Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species*,

Ken-ichi SetsukinaiDagger , Yasuteru UranoDagger , Katsuko Kakinuma§, Hideyuki J. Majima, and Tetsuo NaganoDagger ||

From the Dagger  Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the § Biophotonics Research Project/MMBS, Graduate School of Science, The University of Tokyo, Misaki, Miura, Kanagawa 238-0225, Japan, and the  Kagoshima University Dental School, Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan

Received for publication, September 10, 2002, and in revised form, October 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (·OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (-OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) by using HPF or APF alone, but -OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluorescein, and for the first time we could visualize -OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and -OCl in many biological and chemical applications.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species (ROS)1 play key roles in many pathogenic processes, including carcinogenesis (1), inflammation (2), ischemia-reperfusion injury (3), and signal transduction (4-7). Several methods, including electron spin resonance (8) and chemiluminescence (9), have been developed to detect ROS, but fluorescence detection is superior in terms of high sensitivity and experimental convenience. Experimental studies on Ca2+-dependent signal transduction in cells were greatly facilitated by the development of fluorescent indicators for cytosolic Ca2+ (10, 11). Several fluorescence probes to detect ROS, such as 2',7'-dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123, have also been developed. However, as Hempel and co-workers (12) pointed out, DCFH and dihydrorhodamine 123 can react with various ROS and oxidizing species (superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>), hydrogen peroxide (H2O2), nitric oxide (NO), ferrous ion, and others), and in addition, DCFH is easily autoxidized, resulting in a spontaneous increase in fluorescence upon exposure to light. Therefore, it is not appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2 or NO, but rather they should be considered as detecting a broad range of oxidizing reactions that may be increased during intracellular oxidative stress (12).

There are many species of ROS, as mentioned above, but they tend to be considered collectively as "oxidative stress" when their effects in living cells are discussed. However, we believe that each species of ROS is likely to have a specific role in living cells. There is some evidence for this view. For example, H2O2 is an endothelium-derived hyperpolarizing factor in human and mice (13), p38 mitogen-activated protein kinase mediates caspase-3 activation during apoptosis induced by singlet oxygen (1O2) but not by H2O2 (14), and hydroxyl radical (·OH) plays an important role as a second messenger in T cell activation (15). In addition, each species of ROS has a characteristic chemical reactivity; for example, 1O2 reacts with anthracenes to yield endoperoxides in the Diels-Alder mode (16), whereas ·OH can react directly with aromatic rings to yield hydroxylated products (17), and NO reacts with guanine to yield the deaminated compound (18). However, because of the problems, i.e. lack of selectivity among species and autoxidation (12, 19-20), the roles of an individual species of ROS in living cells remain uncertain. Therefore, we believe that it is very important to be able to detect each species of ROS selectively. If novel fluorescence probes that overcome the above problems were available, they would contribute greatly to the elucidation of the roles of individual ROS in living cells, because we would be able to "see" the generation of specific ROS with high resolution in time and space.

It is known that ·OH participates in various biological processes. For example, HeLa, MW451, and HL-60 cells are induced to undergo apoptosis by ·OH (21). ·OH can damage DNA bases (1) and mediates redox alteration of cell-membrane Ca2+ channels (22). However, because of the lack of effective direct detection methods for ·OH, its participation in these events has been established only indirectly by using inhibitors such as dithioethanol, glutathione, and desferrioxamine (1, 15, 21, 22). Therefore, we wished to develop novel fluorescence probes for highly reactive oxygen species (hROS). Here, we use the term hROS to indicate reactive oxygen species with strong oxidizing power sufficient to directly hydroxylate aromatic rings (for example, ·OH or reactive intermediates of peroxidase).

We report herein the development of novel fluorescence probes for ROS, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), which can specifically detect certain species of ROS in terms of an increase of fluorescence and exhibit complete resistance to autoxidation both in vitro and in vivo. We also describe the visualization of hypochlorite (-OCl) in stimulated neutrophils.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 4-Fluoronitrobenzene, 4-iodophenol, isobutene, 2,2,2-trifluoroethanol, and trifluoromethanesulfonic acid were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Uranine (sodium fluorescein), cuprous chloride, 2,2'-azobis(2-amidinopropane)dihydrochloride, horseradish peroxidase (HRP, EC 1.11.1.7), polyvinylpyrrolidone, and 4beta -phorbol-12-myristate-13-acetate (PMA) were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). Fluorescein was purchased from Aldrich. DCFH-DA and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) were purchased from Molecular Probes (Eugene, OR). 3-(4-Hydroxyphenyl)propionic acid (HPPA) was purchased from Dojindo Laboratories (Kumamoto, Japan). Myeloperoxidase (MPO, EC 1.11.1.7) was purchased from Calbiochem (San Diego, CA). Pyridine, dichloromethane, dimethyl sulfoxide (Me2SO), and methanol were used after distillation. Other materials were of the best grade available and used without further purification.

Instruments-- 1H NMR spectra were recorded on a JEOL JNM-LA300 instrument at 300 MHz. Mass spectra were determined with a JEOL JMS-SX102A mass spectrometer. Fluorescence spectroscopic studies were performed on a Hitachi F4500.

Fluorometric Analysis-- The slit width was 2.5 nm for both excitation and emission. The photomultiplier voltage was 950 V. HPF, APF, and DCFH-DA were dissolved in DMF to obtain 10 mM stock solutions. We obtained DCFH by hydrolyzing DCFH-DA with 0.01 M aqueous NaOH for 30 min at 37 °C in the dark (12).

Cell Lines-- A human hepatocellular carcinoma cell line (HLE) was purchased from the Health Science Research Resources Bank of Japan Health Sciences Foundation (Osaka, Japan). PCR3.1-Uni plasmid (Invitrogen) containing a sense human manganase-superoxide dismutase cDNA insert was a kind gift of Dr. Makoto Akashi (National Institute of Radiological Sciences, Chiba, Japan). The HLE cell line was transfected using the GenePORTER transfection procedure (Gene Therapy Systems, San Diego, CA) according to the previous report by Motoori and co-workers (23). Manganase-superoxide dismutase-transfected HLE cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and 500 µg/ml Genetecin at 37 °C in humidified air containing 5% CO2. Genetecin was removed at least 24 h before the experiments were performed.

Light-induced Autoxidation-- Manganase-superoxide dismutase-transfected HLE cells were seeded 1 day before dye loading onto a glass-bottomed dish. The cells were then rinsed with modified Hanks' balanced salt solution containing 10.0 mM HEPES, 1.0 mM MgCl2, 2.0 mM CaCl2, and 2.7 mM glucose adjusted to pH 7.3 ± 0.05. Then, the cells were loaded with HPF or DCFH-DA (10 µM) by incubation for 30 min at 37 °C in the dark. Fluorescence images were acquired using a CSU-10 confocal laser scanning unit (Yokogawa Electric Co., Tokyo, Japan) coupled to an IX90 inverted microscope with an UPlanAPO ×20 objective lens (Olympus Optical Co.) and a C5810-01 color chilled 3CCD camera (Hamamatsu Photonics K. K.). The excitation wavelength was 488 nm, and the emission was filtered using a 515-nm barrier filter. After loading the dyes, the fluorescence images were acquired. After that, the cells were laser-irradiated at 488 nm for 10 s, and then the fluorescence images were acquired again. The laser power, the exposure time of the 3CCD camera (for acquiring the fluorescence images), and the gain of the amplifier were held at 500 µW, 1 s, and 18 decibels, respectively, to allow quantitative comparisons of the relative fluorescence intensity of the cells between groups.

Preparation of Porcine Neutrophils-- Neutrophils were obtained from 1.8 liters of porcine blood basically according to the method of Wakeyama and co-workers (24) with some modifications (25). Erythrocytes in the buffy coat collected from blood were hemolyzed with a large volume of ice-cold 0.2% NaCl solution for 30 s, and then the preparation was promptly mixed with an equal volume of ice-cold 1.6% NaCl solution to restore the isotonic condition. Neutrophils were separated from platelets and mononuclear cells by the Conray-Ficoll method described previously (26). The neutrophils were suspended in Krebs-Ringer phosphate buffer (114 mM NaCl, 4.6 mM KCl, 2.4 mM MgSO4, 1.0 mM CaCl2, 15 mM NaH2PO4/Na2HPO4, pH 7.4) and kept on ice until use.

Bioimaging of Neutrophils-- Separated porcine neutrophils were seeded onto a glass-bottomed dish. Then, the cells were loaded with HPF or APF (10 µM) by incubation for 30 min at room temperature. Dye-loaded neutrophils were stimulated with PMA (2 ng/ml; 0.1% DMF was contained as a cosolvent). Fluorescence images were acquired twice in each experiment (before and 10 min after the stimulation with PMA) using an LSM510 confocal laser scanning unit (Carl Zeiss Co., Ltd.) coupled to an Axiovert 100M inverted microscope with a Plan-Neofluar ×100/1.3 objective lens (Carl Zeiss Co., Ltd.). The excitation wavelength was 488 nm, and the emission was filtered using a 505-550 nm barrier filter.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design and Synthesis of HPF and APF-- We reported previously that aryloxyphenols are O-dearylated in the ipso-substitution manner by hROS such as ·OH, reactive intermediates of peroxidase, and cytochrome P450, but not by other ROS (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, H2O2, 1O2, and so on) (27, 28). In addition, our recent investigation of the absorption and fluorescence properties of fluorescein derivatives showed that the fluorescence of fluorescein could be quenched by protection of the phenolic hydroxy group at the 6'-position of fluorescein with an electron-rich aromatic ring.2 Therefore, we designed and synthesized two novel fluorescence probes for hROS, HPF and APF, by making use of this fact. We expected that almost nonfluorescent HPF and APF would be O-dearylated upon reaction with hROS to yield strongly fluorescent fluorescein. The putative reaction scheme is shown in Fig. 1. The dynamic range of fluorescence augmentation should be wide, because both molar absorptivity and quantum efficiency values are greatly increased upon O-dearylation, so these compounds should operate as sensitive and selective fluorescence probes for hROS. HPF and APF were obtained in only 3 steps and 2 steps, respectively (see Supplemental Materials).


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Fig. 1.   Scheme of O-dearylation reaction of HPF and APF with hROS. epsilon  is the molar absorptivity, and phi fl is the relative quantum efficiency of fluorescence.

Reactivity of HPF and APF with ·OH-- First, we investigated the reactivity of HPF and APF for chemically generated hROS. We tried to detect ·OH, one of the hROS, formed in the Fenton reaction, using HPF and APF (Fig. 2A). H2O2 was added to buffer solutions of HPF and APF and then ferrous perchlorate was added. The fluorescence intensity did not increase upon the addition of H2O2 alone, but increased substantially upon the addition of ferrous perchlorate in the presence of H2O2. The results clearly showed that both HPF and APF could detect ·OH selectively. In addition, the fluorescence increase caused by the reaction with ·OH was suppressed in buffer solutions of HPF and APF containing Me2SO (a quencher of ·OH), and we confirmed the production of fluorescein in the reaction mixture with ·OH by reverse-phase high-performance liquid chromatography and three-dimensional fluorescence spectroscopy (data not shown). Furthermore, we examined the relation between the concentration of ferrous perchlorate and the fluorescence increase in the Fenton reaction (Fig. 2B). Ferrous perchlorate was added at various concentrations to buffer solutions of HPF and APF containing an excess of H2O2. The results showed that the fluorescence increase is proportional to the concentration of ferrous perchlorate. Therefore, both HPF and APF can detect ·OH formed in the Fenton reaction in terms of a dose-dependent increase of fluorescence.


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Fig. 2.   Detection of ·OH in the Fenton reaction using HPF and APF. A, HPF (lower line) or APF (upper line) (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4). The fluorescence intensity was determined at 515 nm with excitation at 490 nm. H2O2 (final 1 mM) was added at 1, and ferrous perchlorate (final 100 µM) was added two times at 2 and 3. B, relation between the concentrations of added ferrous perchlorate and fluorescence increase in the Fenton reaction. Data are mean ± S.E. (n = 3). HPF (circle) or APF (square) (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4) containing H2O2 (1 mM). The wavelength for measurement was the same as A.

Reactivity of HPF and APF with -OCl-- -OCl has a strong microbicidal activity, and plays a key role in the killing of bacteria by neutrophils (29, 30). Therefore, we investigated the reactivity of HPF and APF with -OCl. The results are shown in Fig. 3. Interestingly, the fluorescence intensity of APF greatly increased upon addition of -OCl, whereas that of HPF did not. In addition, the fluorescence increase of APF was dose-dependent. Therefore, we could detect -OCl selectively by using both HPF and APF together.


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Fig. 3.   Detection of -OCl by HPF and APF. Relation between the concentrations of added NaOCl and fluorescence increase. HPF (square) or APF (circle) (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4). The fluorescence intensity was determined at 515 nm with excitation at 490 nm.

Reactivity of HPF, APF, and DCFH with Various ROS, and the Lability of the Probes to Light-induced Autoxidation-- As mentioned above, DCFH is widely used as a fluorescence probe for ROS, but it lacks specificity among ROS and suffers from autoxidation; that is, the fluorescence increases even in the absence of ROS upon illumination. We compared the reactivities of HPF, APF, and DCFH with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, H2O2, ·OH, 1O2, -OCl, NO, peroxynitrite (ONOO-), and alkylperoxyl radical (ROO·). The observed fluorescence increases are shown in Table I. 3-(1,4-Dihydro-1,4-epidioxy-1-naphthyl)propionic acid thermally generates 1O2 under mild conditions (31) and 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene thermally generates NO under mild conditions (32). 2,2'-Azobis(2-amidinopropane)dihydrochloride is an azo-initiator that forms alkyl radicals as a result of thermal decomposition, and these alkyl radicals can react with molecular oxygen to give alkylperoxyl radicals (33). Under these conditions, DCFH reacted unselectively with all of these reactive species. On the other hand, HPF and APF showed fluorescence augmentation only upon reaction with ·OH, ONOO-, and/or -OCl, and not with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, H2O2, 1O2, NO, ROO·, which are thought to be produced in many biological systems. ONOO- has a strong oxidizing power (34, 35), so it should be included in the category of hROS. Furthermore, DCFH was extensively autoxidized, resulting in a marked increase of the fluorescence intensity, whereas HPF and APF were not autoxidized at all. These results show that HPF and APF have much higher selectivity among ROS and a greater resistance to autoxidation than DCFH.

                              
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Table I
Fluorescence increase of HPF, APF, and DCFH in various ROS generating systems
Dyes (final 10 µM, 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4). The fluorescence intensities of HPF, APF, and DCFH were measured at 515, 515, and 520 nm with excitation at 490, 490, and 500 nm, respectively. DCFH was obtained by the hydrolysis of DCFH-DA with base as mentioned under "Experimental Procedures."

Next, we examined whether light-induced autoxidation would occur under conditions practically used for excitation in fluorescence microscopy. We loaded HPF or DCFH-DA into HLE cells and irradiated the dye-loaded cells for 10 s. Fluorescein and dichlorofluorescein, the autoxidized products, form dianions in the buffer (pH 7.3) (36, 37), and therefore tend to remain in the intracellular medium. The results are shown in Fig. 4. HPF could permeate the cell membrane and enter into cells. DCFH was much more easily autoxidized by light irradiation than HPF in cells under conditions practically used for excitation in fluorescence microscopy.


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Fig. 4.   Light-induced autoxidation in DCFH-DA-loaded HLE cells (A) and HPF-loaded HLE cells (B). HLE cells were loaded with DCFH-DA or HPF (10 µM; 0.1% DMF as a cosolvent) by incubation for 30 min at 37 °C in the dark. Fluorescence images were acquired. After that, the cells were laser-irradiated at 488 nm for 10 s, and fluorescence images were acquired again.

Application of HPF and APF to an Enzymatic System-- We investigated whether HPF and APF could detect hROS generated in an enzymatic system, i.e. the HRP/H2O2 system. In this system, two-electron oxidation of the native enzyme (HRP) to compound I is followed by two one-electron reductions to yield the resting state of HRP (38). H2O2 was added to buffer solutions of HPF and APF containing HRP. As shown in Fig. 5A, the fluorescence intensity increased immediately upon the addition of H2O2. Furthermore, it was found that HPF and APF could detect hROS generated in the HRP/H2O2 system in a dose-dependent manner (Fig. 5B). Thus, the data in Fig. 5 really show that HPF and APF can serve as substrates for horseradish peroxidase, and HPF and APF could detect hROS in a dose-dependent manner not only in a chemical system, but also in an enzymatic system.


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Fig. 5.   Detection of hROS in the HRP/H2O2 system using HPF and APF. A, representative data are shown (n = 3). HPF (lower line) or APF (upper line) (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4) containing HRP (0.2 µM). H2O2 (final 1 µM) was added at the time indicated by the arrow. The fluorescence intensity was determined at 515 nm with excitation at 490 nm. These reactions were performed at 37 °C. B, relation between the amount of added H2O2 and fluorescence increase in the HRP/H2O2 system using HPF (circle) and APF (square). Data are mean ± S.E. (n = 3). Dyes (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4) containing HRP (0.2 µM). The fluorescence intensity was determined at 515 nm with excitation at 490 nm. These reactions were performed at 37 °C for 5 min.

Next, we compared the reactivity for hROS generated in the HRP/H2O2 system and the lability to light-induced autoxidation among our novel fluorescence probe APF and two widely used fluorescence probes for peroxidase (Amplex Red and HPPA) (39, 40). APF had slightly greater reactivity than Amplex Red for hROS generated in the HRP/H2O2 system and much greater reactivity than HPPA (Fig. 6A). Furthermore, APF had much greater resistance to autoxidation than Amplex Red (Fig. 6B). Therefore, APF is superior to the most widely used fluorescence probes for peroxidase.


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Fig. 6.   Comparison between APF, Amplex Red, and HPPA. A, comparison of the sensitivity in the HRP/H2O2 system. Dyes (final 10 µM; 0.1% DMF as a cosolvent) were added to 0.15 M KCl, 25 mM Tris-HCl buffer (pH 7.4) containing HRP (0.2 µM). H2O2 (final 3 µM) was added at the time indicated by the arrow. The fluorescence intensity with APF, Amplex Red, and HPPA was determined at 515, 580, and 405 nm with excitation at 490, 570, and 320 nm, respectively. These reactions were performed at 37 °C. B, comparison of the lability of light-induced autoxidation. Dyes (final 10 µM; 0.1% DMF as a cosolvent) were added to 0.15 M KCl, 25 mM Tris-HCl buffer (pH 7.4). Dyes were placed under a fluorescent lamp for the indicated time.

Furthermore, we applied HPF and APF to the MPO/H2O2/Cl- system. In the presence of Cl-, -OCl is predominantly produced via the reactive intermediate, compound I. The results are shown in Fig. 7. APF showed a dose-dependent fluorescence increase in this system, whereas HPF showed no fluorescence. These results correspond well with the reactivities of HPF and APF for -OCl. Therefore, we succeeded in visualizing the production of -OCl in the MPO/H2O2/Cl- system.


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Fig. 7.   Application of HPF and APF to the MPO/H2O2/Cl- system. A, representative data are shown (n = 3). HPF (lower line) or APF (upper line) (final 10 µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1 M, pH 7.4) containing MPO (11.2 nM) and NaCl (150 mM). H2O2 (final 1 µM) was added at the time indicated by the arrow. The fluorescence intensity was determined at 515 nm with excitation at 490 nm. These reactions were performed at 37 °C. B, relation between the amount of added H2O2 and fluorescence increase in the MPO/H2O2/Cl- system using HPF (circle) and APF (square). Data are mean ± S.E. (n = 3). The buffer and wavelength for measurement were same as A. These reactions were performed at 37 °C for 25 min.

Application of HPF and APF to Neutrophils-- Neutrophils are a population of circulating blood cells, and their primary function is host defense against pathogenic microorganisms. Exposure of neutrophils to various stimuli such as PMA (41) and fatty acids (42) activates the "respiratory burst oxidase," NADPH oxidase, to generate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, which is then converted to H2O2 and O2 (43, 44). As neither O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> nor H2O2 is strongly microbicidal, these species are thought to be precursors of more potent oxidizing agents, such as ·OH, -OCl, and 1O2. Neutrophils contain azurophilic granules, in which MPO exists abundantly, and MPO has been shown to catalyze the formation of -OCl from H2O2 and Cl- in vitro (45). Therefore, we tried to apply our novel fluorescence probes to neutrophils. We stimulated HPF- or APF-loaded neutrophils with PMA, and observed the dye-loaded neutrophils without washing out the extracellular medium. The results are shown in Fig. 8. It is noteworthy that the fluorescence intensity of HPF-loaded neutrophils did not change upon stimulation with PMA, whereas that of APF-loaded neutrophils greatly increased. Our results suggest that MPO produces -OCl in the presence of both Cl- and H2O2, which is generated by the stimulation with PMA, and we could identify this -OCl production by using HPF and APF together. In other words, we could for the first time visualize -OCl selectively, distinguishing it from other ROS, by using HPF and APF together, even in a biological system.


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Fig. 8.   Fluorescence images of HPF- or APF-loaded neutrophils. HPF or APF (final 10 µM; 0.1% DMF as a cosolvent) were loaded into neutrophils for 30 min at room temperature, and the dye-loaded neutrophils were stimulated with PMA (2 ng/ml). Fluorescence images were acquired before and 10 min after the stimulation with PMA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have succeeded in developing novel autoxidation-resistant fluorescence probes, HPF and APF, that can reliably detect hROS and/or -OCl selectively. Because it is likely that individual ROS have distinct roles in biological systems, the availability of selective fluorescence probes will be extremely useful. For example, by using HPF or APF, we can distinguish ·OH from NO. This is very important, because DCFH reacts with both ·OH and NO and so cannot be used reliably to study the biological role of ·OH. In addition, the mere production of H2O2 is completely different in terms of cell damage from the situation in which H2O2 is converted into hROS in the presence of low-valent metal ions. We feel our probes are useful here, because they can distinguish these two situations. Furthermore, we can also distinguish ONOO- from NO or O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. It has been reported that ONOO- can be generated from NO and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in vitro and in vivo (46, 47), and therefore we will be able to visualize the production of ONOO- with a clear distinction from that of NO or O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, and this will allow a reliable evaluation of the role of ONOO- in various processes. Furthermore, we could detect -OCl selectively by using HPF and APF together, because HPF shows no fluorescence increase with -OCl, whereas APF shows a dose-dependent increase. The ability to selectively detect individual species of ROS represents a major advance.

As shown in Table I and Fig. 4, the currently used fluorescence probe DCFH is easily autoxidized by light irradiation. This means that precautions must be taken to exclude light during incubation to load DCFH-DA into cells, and it is necessary to change the visual field often during observations. However, HPF and APF are not autoxidized at all, as shown in Table I and Fig. 4. Therefore, we believe HPF and APF will contribute greatly to the elucidation of the roles of ROS in living cells by making it possible to see the generation of specific ROS with high resolution in time and space. Although the sensitivity of HPF and APF is inferior to that of DCFH (Table I), lability to autoxidation and selectivity among ROS, rather than sensitivity, are considered to be critical for fluorescence probes for ROS.

The question arises, why are HPF and APF selective for hROS, unlike DCFH? DCFH is nonfluorescent, and HPF and APF possess low fluorescence quantum efficiency, and all of them are converted to strongly fluorescent compounds, dichlorofluorescein or fluorescein, by oxidation. However, DCFH is converted to dichlorofluorescein, initially via abstraction of the hydrogen atom at the 9'-position, whereas HPF and APF are converted to fluorescein, initially via abstraction of the hydrogen atom of the phenolic hydroxy group or abstraction of one electron from the nitrogen atom. The hydrogen atom at the 9'-position of DCFH is readily abstracted because this hydrogen atom can be considered as being located at the central carbon of a triphenylmethane. It is therefore vulnerable even to a weakly oxidizing species, and this is the reason why DCFH lacks the selectivity among ROS. However, a strongly oxidizing species is required for the ipso-substitution reaction of HPF and APF. Therefore, we conclude that the difference of oxidizing power required for oxidation reaction used for detection causes the difference of selectivity among ROS. Furthermore, the fact that HPF shows no fluorescence increase with -OCl, whereas APF does (Fig. 3 and Table I), reflects the difference in lability to oxidation between an aryloxyphenol and an aryloxyaniline.

HPF and APF could detect hROS generated in the HRP/H2O2 system (Fig. 5). HRP is often used as an enzyme label in immunohistochemical studies, 3,3-diaminobenzidine is commonly used as a substrate for measurement of the peroxidase activity. However, 3,3-diaminobenzidine can be detected only by absorbance measurement and is easily autoxidized by light irradiation. Because HPF and APF permit fluorescence detection, which has higher sensitivity than absorbance detection, and they are not autoxidized by light irradiation at all, they are likely to be more effective reagents for immunohistochemistry using peroxidase than 3,3-diaminobenzidine and related compounds.

We also used HPF and APF to visualize the production of -OCl from neutrophils (Fig. 8). Dye-loaded neutrophils weakly fluoresced before the stimulation with PMA, because the dyes were taken up by pinocytosis and MPO was slightly released into pinocytic vacuoles. Nevertheless, the fluorescence intensity of APF-loaded neutrophils markedly increased, in contrast to little fluorescence increase of HPF-loaded cells upon stimulation with PMA. -OCl is believed to play important roles not only in bacterial killing bacteria by neutrophils but also in injury to the venular endothelial surface in platelet-activating factor-induced microvascular damage (48). However, it has been difficult to draw firm conclusions concerning direct participation of -OCl because a completely selective detection method for -OCl has never been developed. Therefore, our finding that we could detect -OCl selectively by using HPF and APF together will make it possible for the first time to elucidate reliably the roles of -OCl in biological systems such as neutrophils.

In summary, we have developed novel fluorescence probes, HPF and APF, that can selectively and dose dependently detect certain species among ROS and that are highly resistant to autoxidation. They can be used in enzymatic and cellular systems. They are greatly superior to the existing fluorescence probes for ROS, and are expected to have many chemical and biological applications.

    ACKNOWLEDGEMENT

We thank Dr. Hidehiko Nakagawa (National Institute of Radiological Sciences, Chiba, Japan) for providing peroxynitrite solution.

    FOOTNOTES

* This work was supported by Ministry of Education, Science, Sports and Culture of Japan Research Grants 11794026, 12470475, 12557217 (to T. N.), 10771238 and 12771349 (to Y. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains text and additional references.

|| To whom correspondence should be addressed. Tel.: 81-3-5841-4850; Fax: 81-3-5841-4855; E-mail: tlong@mol.f.u-tokyo.ac.jp.

Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209264200

2 K. Setsukinai, Y. Urano, and T. Nagano, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; DCFH, 2',7'-dichlorodihydrofluorescein; HPF, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid; APF, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid; HRP, horseradish peroxidase; PMA, 4beta -phorbol-12-myristate-13-acetate; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; Amplex Red, 10-acetyl-3,7-dihydroxyphenoxazine; HPPA, 3-(4-hydroxyphenyl)propionic acid; MPO, myeloperoxidase; DMF, N,N-dimethylformamide; HLE, human hepatocellular carcinoma cell line; CCD, charge coupled device; hROS, highly reactive oxygen species; ROO·, alkylperoxyl radical; NO, nitric oxide; O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, superoxide radical.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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