Free Radical Biology & Medicine 40 (2006) 968 – 975
www.elsevier.com/locate/freeradbiomed
Original Contribution
The oxidation of 2V,7V-dichlorofluorescin to reactive oxygen species:
A self-fulfilling prophesy?
Marcelo G. Bonini a,*, Cristina Rota b, Aldo Tomasi b, Ronald P. Mason a
a
b
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC 27709, USA
Department of Biomedical Sciences and Free Radical Metabolite Section, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy
Received 12 September 2005; revised 17 October 2005; accepted 18 October 2005
Available online 4 November 2005
Abstract
The oxidation of 2V,7V-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both
DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build
up long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system.
S
Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of
H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate
S
S
form with the concomitant formation of DCF semiquinone and DCFHDA-derived radicals. These are oxidized by O2, producing O2 (as
demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA
oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH,
presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a
variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure;
therefore, its use in biological systems should be approached with caution.
Published by Elsevier Inc.
Keyword: Peroxynitrite; Peroxidase; DCF; DCFH; EPR; Free radicals
Introduction
2V,7V-Dichlorofluorescin (DCFH) and its diacetate form
(DCFHDA) are widely used to measure oxidative stress in
cells due to the high sensitivity of fluorescence-based assays.
The assay consists of the oxidation of DCFH (after hydrolysis
of the diacetate form) to fluorescein by ferryl-type intermedi-
S
Abbreviations: DCF, 2V,7V-dichlorofluorescein; DCF , DCF semiquinone
free radical; DCFH, 2V,7V-dichlorofluorescin; DCFHDA, 2V,7V-dichlorofluorescin diacetate; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DTPA, diethylenetriamine pentaacetic acid; OH, hydroxyl free radical; OOH, superoxide free
radical; peroxynitrite/PN, both peroxynitrous acid (ONOOH) and peroxynitrite
anion (ONOO), which are in equilibrium at near neutral pH (pK a 6.6), unless
otherwise specified.
* Corresponding author. Fax: +1 919 541 1043.
E-mail address: [email protected] (M.G. Bonini).
S
S
0891-5849/$ - see front matter. Published by Elsevier Inc.
doi:10.1016/j.freeradbiomed.2005.10.042
ates and/or oxygen and nitrogen reactive species, whose
fluorescence can be measured at 522 nm [1].
However, there are many controversies regarding its
validity. The oxidation of DCFH alone is not enough to
establish the formation of specific reactive species nor to probe
the mechanisms by which such oxidation occurred. LeBel et al.
[2] emphasized that the interpretation of specific reactive
oxygen species involved in the oxidation of DCFH to DCF in
biological systems should be approached with caution. In our
laboratory, we demonstrated that the photoreduction of DCF
results in the formation of the DCF semiquinone free radical
S
(DCF ) which, under aerobic conditions, is oxidized by
oxygen to its parent dye, DCF, concomitantly forming
superoxide radical [3]. We also reported that DCFH, upon
reacting with horseradish peroxidase compound I or compound
S
II, was oxidized to DCF , which was subsequently airoxidized to DCF with the generation of superoxide radical [4].
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
More recently we demonstrated that the oxidation of the
fluorescent dye DCF by horseradish peroxidase resulted in the
S
formation of the oxidizing DCF phenoxyl free radical (DCF )
[5]. Interestingly, DCFH and structurally similar fluorogenic
probes such as dihydrorhodamine 123 have been invoked as
specific probes to measure nitrogen reactive species, in
particular, peroxynitrite [6 –9].
In the present paper, the reaction of DCFH with
horseradish peroxidase and peroxynitrite was studied. Our
main goal was to evaluate the validity of this technique to
assess peroxynitrite production in complex systems. Our
results demonstrate that peroxynitrite indeed oxidized HRP
to HRP compound I (as previously demonstrated [10]), which
was probably responsible for the initial oxidation of DCFH
and DCFHDA to fluorescent products. Interestingly,
DCFHDA was considerably more resistant to peroxynitritemediated oxidation than its hydrolyzed counterpart, confirming previous observations [8]. However, DCFH and
DCFHDA oxidation proceeded for prolonged incubation
periods far beyond peroxynitrite’s lifetime, producing exceedingly high yields of fluorescein. More important, fluorescence
buildup was inhibited by catalase and superoxide dismutase.
Taken together, these findings suggest that DCFH/DCFHDAderived oxidants were produced, which powered the system at
prolonged incubation times. In the cell biology literature,
inhibition of DCF formation by superoxide dismutase and/or
catalase is routinely interpreted as proof that superoxide and/
or hydrogen peroxide is formed in the biological system in
question. This investigation proved that the formation of these
species is intrinsic to the oxidation of DCFH or DCFHDA by
peroxynitrite in the presence of horseradish peroxidase and,
presumably, in the presence of any hemoprotein with
peroxidase activity.
Experimental procedures
Chemicals
Horseradish peroxidase type VI-A (EC 1.11.1.7), diethylenetriamine pentaacetic acid (DTPA), and the spin trap 5,5dimethyl-1-pyrroline N-oxide (DMPO) were purchased from
Sigmas Co. (St. Louis, MO, USA). DMPO was purified by
vacuum distillation at ambient temperature and stored at
70-C until use. DCFHDA was purchased from Molecular
Probes (Eugene, OR, USA). DCFH was produced immediately
before each assay by incubating DCFHDA in NaOH (0.1 M)
for 20 min. After incubation, basic DCFH solutions were
diluted in phosphate buffer for the experiments. Sodium
phosphate was purchased from Mallin Krodt Aker, Inc. (Paris,
KY, USA). Chelex 100 resin was purchased from Bio-Rad
Laboratories (Hercules, CA, USA). Catalase (from beef liver,
65,000 U/mg) (EC 1.11.1.6) and superoxide dismutase (from
bovine erythrocytes, 5000 U/mg) (EC 1.15.1.1) were purchased from Roche Molecular Biochemicals (Indianapolis, IN,
USA). All the reactions were carried out in 150 mM sodium
phosphate buffer, pH 7.4. The buffer was treated with Chelex
100 resin to remove traces of transition metal ions and
969
contained 100 AM DTPA to minimize the possibility of trace
metal interference.
Peroxynitrite synthesis
Peroxynitrite was synthesized as previously described [11].
Briefly, NaNO2 (0.65 M) was reacted with H2O2 (0.70 M) in a
quenched flow reactor. Excess H2O2 was removed by MnO2
addition. Typically, freshly synthesized peroxynitrite solutions
were stirred on MnO2 for 30 min on ice. Concentrations of
H2O2 and NO2 in stock solutions (measured at 240 nm ( =
43.6 M1 cm1 and 254 nm ( = 23.6 M1 cm1, respectively,
after peroxynitrite decomposition) did not exceed 1 and 35%,
respectively. Stock concentrations of peroxynitrite were determined before each set of experiments by using the extinction
coefficient ( = 1670 M1 cm1 at 302 nm [12,13].
Electron paramagnetic resonance experiments
EPR spectra were recorded on a Bruker EMX EPR
spectrometer (Billerica, MA, USA) operating at 9.81 GHz
with a modulation frequency of 100 kHz and equipped with an
ER 4122 SHQ cavity. All experiments were performed at room
temperature with a 17-mm quartz flat cell. The data analysis
and spectral simulation were performed using programs
developed in our laboratory and available through the Internet
(http://www.epr.niehs.nih.gov/). The details of the program
have been described elsewhere [14].
Fluorescence spectroscopy
Fluorometric measurements were made with a TECAN
Spectrafluor Plus. Fluorescence levels were measured immediately before peroxynitrite addition and then continuously up
to 50 min. Optical filters were used to select the excitation
wavelength at 492 nm, and the emission was measured at
530 nm.
Horseradish peroxidase UV – visible spectra
All optical measurements were carried out with a Varian
Cary 100 Bio spectrophotometer.
Oxygen uptake experiments
Oxygen uptake measurements were made with a Clark-type
oxygen electrode fitted to a 1.8-ml Gilson sample cell and
monitored by a Model 53 oxygen monitor (Yellow Springs
Instruments, Yellow Springs, OH, USA). The point of reagent
addition during the course of the experiment is marked with an
arrow. After establishing a baseline measurement, reactions
were initiated by adding peroxynitrite to the reaction chamber.
Oxygen concentration in the samples as a function of time was
recorded by a PC interfaced to the oxygen monitor with a Data
Translation DT2801 data acquisition board. The reaction
chamber had its temperature controlled and set to 37-C. At
this temperature the saturating concentration of O2 corres-
970
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
ponding to the full electrode response at 100% was taken as
211 AM [15].
Calculations
The first-order rate constant (k) for peroxynitrite decomposition at pH 7.4 has been calculated to be 0.17 s1 [16], giving
a half-life of approximately 4 s under these conditions:
k ¼ 1:2½H þ =ðKa þ ½H þ Þ:
Results
Fluorescence studies
DCFHDA is commonly used as a probe to detect the
formation of reactive oxygen and nitrogen species in complex
systems because of the high sensitivity of fluorimetric assays.
Particularly, DCFHDA or its hydrolyzed form, DCFH, has
been suggested as an adequate probe to detect peroxynitrite
production in cell cultures. Because of that, we examined the
oxidation of the probes mediated by peroxynitrite/HRP under
the conditions typically employed in the presence and in the
absence of catalase and superoxide dismutase. As shown in
Fig. 1, trace A, addition of peroxynitrite (1 AM) to solutions
containing HRP (1 AM) and DCFHDA (5 AM) produced a
sustained increase in the fluorescence measured at 530 nm. The
increase was observed for periods of almost 1 h, long after
peroxynitrite completed decomposition (see Experimental
procedures, Calculations). Addition of Cu,Zn-SOD (30 AM)
slightly reduced the rate of fluorescence buildup (Fig. 1, trace
B). In contrast, addition of catalase (20 AM) dramatically
inhibited DCFHDA oxidation, demonstrating H2O2 production
in the system (Fig. 1, trace C). A combination of catalase and
Cu,Zn-SOD inhibited DCFHDA oxidation additively (Fig. 1,
trace D). No fluorescence was detected when predecomposed
peroxynitrite was used (Fig. 1, trace E). Similarly, the removal
of HRP from the incubations or the use of the apoenzyme
protected DCFHDA from oxidation, implying a minor role for
Fig. 1. Representative kinetic traces of the fluorescence development emitted at
530 nm (excitation at 492 nm) (trace A) in incubations of DCFHDA (5 AM),
peroxynitrite (1 AM), and HRP (1 AM) in the presence of DTPA (0.1 mM) in
phosphate buffer (150 mM, pH 7.2) at room temperature. In trace B, SOD
(30 AM) was included. Trace C contained catalase (20 AM). Trace D
contained catalase and SOD. In trace E, peroxynitrite was replaced with
predecomposed peroxynitrite.
Fig. 2. Visible spectra of incubations of HRP (1.5 AM) in the absence and in the
presence of peroxynitrite (3.0 AM) and DCFH (10 AM). Trace A represents the
native ferric enzyme; trace B was recorded immediately after peroxynitrite
addition; traces C and D were recorded 3 and 10 min after peroxynitrite
addition, respectively.
S
S
peroxynitrite-derived radicals ( OH and NO2) in the DCFHDA
conversion to fluorescent products in this system (data not
shown). Confirming previous observations [8], DCFHDA was
not significantly oxidized by peroxynitrite in the absence of HRP
(data not shown). Taken together, these data indicate that
DCFHDA initial oxidation by HRP/peroxynitrite leads to the
production of H2O2 through dissolved oxygen reduction, which
supports its further oxidation after the disappearance of
peroxynitrite.
UV – visible spectroscopy studies
Oxidation of DCFH, however, was considerably faster than
that observed in the case of DCFHDA. As shown in Fig. 2, the
addition of peroxynitrite (3 AM) to a solution containing HRP
(1.5 AM) and DCFH (10 AM) produced an intense absorption
peak at 500 nm consistent with that expected for fluorescein
formation (Fig. 2, trace B). Fluorescein absorption increase was
sustained for about 10 min after the disappearance of
peroxynitrite. Horseradish peroxidase compound I, which is
produced by its reaction with peroxynitrite (maximum at 403
nm), could not be observed, proving that DCFH is an excellent
substrate for compound I of HRP. Instead, HRP compound II
(maximum at 420 nm) could be observed immediately after
peroxynitrite addition (Fig. 2, trace B). At more prolonged
incubation times, the HRP Soret band blue-shifted concomitant
with fluorescein peak development, indicating superoxide
formation, whose trapping by ferric HRP leads to compound
III formation (maximum at 417 nm) (Fig. 2, trace C). Similar
behavior was observed for DCFHDA. The spectrum of resting
HRP (2.4 AM) was recorded and is shown in Fig. 3, inset, trace
A (maximum at 403 nm). Peroxynitrite addition (5 AM)
resulted in the detection of a spectrum in which features from
HRP compounds I and II, that is, maxima around 403 and 420
nm, respectively, were clear (Fig. 3, inset trace B). It is not
surprising that the pure compound I spectrum was not seen
because the peroxynitrite contaminant NO2 can serve as a oneelectron reductant to compound I of peroxidases [17 – 20].
Nevertheless, the addition of DCFHDA (10 AM) instantly led
to a marked conversion of the spectrum to that of compound II
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
Fig. 3. Visible spectra of incubations of HRP (2.4 AM) in the absence and in
the presence of peroxynitrite (5.0 AM) and DCFHDA (10 AM). Inset shows
the spectra of resting ferric HRP (trace A) and HRP 1 min after peroxynitrite
addition (trace B). Main graph shows incubations right after DCFHDA
addition (dashed line) and 15 and 30 min after DCFH addition (solid and
dotted lines, respectively).
(maximum around 420 nm) (Fig. 3, dashed line), with the
obligate formation of DCFHDA-derived radical. At prolonged
incubation times, a peak at 500 nm characteristic of fluorescein-type products could be clearly observed (Fig. 3, solid and
dotted lines) although its increase over time was far more
discrete than in the case of DCFH. Also, in this case, formation
of HRP compound III could be noticed at prolonged incubation
times (Fig. 3, solid line), providing evidence that the oxidation
S
of DCFH and its diacetate form leads to O2 production.
DCFHDA, which was proven to be more resistant to
oxidation by HRP/peroxynitrite, was also evaluated as a
substrate for compound II. After peroxynitrite (5 AM) addition
to HRP (2.4 AM), compounds I and II were produced.
However, compound I decayed within 90 min to produce an
almost pure compound II spectrum (Fig. 4, dashed line).
DCFHDA (10 AM) slowly shifted the spectrum toward a
maximum at 403 nm, demonstrating compound II reduction to
the ferric enzyme. Once again, prolonged incubations allowed
the observation of fluorescein peak development without the
need of adding extra peroxynitrite (Fig. 4, dotted line).
971
Fig. 4. Visible spectra of 90-min incubations of HRP (2.4 AM) and
peroxynitrite (5.0 AM). Ninety minutes after peroxynitrite addition to HRP,
DCFHDA (10 AM) was included in the incubations. Dashed trace shows the
incubation in the absence of DCFHDA; solid and dotted traces show the spectra
registered 10 and 20 min after DCFHDA addition, respectively.
(Fig. 5, trace C). Catalase alone slightly inhibited O2 consumption and, indeed, was far less efficient than in the presence of
SOD, possibly because of its conversion to the inactive
S
catalase –O2 complex (compound III) by O2 (Fig. 5, trace
D). If H2O2 had accumulated significantly, the addition of
catalase would have resulted in the production of O2, which
would appear as oxygen rebound when catalase was included.
Apparently DCFH is a good enough substrate that H2O2 does not
accumulate even though DCFH oxidation leads to its production. Interestingly, substituting DCFHDA for DCFH (1 mM)
resulted in a marked diminishment of O2 uptake by the HRP/
peroxynitrite system, further indicating the increased resistance
of the diacetate form to oxidation by HRP ferryl intermediates
(Fig. 5, trace A).
Oxygen uptake experiments
Oxygen uptake is often a consequence of free radical
production. Because of this, we evaluated the oxygen uptake
by incubations of DCFH or DCFHDA in the presence of
peroxynitrite and HRP. According to Fig. 5, in the presence of
HRP (4 AM) and DCFH (1 mM), peroxynitrite (15 AM) addition
triggered a sustained oxygen consumption for more than 10 min,
clearly after peroxynitrite total decomposition and initial
compound I reduction (Fig. 5, trace E). Indeed, the use of the
S
O2 scavenger ferric cytochrome c Fe(III) (250 AM) abolished
O2 consumption (Fig. 5, trace B). Catalase (20 AM) plus SOD
(30 AM) addition also inhibited some of the O2 consumption,
S
providing further evidence for an O2 /H2O2-driven mechanism
Fig. 5. Oxygen uptake by DCFH (1 mM) or DCFHDA (1 mM) incubation in
the presence of HRP (4 AM) and peroxynitrite (15 AM). Reactions were started
by adding peroxynitrite to the reaction chamber 1 min after sealing. (Trace A)
DCFHDA was used instead of DCFH, (trace B) cytochrome c Fe(III) (250 AM)
was added, (trace C) catalase (20 AM) plus SOD (30 AM) was added, (trace D)
catalase alone (20 AM) was added, and (trace E) no further additions. All
reactions were performed in phosphate buffer (pH 7.4, 0.1 M). Time points at
which additions were made are marked with an arrow.
972
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
Fig. 6. EPR spectra of DMPO adducts obtained in the incubations of HRP
(6 AM), peroxynitrite (12 AM), DCFH (100 AM), and DMPO (100 mM). All
incubations were performed in phosphate buffer (pH 7.0, 200 mM) at room
temperature. (Trace A) No further additions, (trace B) DCFH was replaced by
DCFHDA (100 AM), (trace C) peroxynitrite was replaced by predecomposed
peroxynitrite, (trace D) in the absence of DCFH, (trace E) in the absence of
HRP, (trace F) in the presence of SOD (30 AM), and (trace G) in the presence of
both SOD and catalase (20 AM). Instrumental conditions were microwave
power, 20 mW; scan rate, 0.3 G/s; time constant, 82 ms; modulation amplitude,
1 G; and receiver gain, 6.32 105.
3, 4, and 5) had demonstrated that some oxidation of DCFHDA
took place when it was incubated with HRP/peroxynitrite.
Because of that, we increased the concentrations of the
reagents, trying to characterize free radical intermediates
produced by DCFHDA exposure to HRP/peroxynitrite. DMPO
again was used as the spin trap. However, due to the slow rate
of DCFHDA oxidation compared to DCFH and the known
sensitivity of DMPO and its radical adducts to peroxynitritederived oxidants, presumably SNO2, we included an antioxidant, urate, in the incubations, a strategy used before to prove
SOH production from peroxynitrite [21].
As shown in Fig. 7, the incubation of DCFHDA (100 AM)
with HRP (60 AM) and peroxynitrite (60 AM) in the presence
of DMPO (100 mM) and urate (50 AM) led to detection of a
S
spectra dominated by DMPO/ OH (a N = 14.9 G and a Hh =
14.9 G; marked with X in Fig. 7A). Also a carbonyl radical
adduct could be detected (Fig. 7, marked with O). This
carbonyl radical adduct (a N = 15.2 G and a Hh = 19.4 G) has
been previously characterized in the reaction of uric acid with
decomposing peroxynitrite in the presence of DMPO [26]. As
S
before, SOD and catalase inhibited DMPO/ OH formation,
S
and H2O2 production in the system,
demonstrating O2
respectively (Figs. 7B and 7C). In the absence of DCFHDA
S
(Fig. 7E), the DMPO/ OH radical adduct signal was barely
detectable, revealing the need for the intermediate DCFHDAS
derived radical in the production of O2 (which ultimately led
EPR spin-trapping studies
The EPR spin trapping technique was employed to
characterize the free radical intermediates produced during
DCFH/DCFHDA oxidation by HRP/peroxynitrite. Indeed,
using DMPO as the spin trap, we could detect a relatively
intense spectrum arising from DCFH/HRP/peroxynitrite incuS
bations largely dominated by DMPO/ OH radical adduct (a N =
H
14.9 G and a h = 14.9 G, marked with ) (Fig. 6A). The signal
was inhibited by SOD and SOD plus catalase additions (Figs.
6F and 6G, respectively) and could not be detected in the
absence of DCFH (Fig. 6D) or when predecomposed
peroxynitrite was used (Fig. 6C). In the absence of HRP, a
S
less intense DMPO/ OH spectrum could be observed,
probably arising from the peroxynitrous acid spontaneous
S
decomposition to OH, which is expected to happen when the
oxidant decomposes in the absence of target reactants (Fig.
6E) [21 – 25]. The fact that SOD and SOD plus catalase
S
additions inhibited DMPO/ OH production strongly argues in
S
favor of O2 trapping by DMPO. Indeed, DMPO/SOOH is
not particularly stable and spontaneously decays by first-order
kinetics. The replacement of DCFH with DCFHDA, not
surprisingly, did not lead to any detectable EPR signal under
these experimental conditions, further demonstrating the
increased resistance of DCFHDA to oxidation compared to
DCFH (Fig. 6B).
Nevertheless, oxygen consumption experiments as well as
UV –Vis spectrophotometry and fluorescence studies (Figs. 1,
&
Fig. 7. EPR spectra of DMPO adducts obtained in the incubations of HRP
(60 AM), peroxynitrite (60 AM), DCFHDA (100 AM), urate (50 AM), and DMPO
(100 mM). All incubations were performed in phosphate buffer (pH 7.0, 200 mM)
at room temperature. (Trace A) No further additions, (trace B) in the presence of
catalase (20 AM), (trace C) in the presence of SOD (30 AM), (trace D) in the
presence of both SOD and catalase, (trace E) in the absence of DCFHDA, and
(trace F) peroxynitrite replaced by predecomposed peroxynitrite. Instrumental
conditions were microwave power, 20 mW; scan rate, 0.15 G/s; time constant, 327
ms; modulation amplitude, 1 G; and receiver gain, 6.32 105.
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
to DMPO/SOH production) and H2O2 that powered the
peroxidase system when peroxynitrite was no longer available
(k = 0.17 s1, pH 7.4) ([23,27], see also Calculations under
Experimental procedures). As expected, predecomposed peroxynitrite had no effect on the initiation of the radical chain
reactions (Fig. 7F). The EPR experiments demonstrate the
S
intermediate formation of O2 by one-electron oxidized
DCFH/DCFHDA, which reduces molecular oxygen. Clearly,
taken together the data shown herein demonstrate that most of
DCFH/DCFHDA oxidation occurred by mechanisms independent of the initially added peroxynitrite.
Discussion
Previous studies demonstrated the formation of DCF
semiquinone free radical by photoreduction of DCF [3] or by
horseradish peroxidase-catalyzed oxidation of the reduced
nonfluorescent compound DCFH [4] and the formation of
DCF phenoxyl free radical after oxidation of DCFH by
horseradish peroxidase [5]. These studies clearly showed that
DCFH cannot be used to conclusively measure superoxide or
hydrogen peroxide formation in cells undergoing oxidative
stress because the dye itself is able to generate reactive oxygen
species in aerobic environments.
In the present study, we analyzed the reaction of DCFH with
horseradish peroxidase in the presence of peroxynitrite, but the
initiator could have been any peroxide substrate for HRP
because superoxide radical anion and, consequently, hydrogen
peroxide formation are an unavoidable consequence of DCFH
oxidation. Comparatively, we evaluated the oxidation of the
commercially available DCFH precursor, DCFHDA, under the
same experimental conditions. Although more resistant to
oxidation by peroxynitrite and HRP compounds I and II,
DCFHDA has also been proven to be a substrate for peroxidase
hypervalent intermediates and suffers from the same drawbacks
of DCFH concerning the amplification of the oxidative process
S
by self-induced O2 /H2O2 production. Clearly, the many
reports of the inhibition of DCFH oxidation by superoxide
dismutase and catalase [28 –36] in cellular systems, which have
interpreted these effects as evidence of cellular ROS formation,
need to be addressed with caution.
UV –visible spectrophotometry demonstrated the formation
of horseradish peroxidase compounds I and II upon reaction
of the peroxynitrite with HRP. Immediate addition of DCFH
or DCFHDA to the system led to the production of HRP
S
compound II with the obligate formation of DCF semiquinone radical or DCFHDA radical, respectively (Figs.
2, 3, and 4). At prolonged incubation periods, fluorescein
product formation was clear and increased with time, long
after the complete decomposition of peroxynitrite. Late
addition (90 min) of DCFHDA to HRP compound II rendered
similar results, demonstrating that DCFHDA serves as a
reductant for both HRP compounds I and II, analogous to
DCFH itself (Fig. 4).
The production of H2O2 during DCFH or DCFHDA
oxidation by the peroxidase system was further demonstrated
by the inhibition of fluorescein product formation and
973
DMPO/SOH production by catalase (Figs. 1, 6, and 7).
S
Furthermore, SOD addition markedly inhibited DMPO/ OH
formation (Figs. 6 and 7), but had little effect on the rate of
DCFH (data not shown) or DCFHDA oxidation (Fig. 1). This
further confirms that H2O2 production was a consequence of
S
O2 dismutation, which, in turn, was produced by the
S
reduction of molecular oxygen by the DCF semiquinone
radical or DCFHDA radical (see Scheme 1). The initiation of
peroxidase-catalyzed DCFH oxidation is not limited to HRP,
but will occur with any heme-containing protein with
peroxidase activity such as myoglobin [37], hemoglobin [37],
cytochrome c [37,38], prostaglandin H synthase [39], and even
heme itself [37]. The peroxidase radical oxidants (compound I
and the globin radical) and ferryl are necessary to support the
amplification of DCFH radical formation, because neither
superoxide radical anion nor hydrogen peroxide by itself
oxidizes DCFH [8,40].
From the present study and the results obtained earlier, we
conclude that the use of DCFH/DCFHDA (or fluorogenic
probes with similar structure, such as dihydrorhodamine 123)
in complex systems possessing peroxidase activity may
generate a number of misleading results arising from the
ability of probe-derived radical to produce reactive oxygen
species, which are responsible for the vast majority of the
observed fluorescence regardless of the initial oxidant.
Clearly, in our experiments the inhibition of DCF/DCF-like
product formation by catalase and superoxide dismutase was
the result of the inhibition of the propagation reactions which
occurred long after the initial reaction of peroxynitrite with
horseradish peroxidase. In other words, the inhibition of DCF/
DCF-like product formation by catalase and, to a lesser
S
extent, by superoxide dismutase evinces that O2 and H2O2
production is inherent in the oxidation of DCFH, which,
therefore, cannot be used to prove the formation of reactive
oxygen species.
Scheme 1. Schematic representation of DCFH oxidation by HRP initiated by
peroxides. DCF semiquinone radical reduces molecular oxygen to
superoxide radical anion (a source of H2O2), consequently forming the
fluorescent dye (DCF). Hydrogen peroxide, in turn, reacts with HRP, initiating
another oxidative cycle.
S
974
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
Acknowledgments
The authors thank Drs. Arno Siraki and Dario Ramirez for
helpful discussion and Mary J. Mason for her assistance during
the preparation of the manuscript. This work was supported by
the Intramural Research Program of the NIH, NIEHS.
References
[1] Tsuchiya, M.; Suematsu, M.; Suzuki, H. In vivo visualization of oxygen
radical-dependent photoemission. Methods Enzymol. 233:128 – 140;
1994.
[2] LeBel, C. P.; Ischiropoulos, H.; Bondy, S. C. Evaluation of the probe
2V,7V-dichlorofluorescin as an indicator of reactive oxygen species
formation and oxidative stress. Chem. Res. Toxicol. 5:227 – 231; 1992.
[3] Marchesi, E.; Rota, E.; Fann, Y. C.; Chignell, C. F.; Mason, R. P.
Photoreduction of the fluorescent dye 2V-7V-dichlorofluorescein: a spin
trapping and direct electron spin resonance study with implications for
oxidative stress measurements. Free Radic. Biol. Med. 26:148 – 161;
1999.
[4] Rota, C.; Chignell, C. F.; Mason, R. P. Evidence for free radical
formation during the oxidation of 2V-7V-dichlorofluorescin to the
fluorescent dye 2V-7V-dichlorofluorescein by horseradish peroxidase:
possible implications for oxidative stress measurements. Free Radic.
Biol. Med. 27:873 – 881; 1999.
[5] Rota, C.; Fann, Y. C.; Mason, R. P. Phenoxyl free radical formation
during the oxidation of the fluorescent dye 2V,7V-dichlorofluorescein by
horseradish peroxidase: possible consequences for oxidative stress
measurements. J. Biol. Chem. 274:28161 – 28168; 1999.
[6] Ischiropoulos, H.; Gow, A.; Thom, S. R.; Kooy, N. W.; Royall, J. A.;
Crow, J. P. Detection of reactive nitrogen species using 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123. Methods Enzymol.
301:367 – 373; 1999.
[7] Possel, H.; Noack, H.; Augustin, W.; Keilhoff, G.; Wolf, G. 2,7Dihydrodichlorofluorescein diacetate as a fluorescent marker for
peroxynitrite formation. FEBS Lett. 416:175 – 178; 1997.
[8] Kooy, N. W.; Royall, J. A.; Ischiropoulos, H. Oxidation of 2V,7Vdichlorofluorescin by peroxynitrite. Free Radic. Res. 27:245 – 254; 1997.
[9] Crow, J. P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are
sensitive indicators of peroxynitrite in vitro: implications for intracellular
measurement of reactive nitrogen and oxygen species. Nitric Oxide
1:145 – 157; 1997.
[10] Floris, R.; Piersma, S. R.; Yang, G.; Jones, P.; Wever, R. Interaction of
myeloperoxidase with peroxynitrite: a comparison with lactoperoxidase,
horseradish peroxidase and catalase. Eur. J. Biochem. 215:767 – 775;
1993.
[11] Bonini, M. G.; Radi, R.; Ferrer-Sueta, G.; Ferreira, A. M.; Augusto, O.
Direct EPR detection of the carbonate radical anion produced from
peroxynitrite and carbon dioxide. J. Biol. Chem. 274:10802 – 10806;
1999.
[12] Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman,
B. A. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc.
Natl. Acad. Sci. USA 87:1620 – 1624; 1990.
[13] Denicola, A.; Freeman, B. A.; Trujillo, M.; Radi, R. Peroxynitrite
reaction with carbon dioxide/bicarbonate: kinetics and influence on
peroxynitrite-mediated oxidations. Arch. Biochem. Biophys. 333:49 – 58;
1996.
[14] Duling, D. R. Simulation of multiple isotropic spin-trap EPR spectra.
J. Magn. Reson. B 104:105 – 110; 1994.
[15] Holtzman, J. L. Calibration of the oxygen polarograph by the depletion
of oxygen with hypoxanthine – xanthine oxidase-catalase. Anal. Chem.
48:229 – 230; 1976.
[16] Goldstein, S.; Czapski, G. Viscosity effects on the reaction of
peroxynitrite with CO2: evidence for radical formation in a solvent cage.
J. Am. Chem. Soc. 121:2444 – 2447; 1999.
[17] van der Vliet, A.; Eiserich, J. P.; Halliwell, B.; Cross, C. E. Formation of
reactive nitrogen species during peroxidase-catalyzed oxidation of
nitrite: a potential additional mechanism of nitric oxide-dependent
toxicity. J. Biol. Chem. 272:7617 – 7625; 1997.
[18] van Dalen, C. J.; Winterbourn, C. C.; Senthilmohan, R.; Kettle, A. J.
Nitrite as a substrate and inhibitor of myeloperoxidase: implications for
nitration and hypochlorous acid production at sites of inflammation.
J. Biol. Chem. 275:11638 – 11644; 2000.
[19] Sampson, J. B.; Ye, Y.; Rosen, H.; Beckman, J. S. Myeloperoxidase and
horseradish peroxidase catalyze tyrosine nitration in proteins from nitrite
and hydrogen peroxide. Arch. Biochem. Biophys. 356:207 – 213; 1998.
[20] Gebicka, L. Kinetic studies on the oxidation of nitrite by horseradish
peroxidase and lactoperoxidase. Acta Biochim. Pol. 46:919 – 927; 1999.
[21] Augusto, O.; Gatti, R. M.; Radi, R. Spin-trapping studies of peroxynitrite
decomposition and of 3-morpholinosydnonimine N-ethylcarbamide
autooxidation: direct evidence for metal-independent formation of free
radical intermediates. Arch. Biochem. Biophys. 310:118 – 125; 1994.
[22] Lymar, S. V.; Khairutdinov, R. F.; Hurst, J. K. Hydroxyl radical
formation by O – O bond homolysis in peroxynitrous acid. Inorg. Chem.
42:5259 – 5266; 2003.
[23] Merényi, G.; Lind, J.; Goldstein, S.; Czapski, G. Peroxynitrous acid
homolyzes into OH and NO2 radicals. Chem. Res. Toxicol. 11:
712 – 713; 1998.
[24] Goldstein, S.; Czapski, G.; Lind, J.; Merényi, G. Carbonate radical ion is
the only observable intermediate in the reaction of peroxynitrite with
CO2. Chem. Res. Toxicol. 14:1273 – 1276; 2001.
[25] Coddington, J. W.; Hurst, J. K.; Lymar, S. V. Hydroxyl radical
formation during peroxynitrous acid decomposition. J. Am. Chem. Soc.
121:2438 – 2443; 1999.
[26] Santos, C. X. C.; Anjos, E. I.; Augusto, O. Uric acid oxidation by
peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation. Arch. Biochem. Biophys. 372:285 – 294; 1999.
[27] Radi, R.; Cassina, A.; Hodara, R.; Quijano, C.; Castro, L. Peroxynitrite
reactions and formation in mitochondria. Free Radic. Biol. Med.
33:1451 – 1464; 2002.
[28] Lawler, J. M.; Song, W.; Demaree, S. R. Hindlimb unloading increases
oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free
Radic. Biol. Med. 35:9 – 16; 2003.
[29] Miura, H.; Bosnjak, J. J.; Ning, G.; Saito, T.; Miura, M.; Gutterman,
D. D. Role for hydrogen peroxide in flow-induced dilation of human
coronary arterioles. Circ. Res. 92:e31 – e40; 2003.
[30] van Reyk, D. M.; King, N. J. C.; Dinauer, M. C.; Hunt, N. H. The
intracellular oxidation of 2V,7V-dichlorofluorescin in murine T lymphocytes. Free Radic. Biol. Med. 30:82 – 88; 2001.
[31] Kim, Y. H.; Takahashi, M.; Nogushi, N.; Suzuki, E.; Suzuki, K.;
Taniguchi, N.; Niki, E. Inhibition of c-Jun expression induces
antioxidant enzymes under serum deprivation. Arch. Biochem. Biophys.
374:339 – 346; 2000.
[32] Atlante, A.; Gagliardi, S.; Minervini, G. M.; Ciotti, M. T.; Marra,
E.; Calissano, P. Glutamate neurotoxicity in rat cerebellar granule
cells: a major role for xanthine oxidase in oxygen radical formation.
J. Neurochem. 68:2038 – 2045; 1997.
[33] Oyama, Y.; Hayashi, A.; Ueha, T.; Maekawa, K. Characterization of
2V,7V-dichlorofluorescin fluorescence in dissociated mammalian brain
neurons: estimation on intracellular content of hydrogen peroxide. Brain
Res. 635:113 – 117; 1994.
[34] Puntarulo, S.; Cederbaum, A. I. Role of cytochrome P-450 in the
stimulation of microsomal production of reactive oxygen species by
ferritin. Biochim. Biophys. Acta 1289:238 – 246; 1996.
[35] Reid, M. B.; Haack, K. E.; Franchek, K. M.; Valberg, P. A.; Kobzik,
L.; West, M. S. Reactive oxygen in skeletal muscle. I. Intracellular
oxidant kinetics and fatigue in vitro. J. Appl. Physiol. 73:1797 – 1804;
1992.
[36] Scott, J. A.; Homcy, C. J.; Khaw, B. A.; Rabito, C. A. Quantitation of
intracellular oxidation in a renal epithelial cell line. Free Radic. Biol.
Med. 4:79 – 83; 1988.
[37] Ohashi, T.; Mitzutani, A.; Murakami, A.; Kojo, S.; Ishii, T.; Taketani, S.
Rapid oxidation of dichlorodihydrofluorescin with heme and hemopro-
S
S
M.G. Bonini et al. / Free Radical Biology & Medicine 40 (2006) 968 – 975
teins: formation of the fluorescein is independent of the generation of
reactive oxygen species. FEBS Lett. 511:21 – 27; 2002.
[38] Lawrence, A.; Jones, C. M.; Wardman, P.; Burkitt, M. J. Evidence for the
role of a peroxidase compound I-type intermediate in the oxidation of
glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome
c/H2O2: implications for oxidative stress during apoptosis. J. Biol. Chem.
278:29410 – 29419; 2003.
975
[39] Larsen, L. N.; Dahl, E.; Bremer, J. Peroxidative oxidation of leucodichlorofluorescein by prostaglandin H synthase in prostaglandin
biosynthesis from polyunsaturated fatty acids. Biochim. Biophys. Acta
1299:47 – 53; 1996.
[40] Royall, J. A.; Ischiropoulos, H. Evaluation of 2V,7V-dichlorofluorescin and
dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in
cultured endothelial cells. Arch. Biochem. Biophys. 302:348 – 355; 1993.