Singlet oxygen reacts with 2′,7′-dichlorodihydrofluorescein and contributes to the formation of 2′,7′-dichlorofluorescein

Article abstract of DOI:10.1111/j.1751-1097.2008.00345.x

There are controversial reports in the literature concerning the reactivity of singlet oxygen (¹O₂) with the redox probe 2′,7′-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which ¹O₂ is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2′,7′- dichlorofluorescein (DCF), increases in D₂O and decreases in sodium azide, proving the direct role of ¹O₂ in this process. We have also prepared solutions in which either ¹O₂ or dication (MB^?2+) and semi-reduced (MB^?) radicals of the sensitizer and subsequently super-oxide radical (O₂ ^?-) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O₂^?-, indicating that both ¹O₂ and MB^?2+ react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB^?2+ than in the presence of ¹O₂, considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of ¹O₂ with DCFH is actually larger than that of MB^?2+. We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and ¹O₂.

Full text of DOI:10.1111/j.1751-1097.2008.00345.x

Photochemistry and Photobiology, 2008, 84: 1238–1243
Singlet Oxygen Reacts with 2¢,7¢-Dichlorodihydrofluorescein and
Contributes to the Formation of 2¢,7¢-Dichlorofluorescein
1
Nasser A. Daghastanli , Rosangela Itri and Mauricio S. Baptista*
1
2
1
Instituto de F ´ı sica, Universidade de S a˜ o Paulo, S a˜ o Paulo, Brazil
Departamento de Bioqu ´ı mica, Instituto de Qu ´ı mica, Universidade de S a˜ o Paulo, S a˜ o Paulo, Brazil
2
Received 3 September 2007, accepted 20 February 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00345.x
enzymatically hydrolyzed by intracellular esterases to form the
nonpermeable DCFH (2–7). It was initially thought to be
useful as a specific indicator for intracellular H (2–5).
ABSTRACT
There are controversial reports in the literature concerning the
1
reactivity of singlet oxygen ( O ) with the redox probe 2¢,7¢-
2
O
2
2
However, it has been already demonstrated that DCFH is not
oxidized directly by H O , instead it reacts with other ROS
and NOS generated by intracellular peroxidases (6). Some
dichlorodihydrofluorescein (DCFH). By carefully preparing
is quantitatively generated in the presence
2
2
1
solutions in which O
2
of DCFH, we were able to show that the formation rate of the
fluorescent molecule derived from DCFH oxidation, which is
authors have suggested that it is also oxidized by singlet
1
oxygen ( O ), although this has never been shown experimen-
2
1
tally (2–5,7,8). In fact, Bilski et al. provided evidence that O
2
¢,7¢-dichlorofluorescein (DCF), increases in D
2
O and decreases
2
in this process. We
2
1
in sodium azide, proving the direct role of O
have also prepared solutions in which either O2 or dication
does not directly react with DCFH (9). Considering that
DCFH is a widespread probe for estimating oxidative stress in
1
•2+
•
1
cells, it is important to know whether or not it reacts with O
(MB ) and semi-reduced (MB ) radicals of the sensitizer and
•
subsequently super-oxide radical (O
2
.
Generating ROS species specifically is always a challenge.
)
2
) are generated. The
absence of any effect of SOD and catalase ruled out the DCFH
•
However, we have shown that methylene blue (MB) under
1
photoexcitation can be directed to form either O or semi-
)
1
, indicating that both O
•2+
oxidation by O
2
2
and MB
with DCFH. Although the formation of DCF was 1 order of
react
2
•
•2+
reduced (MB ) and dication (MB ) radicals and subse-
•
2+
2•
)
magnitude larger in the presence of MB
1
than in the presence
O , considering the rate of spontaneous decays of these
quently anion radical superoxide (O ) (10–13). Monomer
and dimer species of MB have different absorption spectra and
the dimerization equilibrium is modulated by the presence of
anionic interfaces including sodium dodecyl sulfate (SDS)
micelles (10,11), other membrane mimetic systems (12) and
mitochondria (13). Depending on the MB local interfacial
concentration either monomers or dimers are favored, shifting
the triplet deactivation mechanism from energy transfer and
of
species in aqueous solution, we were able to conclude that the
1
reactivity of O2 with DCFH is actually larger than that of
2
•2+
MB . We conclude that DCFH can continue to be used as a
probe to monitor general redox misbalance induced in biologic
1
systems by oxidizing radicals and O
2
.
1
•
formation of O
and MB
2
to electron transfer and formation of MB
•)
•
2+
radicals. O
2
is subsequently formed by the
•
reaction of molecular oxygen with MB (Scheme 1) (10,12).
There has been interest in designing inexpensive photody-
namic therapy protocols (PDT) (14–16). DCFH (in its
diacetate form) offers an interesting option to investigate the
INTRODUCTION
The diverse roles of reactive oxygen and nitrogen species in
biology and medicine translate into increased interest in
measuring and understanding redox balance in cells (1).
Although extremely used in biochemistry and in cell biology
to sense oxidative stress, the reactivity of 2¢,7¢-dichlorodihy-
drofluorescein (DCFH) against several reactive oxygen and
nitrogen species (ROS and NOS, respectively) is still not
conclusively defined (2–5).
DCFH is a nonfluorescent compound that after oxidation
gives rise to 2¢,7¢-dichlorofluorescein (DCF), a strong fluores-
cent molecule. Its diacetate form, 2¢,7¢-dichlorodihydrofluo-
rescein diacetate (DCFH-DA), has been widely applied in cell
studies, because it easily penetrates membranes and it is
general oxidative stress induced in cells and tissues by
1
photosensitizers that are generally excellent sources of
(
DCFH reacts with O . By using known controls of the
O
2
14–20). However, it is imperative to know whether or not
1
2
1
photochemical events related with
photochemistry of MB in the presence of anionic interfaces, we
2
O and the controlled
were able to set up experimental protocols to elucidate whether
1
or not DCFH reacts with O and with other radicals derived
from MB photoexcitation.
2
MATERIALS AND METHODS
2
Sodium azide, SDS, deuterium oxide (D O), superoxide dismutase
CuZnSOD (SOD) and catalase were purchased from Sigma and used
as received. Methylene blue (MB-Aldrich) was double recrystallized
from ethanol. Solutions were prepared in Milli-Q water (pH ꢀ 6.5).
*
ꢀ
Corresponding author email: baptista@iq.usp.br (Mauricio S. Baptista)
2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/08
1
238

Photochemistry and Photobiology, 2008, 84 1239
Scheme 1. Methylene blue (MB) photochemical reaction routes where
MB, MB , MB* are MB ground state, singlet and triplet excited
*
3
•
•2+
states, respectively, MB and MB
dication radicals, respectively. Numeric pathways correspond to: (1)
are MB semi-reduced and
Figure 1. Absorption spectra of solutions containing 2¢,7¢-dichloro-
dihydrofluorescein (40 lM) and methylene blue (MB; 3 lM) after
irradiation with a diode laser (50 mW) at k = 664 nm. Spectra
obtained from 0 ( ) to 430 (h) s of irradiation in air-saturated
solution. Formation of 2¢,7¢-dichlorofluorescein and photo-bleaching
of MB are evident by following the absorption peaks at 502 nm (arrow
A) and 664 nm (arrow B), respectively.
light absorption of monomeric MB species (kmax = 664 nm), (2)
intersystem crossing, (3) reaction of MB* with molecular oxygen
3
1
forming singlet oxygen ( O
oxygen returning the ground state dye and forming anion superoxide
•
2
), (4) oxidation of MB by molecular
•)
3
radical (O
2
), (5) redox suppression of ( MB- - - - -MB)* after exciting
ground state dimers, (6) light absorption of dimeric MB species
(
position of the species presented in this scheme does not represent
kmax = ꢀ590 nm), (7) ground state dimerization. The relative
their actual energy levels. Modified from Junqueira et al. (10).
that lead to its oxidation may change depending on the type of
excitation light and on the relative concentrations of species in
the excited and ground states.
We will start by showing a condition in which DCFH seems
. Fig. 1 shows the absorption spectra of
DCFH (D6883; SIGMA) was obtained by hydrolysis of its
diacetate form (DCFH-DA, 2¢, 7¢-dichlorodihydrofluorescein diace-
tate). Briefly, an appropriate aliquot from a DCFH-DA stock solution
1
not to react with O
2
an aqueous solution containing 3 lM of MB (only MB
monomers are present at such concentration) in the presence
of 40 lM DCFH for several irradiation times (664 nm diode
laser). One can observe the DCF formation, characterized by
the increase in the absorption band at ꢀ500 nm (arrow A). It is
important to note that there is also a decrease in the MB
absorption band at 664 nm (arrow B), indicating MB photo-
bleaching, previously shown to result in the formation of the
leuco-form (10,12,13). We also observed a six-fold increase in
DCF formation rate by decreasing the concentration of
molecular oxygen (20 min of nitrogen purging, data not
shown) compared to the experiment performed in air-saturated
(200 lM) was added to an aliquot of NaOH (0.5 M) in a fluorescence
quartz cuvette in the dark and kept on ice for 30 min and used the
same day. The hydrolyzed solution was then neutralized with an
appropriate volume of sulfuric acid to pH 7.2. The pH value was
measured by means of a glass electrode.
Aqueous solutions composed of either monomers or dimers of MB
were prepared by mixing MB into 30 or 1 mM SDS, respectively
(10,12). MB molar concentrations and aggregation state were checked
by absorption spectra measurements (Shimadzu UV–VIS 2400-PC
spectrophotometer).
DCF formation. DCFH and MB-containing samples were irradiated
by a CW 664 nm diode laser, 50 mW (Morgotron, Brazil), in a stirred
quartz cuvette (10 mm optical path length) that was assembled in the
cuvette-holder of a SPEX FLUOROG spectrofluorimeter. Fluores-
cence spectra were obtained in the right-angle mode, using the
excitation wavelength of 498 nm. All measurements were made just
after a specific irradiation procedure, at room temperature. Spectral
data were further manipulated with a 386 GRAMS (Galatic, Inc.)
solution. The presence of D
reported earlier by Bilski et al. (9). However, instead of
2
O and azide shows little effects as
1
considering that DCFH does not react with O , we think that
2
there is another explanation to such results. In fact, they can be
simply understood if we consider that the irradiation of MB at
64 nm induces formation of MB triplets (10–13), which then
and ⁄ or Origin (Microcal) softwares.
1
2
O phosphorescence NIR emission produced by photo-excitation
6
of MB was obtained as previously described (12,13). Briefly, a
Nd:YAG (10 mJ ⁄ pulse; 5 ns ⁄ pulse) Surelite III laser (Continuum)
excites MB species at 532 nm, into a fluorimeter (Edinburgh Analytical
react directly with DCFH molecules at this relatively large
DCFH concentration, promoting its oxidation to DCF and
the reduction of MB to MBH (a two electron plus proton
reduction). The isosbestic point at 538 nm further substanti-
ates this claim. Of note, the reduction of MB to MBH can only
be explained by the consecutive oxidation of DCFH. The six-
fold increase in the oxidation rate in the absence of oxygen
agrees with this mechanism because oxygen deactivates triplet
species, decreasing the efficiency of the reaction between
reducing substrates (DCFH in this case) and triplets (22,23).
Instruments) that is connected to a near-infrared-PMT (R5509 from
was recorded
lifetime was
1
Hamamatsu Co.). The phosphorescence emission of O
2
1
in the 1200–1310 nm spectral region, and the
O
2
monitored at 1280 nm (10–13,18–20), using both a silicon cutoff filter
and a monochromator.
RESULTS AND DISCUSSION
Although DCFH is extensively used as a probe of redox state,
it has some chemical and photochemical characteristics that
make it difficult to define its reactivity. For example, it is a
good reducing agent, it can engage in photoinduced auto-
oxidation reactions and the oxidation product is photoactive
and absorbs visible light (2,9,21). Therefore, the mechanisms
The fact that azide and D O have no effect is also easily
2
explained. Under these conditions DCFH molecules react
1
directly with MB triplets, without the intermediacy of O .
2
Other authors have also reported the oxidation of DCFH by
direct reaction with sensitizers (9,21).

1
240 Nasser A. Daghastanli et al.
With the aim of investigating whether or not O
1
1
istic of O
2
reacts with
2
in aqueous solutions (18–20). In the presence of
•
•)
DCFH, we changed the experimental conditions decreasing
the DCFH concentration (DCFH = 100 nM) to avoid its
direct reaction with photosensitizer triplets, i.e. under this
condition the rate of reaction between sensitizer triplets and
dimers in air-saturated solutions, MB is transformed in O
•
2
2+
in the microsecond time scale (10–13). MB
is a relatively
stable cation radical (lifetime of 300 ls in water), which reacts
with reducing agents, i.e. second-order rate constant with
DABCO was measured to be 6 · 10
presents the corresponding DCF fluorescence emission spectra
from the solutions described above. Note that the fluorescence
intensity of the solution containing monomeric MB, which
6
)1 )1
oxygen is much larger than the rate of reaction with DCFH.
1
Photochemistry of MB was utilized to generate specifically O
M
s
(10). Figure 2B
2
•
2+
or MB and MB ⁄ O
species against DCFH.
•
•)
2
and to test the reactivity of these
1
Figure 2A shows the O phosphorescence emission spectra
1
generates mainly O under irradiation, increases considerably
2
2
in the presence of MB monomers (excess of micelles) and its
absence in the presence of dimers, which leads to the formation
of MB and MB (10–13). The emission band centered at
ꢀ1280 nm with lifetime of 4.6 ls (Fig. 2A inset) is character-
compared with nonirradiated samples, suggesting that DCFH
1
does react with O
2
. It is also interesting to note that in the
conditions in which radical formation is favored (MB dimers),
1
•
2+
•
DCF oxidation is larger than in the solution in which only O
is generated.
2
It is not expected that DCFH partitions in the micelle
pseudo-phase because both DCFH and SDS have negative
charges. MB partitions in the micelle pseudo-phase and
because of the low MB concentrations (micromolar) compared
to the SDS concentrations (millimolar), MB is totally bound in
1
the micelles (10,12). Because the diffusion length of
much larger than the average size of the micelles, DCFH
2
O is
oxidation can occur in the bulk of the solution, where DCFH
1
is preferentially located (24). The extent of the reaction of O
2
with a given target will depend on its local steady-state
concentration and on the bimolecular rate constant (24).
1
In order to study the reaction of O with DCFH, usual
2
2
controls with deuterated water (D O) and sodium azide were
performed. By following the increase in DCF fluorescence as a
function of irradiation time in water and in D O, it is clear that
the process of photo-oxidation is faster (rate of DCF forma-
2
tion in D
efficient (amount of formed DCF is three times larger) in D O
2
O is ꢀ3.6 times faster than in water) and more
2
(
Fig. 3A). These results can be explained by the fact that the
1
O
2
lifetime in water is ca 4.5 ls and in ꢀ100% D
2
O is about
3
5–40 ls (20). Assuming that no other interference occurs, the
1
steady-state concentration of O
giving consequently ꢀ10 times faster reaction rate in ꢀ100%
O. In our experiments we observed only ꢀ3.6 times faster
rate. However, it is important to emphasize that we were not
O in our experiments because DCFH is
2
would be ꢀ10 times larger
D
2
using ꢀ100% D
2
prepared in situ by using aqueous NaOH solution. Further-
more, when the aqueous solution was irradiated in the
1
presence of sodium azide (a known O
2
quencher), the DCF
formation rate was ꢀ5-fold reduced and the final DCF
formation was three-fold decreased (Fig. 3B). Therefore,
1
photo-oxidation of DCFH to DCF via O
2
indeed takes place
O.
Another result that is in agreement with the reaction of O
and it was diminished by azide and increased in D
2
1
2
1
with DCFH was obtained when the concentration of SDS was
changed. It can be observed that the DCF formation rate
increases with the increase in SDS concentration (Fig. 3C).
Figure 2. (A)
2
O phosphorescence emission of aqueous solution of
MB = 20 lM in the presence of MB monomers (SDS = 30 mM, d)
1
and dimers (SDS = 1 mM, h) with DCFH = 100 nM. Inset:
phosphorescence decay in the presence of MB monomers
MB = 20 lM, SDS = 30 mM, DCFH = 100 nM). The lifetime of
.6 ls was calculated by an exponential fit (solid line). (B) DCF
fluorescence emission (maximum at 522 nm) in aqueous solution
pH = 7) with DCFH = 100 nM and MB = 20 lM before irradiation
in the monomeric (4, SDS = 30 mM) and dimeric ( , SDS = 1 mM)
forms and after 3 min irradiation with 50 mW diode laser at
k = 664 nm, in the presence of MB dimers ( ) and monomers (s).
Inset: absorption spectra of MB dimer ( ) and monomer (4). The
absorbance of all samples did not change during irradiation (kexc for
DCF = 498 nm).
O
2
Because the bulk solvent is the same (water) and the reactants
1
are the same ( O
(
4
2
and DCFH), the bimolecular rate should
also be the same. Consequently, the steady-state concentration
1
of O2 must have increased with the increase in SDS
(
1
concentration. Under this condition the rate of O
2
production
depends on the oxygen concentration in the micelle micro-
environment, which is increased with the increase in SDS
concentration (10,12,24). In fact, we and others have observed
1
increases in
2
O production with the increase in micelle

Photochemistry and Photobiology, 2008, 84 1241
concentration as well as with the increase in the molar fraction
of the organic phase in other micro-heterogeneous systems
(
10,12,24), which can explain the results shown in Fig. 3C.
•
)
In the presence of monomers of MB, O2 could also be
1
2
formed by a side reaction leading to the reduction of O (25).
Although we had no reason to suspect that the reduction of
1
O was taking place in our experimental conditions, we tested
2
the effect of SOD. It can be observed that in aqueous and in
2
D O solutions both in the presence of SOD (Fig. 4A), the
oxidation of DCFH follows profiles similar to that observed in
the absence of SOD (Fig. 3A), i.e. rate of DCF formation is
1
O, in agreement again with the oxidation by O
faster in D
Therefore, these results confirm that O reacts with DCFH.
2
2
.
1
2
We believe that Bilski et al. (9) did not observe the reaction of
because of the large DCFH concentrations
1
DCFH with O
2
that were used, favoring the direct reaction with the photo-
sensitizer triplets and because the rose bengal (RB) ⁄ DCFH
system has a more complicated photochemistry due to the
closer absorption bands of RB and DCF, which favor DCF
auto-oxidation.
Although the main idea of this work was to test the
1
reactivity of DCFH against
2
O , the reactivity of DCFH
•)
against other species, including O2 , is also controversial (26–
9). Some cell studies have indicated increased DCF formation
in the presence of SOD (26). In cell-free extracts, presence (27)
2
•)
and absence (28) of reactivity against O have been reported.
2
•
)
In our system O2 is generated directly in the presence of
ground state dimers of MB. Experiments aiming to test the
•)
possible role of O
2
in the oxidation of DCFH were realized
by testing the effect of SOD and catalase (Fig. 4B). It is clear
that neither SOD nor SOD plus catalase shows any effect in
the oxidation of DCFH. Therefore, our results also indicate
•)
that DCFH does not react with O2 , which is in agreement
with other authors (2,26,28). The direct reaction with H O
2
2
has already been ruled out (2,21). This result also leads to the
conclusion that the oxidizing species that is reacting with
DCFH when ground state dimers are excited is the radical
5
22
Figure 3. (A) Fluorescence increase (F ⁄ F
o
) at 522 nm as a function
•2+
dication of MB (MB ). Note that in this condition, besides
•)
of irradiation time in H O ( ) and D O (d). (B) Fluorescence increase
2
2
•2+
O
2
, MB
is the only species present in solution few
at 522 nm as a function of irradiation time in aqueous solution in the
presence (4) and absence (h) of 1.43 mM of sodium azide. (C) Rate of
microseconds after light excitation. Triplet species are deacti-
vated in the nanosecond time scale (10,12). Several literature
fluorescence increase in H O in the presence of 20, 30 and 50 mM
2
•2+
of SDS. Irradiations with diode laser at k = 664 nm, 50 mW and kexc
of DCF = 498 nm, DCFH = 100 nM, MB = 3 lM.
reports provide support to this interpretation—MB
reacts
5
22
Figure 4. (A) Fluorescence increase (F ⁄ F
o
) at 522 nm as a function of irradiation time in H
2
O ( ) and D
O in the presence of 1 mM SDS (ꢀ80% of MB
dimers; Refs. [10,12]) ( ), 300 U mL of SOD (d) and 300 U mL of SOD and 300 U mL of catalase ( ), DCFH = 100 nM, MB = 5 lM.
2
O (d) in the presence of SOD
1
522
ꢀ
300 U mL) . (B) Fluorescence increase (F ⁄ F
o
) at 522 nm as a function of irradiation time in H
2
)
1
)1
)1

1
242 Nasser A. Daghastanli et al.
1
The information that O
2
reacts efficiently with DCFH is
1
especially important in the area of PDT, in which
is generated in situ and the amount of ROS (especially O )
O
2
1
2
is related to the efficiency of cell death (33,34). Researchers in
1
other fields should also be aware that O
2
probably contributes
to the total DCF fluorescence increase observed in cells even if
1
O is not directly generated (35,36).
2
CONCLUSIONS
Our results clearly show that DCFH can be directly oxidized by
1
•2+
2
O and MB . We suggest that the efficiency of the reaction
1
of DCFH with O
•2+
2
is larger than that with MB . The fact
1
that DCFH reacts with O , and with many other oxidizing
2
agents, gives confidence that DCFH can continue to be used as
a probe to monitor general redox misbalance in cells.
Figure 5. DCF fluorescence emission rate at 522 nm in the presence of
MB in water solutions (dimers and monomers are present, [dimer]
<1%), in 50 mM SDS where only monomers are present, and in 1 mM
SDS where dimers are favored (dimer ꢀ85%; Refs. [10,12]). Average
and standard deviations of three independent measurements are
shown. kexc = 498 nm, DCFH = 100 nM, MB = 10 lM.
Acknowledgements—Fundac
Sao Paulo is acknowledged for funding the research and for the
fellowship of N.D. Conselho Nacional de Desenvolvimento Cientıfico
Tecnologico is acknowledged for supporting the productivity
˜
¸ ao de Amparo a Pesquisa do Estado de
`
˜
´
e
´
fellowship of R.I. and M.S.B.
with reducing agents like DABCO (10), the DCFH oxidizing
species in cell studies are likely to be oxidizing radicals
generated by peroxidases and DCFH reacts with compound I
of horseradish peroxidase (30–32).
REFERENCES
1. Sies, H. (1997) Oxidative stress: Oxidants and antioxidants. Exp.
Physiol. 82, 291–295.
2. Gomes, A., E. Fernandes and J. L. F. C. Lima (2005) Fluores-
cence probes used for detection of reactive oxygen species.
J. Biochem. Biophys. Methods 65, 45–80.
It is interesting to compare the reactivity of DCFH against
•2+
1
either O
which MB
2
or MB . As mentioned before, in the condition in
•
2+
is formed (MB dimers are excited), DCF
formation is faster and larger. Figure 5 shows the quantitative
estimation of this effect. The rate of increase in DCF fluores-
cence is around 10 times larger in the presence of excess of
dimers (1 mM SDS) than in the presence of monomers (50 mM
SDS) and it is five times larger compared to that of water. As
mentioned before, this rate should depend on the steady-state
local concentration of reactant species and on bimolecular rate
3. Soh, N. (2006) Recent advances in fluorescent probes for the
detection of reactive oxygen species. Anal. Bioanal. Chem. 386,
5
32–543.
4
. Gomes, A., E. Fernandes and J. L. F. C. Lima (2006) Use of
fluorescence probes for detection of reactive nitrogen species: A
review. J. Fluoresc. 16, 119–139.
5. Bartosz, G. (2006) Use of spectroscopic probes for detection of
reactive oxygen species. Clin. Chim. Acta 368, 53–76.
6. Royall, J. A. and H. Ischiropoulos (1993) Evaluation of 2¢,7¢-
dichlorofluorescin and dihydrhorodamine 123 as fluorescent
1
•2+
constants. Knowing that the efficiencies of either O or MB
2
generations are similar in the presence of MB monomers or
dimers (10), respectively, one could think, at first glance, that
probes for intracellular H
Biochem. Biophys. 302, 348–355.
2 2
O in cultured endothelial cells. Arch.
•
2+
7
. Tobi, S. E., N. Paul and T. J. McMillan (2000) Glutathione
modulates the level of free radicals produced in UVA-irradiated
cells. J. Photochem. Photobiol. B, Biol. 57, 102–112.
. Bryska, M., A. Bacia and D. Elbaum (2001) Oxidative and
hydrolytic properties of b-amyloid. Eur. J. Biochem. 268, 3443–3454.
9. Bilski, P., A. G. Belanger and C. F. Chignell (2002) Photosensi-
tized oxidation of 2¢,7¢-dichlorofluorescin: Singlet oxygen does not
contribute to the formation of fluorescent oxidation product 2¢,7¢-
dichlorofluorescein. Free Radic. Biol. Med. 33, 938–946.
10. Junqueira, H. C., D. Severino, L. G. Dias, M. Gugliotti and M. S.
Baptista (2002) Modulation of the methylene blue photochemical
properties based on the adsorption at aqueous micelle interfaces.
Phys. Chem. Chem. Phys. 4, 2320–2328.
1. Estevam, M. L., O. R. Nascimento, M. S. Baptista, P. Di Mascio,
A. Faljoni-Alario, M. R. Zucchi and I. L. Nantes (2004) Changes
in the spin state and reactivity of cytochrome c induced by pho-
tochemically generated singlet oxygen and free radicals. J. Biol.
Chem. 279, 39214–39222.
2. Severino, D., H. C. Junqueira, M. Gugliotti, D. S. Gabrielli and
M. S. Baptista (2003) Influence of negatively charged interfaces on
the ground and excited state properties of methylene blue. Pho-
tochem. Photobiol. 77, 459–468.
3. Gabrielli, D., E. Belisle, D. Severino, A. J. Kowaltowski and M. S.
Baptista (2004) Binding, aggregation and photochemical proper-
ties of methylene blue in mitochondrial suspensions. Photochem.
Photobiol. 79, 227–232.
the reactivity of MB
1
against DCFH is larger than that of
2
. However, before reaching such a conclusion, it is impor-
O
•
2+
1
tant to consider the lifetimes of MB
rate constant for the spontaneous decay of MB
2
and O in solution. The
8
•2+
in aqueous
in water it is
lives hundreds of micro-
seconds, while O2 lives only several microseconds. As a
3
)1
1
solution is ꢀ3 · 10 s (10), whereas for O
2
5
)1
•2+
around ꢀ3·10 s (20), i.e. MB
1
1
consequence, the steady-state concentration of O is smaller
2
•
2+
than that of MB . Considering that the final concentration
of oxidized DCF is only 1 order of magnitude larger in the
•
2+
condition in which MB
1
condition in which O
is generated compared to the
1
is generated, one can conclude that O
1
2
2
•
2+
is supposedly more reactive than MB against DCFH.
However, in order to obtain a quantitative estimate of the
1
•2+
relative reactivity of
O
2
and MB
against DCFH, it is
1
necessary to realize a more detailed kinetic study in order to
calculate the second-order rate constants. In cells in the case of
redox misbalance caused by photoinduced processes, DCFH
1
1
may help detect the generation of
2
O . However, during
photochemical reactions it is always important to also consider
the DCFH oxidation by oxidizing radicals and triplet species
(
9,21).

Photochemistry and Photobiology, 2008, 84 1243
27. Zhu, H., G. L. Bannenberg, P. Moldeus and H. G. Shertzer (1994)
1
4. Tardivo, J. P., A. Del Giglio, C. S. de Oliveira, D. S. Gabrielli, H.
C. Junqueira, D. B. Tada, D. Severino, R. F. Turchiello and M. S.
Baptista (2006) Methylene blue in photodynamic therapy: From
basic mechanisms to clinical applications. Photodiagn. Photodyn.
Ther. 2, 165–238.
´
Oxidation pathways for the intracellular probe 2¢,7¢-dichloroflu-
orescein. Arch. Toxicol. 68, 582–587.
28. Hempel, S. L., G. R. Buettner, Y. Q. O’Malley, D. A. Wessels and
D. M. Flaherty (1999) Dihydrofluorescein diacetate is superior for
detecting intracellular oxidants: Comparison with 2¢,7¢-dichloro-
dihydrofluorescein diacetate, 5(6)-carboxy-2¢,7¢-dichlorodihydro
fluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol.
Med. 27, 146–159.
29. LeBel, C. P., H. Ischiropoulos and S. C. Bondy (1992) Evaluation
of the probe 2¢,7¢-dichlorofluorescin as an indicator of reactive
oxygen species formation and oxidative stress. Chem. Res. Toxicol.
5, 227–231.
30. Myhre, O., J. M. Andersen, H. Aarnes and F. Fonnum (2003)
Evaluation of the probes 2¢,7¢-dichlorofluorescin diacetate, lumi-
nol, and lucigenin as indicators of reactive species formation.
Biochem. Pharmacol. 65, 1575–1582.
31. Rota, C., C. F. Chignell and R. P. Mason (1999) Evidence for free
radical formation during the oxidation of 2¢,7¢-dichlorofluorescin
to the fluorescent dye 2¢,7¢-dichlorofluorescein by horseradish
peroxidase: Possible implications for oxidative stress measure-
ments. Free Radic. Biol. Med. 27, 873–881.
1
1
1
5. Tardivo, J. P., A. Del Giglio, L. H. Paschoal and M. S. Baptista
(2006) A new PDT protocol to treat AIDS-related Kaposi’s sar-
coma. Photomed. Laser Surg. 24, 528–531.
6. Orth, K., G. Beck, F. Genze and A. Ruck (2000) Methylene blue
mediated photodynamic therapy in experimental colorectal
tumors in mice. J. Photochem. Photobiol. B, Biol. 57, 186–192.
7. Kolarova, H., R. Bajgar, K. Tomankova, P. Nevrelova and J.
Mosinger (2007) Comparison of sensitizers by detecting reactive
oxygen species after photodynamic reaction in vitro. Toxicol. In
Vitro 21, 1287–1291.
1
8. Cadenas, E. and H. Sies (1984) Low-level chemiluminescence as an
indicator of singlet molecular oxygen in biological systems.
Methods Enzymol. 105, 221–231.
1
9. Bonnett, R. (2000) Chemical Aspects of Photodynamic Therapy.
Advanced Chemistry Texts. Gordon & Breach Sci Publisher, UK.
0. Wilkinson, F., W. P. Helman and A. B. Ross (1995) Rate con-
stants for the decay and reactions of the lowest electronically ex-
cited singlet-state of molecular-oxygen in solution—An expanded
and revised compilation. J. Phys. Chem. Ref. Data 24, 663–1021.
1. O’Mailley, Y. Q., K. Reszka and B. E. Britigan (2004) Direct
oxidation of 2¢,7¢-dichlorodihydrofluorescein by pyocyanin and
other redox-active compounds independent of reactive oxygen
species production. Free Radic. Biol. Med. 36, 90–100.
2
32. Bonini, M. G., C. Rota, A. Tomasi and R. P. Mason (2006) The
oxidation of 2¢,7¢-dichlorofluorescin to reactive oxygen species: A
self-fulfilling prophesy? Free Radic. Biol. Med. 40, 968–975.
33. Niedre, M. J., A. J. Secord, M. S. Patterson and B. C. Wilson
(2003) In vitro tests of the validity of singlet oxygen luminescence
measurements as a dose metric in photodynamic therapy. Cancer
Res. 63, 7986–7994.
2
2
2
2
2
2
2. Baptista, M. S. and G. L. Indig (1998) Effect of BSA binding on
photophysical and photochemical properties of triarylmethane
dyes. J. Phys. Chem. B 102, 4678–4688.
34. Redmond, R. W. and I. E. Kochevar (2006) Spatially resolved
cellular responses to singlet oxygen. Photochem. Photobiol. 82,
1178–1186.
35. Holevinsky, K. O. and D. J. Nelson (1995) Simultaneous detection
of free radical release and membrane current during phagocytosis.
J. Biol. Chem. 270, 8328–8336.
36. Gueven, N., O. J. Becherel, O. Howe, P. Chen, J.-F. Haince, M.-E.
Ouellet, G. G. Poirier, N. Waterhouse, M. Fusser, B. Epe, J. M.
Murcia, G. Murcia, C. H. McGowan, R. Parton, C. Mothersill, P.
Grattan-Smith and M. F. Lavin (2007) A novel form of ataxia
oculomotor apraxia characterized by oxidative stress and apop-
tosis resistance. Cell Death Differ. 14, 1149–1161.
3. Baptista, M. S. and G. L. Indig (1997) Mechanism of photoble-
aching of ethyl violet non-covalently bound to bovine serum
albumin. Chem. Commun. 18, 1791–1792.
4. Lissi, E. A., M. V. Encinas, E. Lemp and M. A. Rublo (1993)
) bimolecular processes. Solvent and com-
1
Singlet oxygen O
partmentalization effects. Chem. Rev. 93, 699–723.
2
( 4
g
5. Schweitzer, C. and R. Schmidt (2003) Physical mechanisms of
generation and deactivation of singlet oxygen. Chem. Rev. 103,
1685–1757.
6. Carter, W. O., P. K. Narayanan and J. P. Robinson (1994)
Intracellular hydrogen peroxide and superoxide anion detection in
endothelial cells. J. Leukoc. Biol. 55, 253–258.