlOMe, which was not inhibited by SOD (2 μM) and was only partially inhibited by GSH (5 mM) (Figure 6C). The observed modest effect of 5 mM GSH on the oxidative conversion of FBBE to FlOMe in the X/XO/PAPA-NONOate system cannot be easily explained by the simple competition for peroxynitrite between the probe and GSH (in this case, the effect should be more pronounced). Moreover, that effect was significantly enhanced in the presence of SOD (2 μM). The best explanation is the simultaneous formation of peroxynitrite and other, GSH-derived, oxidants in the X/XO/PAPA-NONOate/GSH system (e.g., GSOOH/ GSOO−). Oxidation of FBBE in Cultured Endothelium. A cell culture study was used to evaluate the applicability of FBBE for the detection of reactive oxygen and nitrogen species in biological environment. The cytotoxicity of the FBBE probe was tested with the use of the MTS assay in EAhy.926 cells to determine nontoxic dose range (see Supporting Information). The probe was nontoxic in the concentration range 0−30 μM (cells were incubated with the probe for 1 and 24 h). To evaluate the applicability of the studied probe for the detection of ROS/RNS in living cells, EAhy.926 cells were treated with doxorubicin for 24 h to induce ROS and RNS 741

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology

Figure 7. Representative fluorescence images of EAhy.926 cells incubated in (A) the absence and (B) the presence of FBBE (5 μM, incubation time = 1 h). (C) Fluorescence images of EAhy.926 cells stimulated with doxorubicin (1 μM, incubation time = 24 h) and then incubated with FBBE (5 μM, incubation time = 1 h). Appropriate control experiments: simultaneous incubation of EAhy.926 cells with doxorubicin (24 h); (D) 1 mM LNAME was added 18 h after the addition of doxorubicin, (E) 100 U/mL PEG-SOD (24 h), and (F) 100 U/mL PEG-CAT (both incubated 24 h). Nuclei were labeled with Hoechst 33342 dye (DAPI channel). Scale bar = 50 μm.

Table 2. Oxidative Conversion of an FBBE Probe in Systems Generating Various ROS/RNS and the Effect of 5 mM GSH on the Observed Oxidation ROS/RNS generated in the system O2•− H2O2 • NO

HNO •

NO/O2•−

ONOO− (bolus addition) HOCl TyrOOH 1 O2

oxidative transformation of FBBE into FlOMe observed; oxidant: H2O2 from the dismutation of O2•− observed; oxidant: H2O2 not observed; •NO, •NO2, and N2O3 cannot oxidatively convert boronates into phenols observed; oxidant: ONOO− formed in the reaction of HNO with molecular oxygen observed; oxidant: ONOO− formed in the reaction of •NO with O2•− observed; oxidant: ONOO− observed; oxidant: HOCl observed; oxidant: TyrOOH not observed

observed effect of GSH (5 mM) on FBBE oxidation Oxidation is almost completely inhibited due to H2O2 scavenging by GSH. as described above In the incubation mixture containing a nitric oxide donor, glutathione, oxygen, and FBBE, the probe is effectively oxidized to FlOMe due to the formation of ONOO− and GSOOH/GSOO− and/or other GSH-derived hydroperoxides. Oxidation is completely inhibited due to HNO scavenging by GSH. Oxidation is only partially inhibited. The probe is effectively oxidized to FlOMe in the reaction with ONOO− and other oxidant/oxidants (GSH-derived), e.g., GSOOH/GSOO− and/or other GSH-derived hydroperoxides. Oxidation is partially inhibited due to ONOO− scavenging by GSH. Oxidation is completely inhibited due to HOCl scavenging by GSH. Oxidation is almost completely inhibited due to TyrOOH scavenging by GSH. In the system generating singlet oxygen, the FBBE probe is effectively oxidized to FlOMe due to the formation of the GSH-derived oxidant (e.g., GSOOH/GSOO−).

742

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology

fluorescein methyl ester formation (FlOMe). This product can be easily detected due to its high fluorescence. Hydrogen peroxide and other amino acids, peptides, and protein derived hydroperoxides can be also detected by the FBBE probe; however, the rate constant of the reaction of FBBE toward H2O2 [(0.96 ± 0.03) M−1 s−1, pH 7.4] is a few orders of magnitudes lower than that toward peroxynitrite. The results of our study on the oxidative conversion of the FBBE probe in systems generating different ROS and RNS are briefly summarized in Table 2. Here, we also briefly discuss the observed effects of GSH. As a proof of the concept of the usefulness of the FBBE probe for the detection of peroxynitrite in cellular microenvironments, we analyzed FBBE oxidation in Ea.hy926 endothelial cells stimulated with doxorubicin. We demonstrated that the incubation of Ea.hy926 endothelial cells with doxorubicin led to FBBE oxidative transformation resulting in the increase in green fluorescence, which was inhibited by LNAME and by PEG-SOD but not by catalase, confirming the ONOO − generation. Obviously, DOX-induced ONOO − formation does not directly imply the formation of reactive oxygen species due to one-electron enzymatic reduction of DOX. ONOO− formation could be due to oxidative stress response triggered by DNA-damage and cellular stress response induced by DOX.41,75 The FBBE probe is suitable for peroxynitrite detection, although distinguishing between different ROS/RNS-dependent FBBE oxidation requires the additional use of several inhibitors (e.g., L-NAME, PEG-SOD, and PEG-catalase). It is important that the FBBE selectivity toward ONOO− increases in cell culture studies due to the presence of glutathione, which effectively scavenges hydrogen peroxide and other hydroperoxides. Among commercially available probes for ROS and RNS, FBBE is favored by its simple synthesis, high reactivity toward peroxynitrite, and ease of detection due to the intensive fluorescence properties of its product.

production and then incubated with 5 μM FBBE for 1 h at 37 °C. Doxorubicin-stimulated cells have shown significant fluorescence response in comparison to the control, where doxorubicin was not present during the incubation (Figure 7B and C, respectively). What is more, we observed that the formation of green fluorescent product was dependent on the doxorubicin concentration (Figure 8). To confirm the identity of the species responsible for FBBE oxidation, we demonstrated that L-NAME, an endothelial nitric oxide synthase inhibitor (Figure 7D), or/and PEG-SOD as a superoxide scavenger (Figure 7E,G), caused significant decrease of fluorescence derived from FBBE oxidation. Both L-NAME and PEG-SOD profoundly inhibited the formation of FlOMe. In turn, simultaneous incubation of cells with doxorubicin and catalase did not affect the oxidation of the probe. Thus, the intracellular oxidation of the probe was not caused by the reaction of the FBBE probe with hydrogen peroxide (Figure 7F,H and Figure 8). This observation led us to the conclusion that the oxidation of FBBE in endothelial cells stimulated by DOX was dependent on the formation of peroxynitrite, originating from the reaction of superoxide and nitric oxide. In order to determine if observed effects are not secondary processes, linked to cell apoptosis, we determined caspase 3 and 7 activities in EA.hy926 cells incubated for 24 h with doxorubicin at concentrations of 0.1, 0.5, 1, and 10 μM (see Supporting Information). DOX at concentration ≤1 μM did not induce the activation of caspases 3 and 7, which suggests that at such DOX concentrations cells do not undergo changes leading to apoptosis (Figure S18). The significant activation of caspases 3 and 7 was observed when cells were incubated with DOX at a concentration of 10 μM (Figure S18).

■

CONCLUSIONS Here, we characterized the novel, fluorescein-based monoboronate probe FBBE. Spectral properties and the reactivity of the newly developed boronate-probe were described. FBBE reacts with peroxynitrite, similarly to other boronates, with a high rate constant [2k = (2.8 ± 0.2) × 105 M−1 s−1, pH 7.4]. The reaction with ONOO− is a two-step reaction resulting in

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00431. Synthesis of bis[(4-pinacol boronate)benzyl)]fluorescein (FbisBBE) and 4-(pinacol boronate)benzyl-derivative of fluorescein methyl ester (FBBE); excitation and emission spectra of the FBBE solutions after bolus addition of peroxynitrite or hypochlorite, the kinetics of the reaction of FBBE with peroxynitrite and hypochlorous acid− competition kinetics approach; oxidation of the FBBE probe in the aerated solutions of SIN-1 and HNO donors; the kinetics of the reaction of FBBE with hydrogen peroxide; FBBE acid−base equilibrium; MTS cell proliferation colorimetric assay; determination of the caspases 3 and 7 activities in EA.hy926 cells incubated for 24 h with doxorubicin; and the methodology and results of DFT calculations (PDF)

Figure 8. Summarized quantitative measurements of the intensity of green fluorescence (FITC channel) derived from the oxidative transformation of FBBE (5 μM, incubation time = 1 h) in EAhy.926 endothelial cells stimulated with doxorubicin (incubation time = 24 h) in the absence or presence of inhibitors as indicated: PEG-SOD (100 U/mL), PEG-CAT (100 U/mL), and L-NAME (1 mM). Incubation times were the same as indicated in Figure 7. The error bars represent standard deviation. *P < 0.05 and **P < 0.01 vs the control; #P < 0.05 vs DOX 1 μM. 743

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology

■

(7) Debowska, K., Debski, D., Hardy, M., Jakubowska, M., Kalyanaraman, B., Marcinek, A., Michalski, R., Michalowski, B., Ouari, O., Sikora, A., Smulik, R., and Zielonka, J. (2015) Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes–Limitations, progress, and perspectives. Pharmacol. Rep. 67, 756−764. (8) Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. 87, 1620−1624. (9) Pacher, P., Beckman, J. S., and Liaudet, L. (2007) Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315−424. (10) Nagano, T. (2009) Bioimaging probes for reactive oxygen species and reactive nitrogen species. J. Clin. Biochem. Nutr. 45, 111− 124. (11) Ueno, T., Urano, Y., Kojima, H., and Nagano, T. (2006) Mechanism-based molecular design of highly selective fluorescence probes for nitrative stress. J. Am. Chem. Soc. 128, 10640−10641. (12) Yang, D., Wang, H. L., Sun, Z. N., Chung, N. W., and Shen, J. G. (2006) A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells. J. Am. Chem. Soc. 128, 6004− 6005. (13) Sun, Z. N., Wang, H. L., Liu, F. Q., Chen, Y., Tam, P. K., and Yang, D. (2009) BODIPY-based fluorescent probe for peroxynitrite detection and imaging in living cells. Org. Lett. 11, 1887−1890. (14) Peng, T., and Yang, D. (2010) HKGreen-3: a rhodol-based fluorescent probe for peroxynitrite. Org. Lett. 12, 4932−4935. (15) Xu, K., Chen, H., Tian, J., Ding, B., Xie, Y., Qiang, M., and Tang, B. (2011) A near-infrared reversible fluorescent probe for peroxynitrite and imaging of redox cycles in living cells. Chem. Commun. (Cambridge, U. K.) 47, 9468−9470. (16) Yu, F., Li, P., Li, G., Zhao, G., Chu, T., and Han, K. (2011) A near-IR reversible fluorescent probe modulated by selenium for monitoring peroxynitrite and imaging in living cells. J. Am. Chem. Soc. 133, 11030−11033. (17) Yu, F., Song, P., Li, P., Wang, B., and Han, K. (2012) A fluorescent probe directly detect peroxynitrite based on boronate oxidation and its applications for fluorescence imaging in living cells. Analyst (Cambridge, U. K.) 137, 3740−3749. (18) Sieracki, N. A., Gantner, B. N., Mao, M., Horner, J. H., Ye, R. D., Malik, A. B., Newcomb, M. E., and Bonini, M. G. (2013) Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin. Free Radical Biol. Med. 61, 40−50. (19) Zielonka, J., Sikora, A., Hardy, M., Joseph, J., Dranka, B. P., and Kalyanaraman, B. (2012) Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chem. Res. Toxicol. 25, 1793−1799. (20) Zielonka, J., Sikora, A., Adamus, J., and Kalyanaraman, B. (2015) Detection and differentiation between peroxynitrite and hydroperoxides using mitochondria-targeted arylboronic acid. Methods Mol. Biol. (N. Y., NY, U. S.) 1264, 171−181. (21) Zielonka, J., Sikora, A., Joseph, J., and Kalyanaraman, B. (2010) Peroxynitrite is the major species formed from different flux ratios of co-generated nitric oxide and superoxide: direct reaction with boronate-based fluorescent probe. J. Biol. Chem. 285, 14210−14216. (22) Sikora, A., Zielonka, J., Adamus, J., Debski, D., DybalaDefratyka, A., Michalowski, B., Joseph, J., Hartley, R. C., Murphy, M. P., and Kalyanaraman, B. (2013) Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications. Chem. Res. Toxicol. 26, 856−867. (23) Zhang, H., Liu, J., Sun, Y. Q., Huo, Y., Li, Y., Liu, W., Wu, X., Zhu, N., Shi, Y., and Guo, W. (2015) A mitochondria-targetable fluorescent probe for peroxynitrite: fast response and high selectivity. Chem. Commun. (Cambridge, U. K.) 51, 2721−2724. (24) Zhou, X., Kwon, Y., Kim, G., Ryu, J. H., and Yoon, J. (2015) A ratiometric fluorescent probe based on a coumarin-hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite. Biosens. Bioelectron. 64, 285−291.

AUTHOR INFORMATION

Corresponding Author

*Institute of Applied Radiation Chemistry, Lodz University of Technology, Ż eromskiego 116, 90-924 Lodz, Poland. Phone: +48-42-631-3170. E-mail: [email protected]. Funding

A.S. was supported by a grant IP2011 049271 from Polish Ministry of Science and Higher Education within the “Iuventus Plus” program. R.M. was supported by a grant from the Foundation for Polish Science (FNP) within the “Homing Plus” program supported by the European Union within European Regional Development Fund, through the Innovative Economy Program. Support from a grant coordinated by JCET, No. POIG.01.01.02-00-069/09 (supported by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme) is also acknowledged. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Access to supercomputing facilities at Cyfronet (Poland; MNiSW/IBM_BC_HS21/PLodzka/045/2013 and MNiSW/ SGI3700/PLodzka/045/2013) is gratefully acknowledged.

■

ABBREVIATIONS CAT, catalase; CREB3L1, element binding protein 3-like 1; DOX, doxorubicin; DPBS, Dulbecco’s phosphate-buffered saline; dtpa, diethylenetriaminepentaacetic acid; EAhy.926, human umbilical vein endothelial cells; FBBE, 4-(pinacol boronate)benzyl-derivative of fluorescein methyl ester; FlOMe, fluorescein methyl ester; L-NAME, L-Nω-nitroarginine methyl ester; NOS, nitric oxide synthase; PB, phosphate buffer; PC1, 3-oxo-3H-phenoxazin-7-yl pinacolatoboron; PEG, polyethylene glycol; PMT, photomultiplier; SOD, superoxide dismutase; RNS, reactive nitrogen species; ROS, reactive oxygen species; R-SH, thiol; RSOOH, thiol hydroperoxide; RSOO−, deprotonated form of thiol hydroperoxide; RS(H)+OO−, zwitterion formed on -SH group upon reaction with 1O2; TopIIβ, topoisomerase II isoform; X, xanthine; XO, xanthine oxidase

■

REFERENCES

(1) Kalyanaraman, B. (2013) Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms. Redox Biol. 1, 244−257. (2) Radi, R. (2013) Peroxynitrite, a stealthy biological oxidant. J. Biol. Chem. 288, 26464−26472. (3) Wardman, P. (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radical Biol. Med. 43, 995−1022. (4) Zielonka, J., Zielonka, M., Sikora, A., Adamus, J., Joseph, J., Hardy, M., Ouari, O., Dranka, B. P., and Kalyanaraman, B. (2012) Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses. J. Biol. Chem. 287, 2984− 2995. (5) Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., Mann, G. E., Moore, K., Roberts, L. J., and Ischiropoulos, H. (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biol. Med. 52, 1−6. (6) Winterbourn, C. C. (2014) The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta, Gen. Subj. 1840, 730−738. 744

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology (25) Li, X., Tao, R. R., Hong, L. J., Cheng, J., Jiang, Q., Lu, Y. M., Liao, M. H., Ye, W. F., Lu, N. N., Han, F., Hu, Y. Z., and Hu, Y. H. (2015) Visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury. J. Am. Chem. Soc. 137, 12296−12303. (26) Peng, T., Wong, N. K., Chen, X., Chan, Y. K., Ho, D. H., Sun, Z., Hu, J. J., Shen, J., El-Nezami, H., and Yang, D. (2014) Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues. J. Am. Chem. Soc. 136, 11728−11734. (27) Chang, M. C., Pralle, A., Isacoff, E. Y., and Chang, C. J. (2004) A selective, cell-permeable optical probe for hydrogen peroxide in living cells. J. Am. Chem. Soc. 126, 15392−15393. (28) Lo, L. C., and Chu, C. Y. (2003) Development of highly selective and sensitive probes for hydrogen peroxide. Chem. Commun. (Cambridge, U. K.), 2728−2729. (29) Sikora, A., Zielonka, J., Lopez, M., Joseph, J., and Kalyanaraman, B. (2009) Direct oxidation of boronates by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite. Free Radical Biol. Med. 47, 1401−1407. (30) Sikora, A., Zielonka, J., Lopez, M., Dybala-Defratyka, A., Joseph, J., Marcinek, A., and Kalyanaraman, B. (2011) Reaction between peroxynitrite and boronates: EPR spin-trapping, HPLC Analyses, and quantum mechanical study of the free radical pathway. Chem. Res. Toxicol. 24, 687−697. (31) Broniowska, K. A., Mathews, C. E., and Corbett, J. A. (2013) Do beta-cells generate peroxynitrite in response to cytokine treatment? J. Biol. Chem. 288, 36567−36578. (32) Zielonka, J., Joseph, J., Sikora, A., and Kalyanaraman, B. (2013) Real-time monitoring of reactive oxygen and nitrogen species in a multiwell plate using the diagnostic marker products of specific probes. Methods Enzymol. 526, 145−157. (33) Michalski, R., Zielonka, J., Gapys, E., Marcinek, A., Joseph, J., and Kalyanaraman, B. (2014) Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid. J. Biol. Chem. 289, 22536−22553. (34) Lippert, A. R., Van de Bittner, G. C., and Chang, C. J. (2011) Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems. Acc. Chem. Res. 44, 793−804. (35) Kim, J., Park, J., Lee, H., Choi, Y., and Kim, Y. (2014) A boronate-based fluorescent probe for the selective detection of cellular peroxynitrite. Chem. Commun. (Cambridge, U. K.) 50, 9353−9356. (36) Xu, J., Li, Q., Yue, Y., Guo, Y., and Shao, S. (2014) A watersoluble BODIPY derivative as a highly selective ″Turn-On″ fluorescent sensor for H2O2 sensing in vivo. Biosens. Bioelectron. 56, 58−63. (37) Daniel, K. B., Agrawal, A., Manchester, M., and Cohen, S. M. (2013) Readily accessible fluorescent probes for sensitive biological imaging of hydrogen peroxide. ChemBioChem 14, 593−598. (38) Yuan, L., Lin, W., Xie, Y., Chen, B., and Zhu, S. (2012) Single fluorescent probe responds to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals. J. Am. Chem. Soc. 134, 1305− 1315. (39) Pichorner, H., Metodiewa, D., and Winterbourn, C. C. (1995) Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione. Arch. Biochem. Biophys. 323, 429− 437. (40) Winterbourn, C. C., Pichorner, H., and Kettle, A. J. (1997) Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils. Arch. Biochem. Biophys. 338, 15−21. (41) Wojcik, T., Buczek, E., Majzner, K., Kolodziejczyk, A., Miszczyk, J., Kaczara, P., Kwiatek, W., Baranska, M., Szymonski, M., and Chlopicki, S. (2015) Comparative endothelial profiling of doxorubicin and daunorubicin in cultured endothelial cells. Toxicol. In Vitro 29, 512−521. (42) Myers, C. E., McGuire, W. P., Liss, R. H., Ifrim, I., Grotzinger, K., and Young, R. C. (1977) Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science 197, 165−167.

(43) Davies, K. J., and Doroshow, J. H. (1986) Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase. J. Biol. Chem. 261, 3060−3067. (44) Doroshow, J. H., and Davies, K. J. (1986) Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical. J. Biol. Chem. 261, 3068−3074. (45) Kalyanaraman, B., Perez-Reyes, E., and Mason, R. P. (1980) Spin-trapping and direct electron spin resonance investigations of the redox metabolism of quinone anticancer drugs. Biochim. Biophys. Acta, Gen. Subj. 630, 119−130. (46) Yen, H. C., Oberley, T. D., Vichitbandha, S., Ho, Y. S., and St Clair, D. K., St (1996) The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J. Clin. Invest. 98, 1253−1260. (47) Vasquez-Vivar, J., Martasek, P., Hogg, N., Masters, B. S., Pritchard, K. A., Jr., and Kalyanaraman, B. (1997) Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin. Biochemistry 36, 11293−11297. (48) Weinstein, D. M., Mihm, M. J., and Bauer, J. A. (2000) Cardiac peroxynitrite formation and left ventricular dysfunction following doxorubicin treatment in mice. J. Pharmacol. Exp. Ther. 294, 396−401. (49) Shuai, Y., Guo, J. B., Peng, S. Q., Zhang, L. S., Guo, J., Han, G., and Dong, Y. S. (2007) Metallothionein protects against doxorubicininduced cardiomyopathy through inhibition of superoxide generation and related nitrosative impairment. Toxicol. Lett. 170, 66−74. (50) Chaiswing, L., Cole, M. P., St. Clair, D. K., St, Ittarat, W., Szweda, L. I., and Oberley, T. D. (2004) Oxidative damage precedes nitrative damage in adriamycin-induced cardiac mitochondrial injury. Toxicol. Pathol. 32, 536−547. (51) Pacher, P., Liaudet, L., Bai, P., Mabley, J. G., Kaminski, P. M., Virag, L., Deb, A., Szabo, E., Ungvari, Z., Wolin, M. S., Groves, J. T., and Szabo, C. (2003) Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction. Circulation 107, 896−904. (52) Mihm, M. J., Coyle, C. M., Schanbacher, B. L., Weinstein, D. M., and Bauer, J. A. (2001) Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure. Cardiovasc. Res. 49, 798−807. (53) Mihm, M. J., Yu, F., Weinstein, D. M., Reiser, P. J., and Bauer, J. A. (2002) Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase. Br. J. Pharmacol. 135, 581−588. (54) Mukhopadhyay, P., Rajesh, M., Batkai, S., Kashiwaya, Y., Hasko, G., Liaudet, L., Szabo, C., and Pacher, P. (2009) Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am. J. Physiol.: Heart Circ. Physiol. 296, H1466−H1483. (55) Majzner, K., Wojcik, T., Szafraniec, E., Lukawska, M., Oszczapowicz, I., Chlopicki, S., and Baranska, M. (2015) Nuclear accumulation of anthracyclines in the endothelium studied by bimodal imaging: fluorescence and Raman microscopy. Analyst (Cambridge, U. K.) 140, 2302−2310. (56) Patel, A. G., and Kaufmann, S. H. (2012) How does doxorubicin work? eLife 1, e00387. (57) Miller, E. W., Tulyathan, O., Isacoff, E. Y., and Chang, C. J. (2007) Molecular imaging of hydrogen peroxide produced for cell signaling. Nat. Chem. Biol. 3, 263−267. (58) Kissner, R., Beckman, J. S., and Koppenol, W. H. (1999) Peroxynitrite studied by stopped-flow spectroscopy. Methods Enzymol. 301, 342−352. (59) Bohle, D. S., Glassbrenner, P. A., and Hansert, B. (1996) Syntheses of pure tetramethylammonium peroxynitrite. Methods Enzymol. 269, 302−311. (60) Hrabie, J. A., Klose, J. R., Wink, D. A., and Keefer, L. K. (1993) New Nitric Oxide-Releasing Zwitterions Derived from Polyamines. J. Org. Chem. 58, 1472−1476. (61) Massey, V. (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim. Biophys. Acta 34, 255− 256. 745

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746

Article

Chemical Research in Toxicology (62) Pattison, D. I., Rahmanto, A. S., and Davies, M. J. (2012) Photooxidation of proteins. Photochem. Photobiol. Sci. 11, 38−53. (63) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J. A., Jr., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, O., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2009) Gaussian 09, revision A.02, Gaussian, Inc., Wallingford, CT. (64) Hargreaves, J. S., and Webber, S. E. (1985) Photon harvesting polymers - intracoil energy-transfer in anthryl-tagged and fluoresceintagged polystyrene. Can. J. Chem. 63, 1320−1327. (65) Tadic, D., and Brossi, A. (1990) Chiral prodyes - ethers and esters of dihydrofluorescein. I. Dibenzyldihydrofluorescein (DBDF) a new reagent. Heterocycles 31, 1975−1982. (66) Smulik, R., Debski, D., Zielonka, J., Michalowski, B., Adamus, J., Marcinek, A., Kalyanaraman, B., and Sikora, A. (2014) Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological Implications. J. Biol. Chem. 289, 35570− 35581. (67) Storkey, C., Davies, M. J., and Pattison, D. I. (2014) Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine, methionine, and peptide derivatives using a new competition kinetic approach. Free Radical Biol. Med. 73, 60−66. (68) Winterbourn, C. C., and Metodiewa, D. (1999) Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radical Biol. Med. 27, 322−328. (69) Trujillo, M., and Radi, R. (2002) Peroxynitrite reaction with the reduced and the oxidized forms of lipoic acid: new insights into the reaction of peroxynitrite with thiols. Arch. Biochem. Biophys. 397, 91− 98. (70) Davies, M. J. (2004) Reactive species formed on proteins exposed to singlet oxygen. Photochem. Photobiol. Sci. 3, 17−25. (71) Devasagayam, T. P., Sundquist, A. R., Di, M. P., Kaiser, S., and Sies, H. (1991) Activity of thiols as singlet molecular oxygen quenchers. J. Photochem. Photobiol., B 9, 105−116. (72) Aubry, J. M., Pierlot, C., Rigaudy, J., and Schmidt, R. (2003) Reversible binding of oxygen to aromatic compounds. Acc. Chem. Res. 36, 668−675. (73) Rougee, M., Bensasson, R. V., Land, E. J., and Pariente, R. (1988) Deactivation of singlet molecular oxygen by thiols and related compounds, possible protectors against skin photosensitivity. Photochem. Photobiol. 47, 485−489. (74) Keszler, A., Zhang, Y., and Hogg, N. (2010) Reaction between nitric oxide, glutathione, and oxygen in the presence and absence of protein: How are S-nitrosothiols formed? Free Radical Biol. Med. 48, 55−64. (75) Wojcik, T., Szczesny, E., and Chlopicki, S. (2015) Detrimental effects of chemotherapeutics and other drugs on the endothelium: A call for endothelial toxicity profiling. Pharmacol. Rep. 67, 811−817.

746

DOI: 10.1021/acs.chemrestox.5b00431 Chem. Res. Toxicol. 2016, 29, 735−746