From the 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
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ABSTRACT |
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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
( 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 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 ( 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
4 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.
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 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.
Reactivity of HPF and APF with
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
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.
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.
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.
Furthermore, we applied HPF and APF to the
MPO/H2O2/Cl 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 We have succeeded in developing novel autoxidation-resistant
fluorescence probes, HPF and APF, that can reliably detect hROS and/or
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
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
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.
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
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
OCl) in
stimulated neutrophils.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Scheme of O-dearylation
reaction of HPF and APF with hROS. is the molar absorptivity,
and
fl is the relative quantum efficiency 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.
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.
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
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.
Fluorescence increase of HPF, APF, and DCFH in various ROS generating
systems
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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.
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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.
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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.
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.
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
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
can be generated from NO and
O
with a clear distinction from that of NO or
O
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.
OCl, whereas APF does (Fig. 3 and Table I), reflects the
difference in lability to oxidation between an aryloxyphenol and an aryloxyaniline.
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.
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ACKNOWLEDGEMENT |
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We thank Dr. Hidehiko Nakagawa (National Institute of Radiological Sciences, Chiba, Japan) for providing peroxynitrite solution.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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, 4-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
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