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181
Original Article J. Clin. Biochem. Nutr., 47, 181–190, November 2010
Advance Publication
JCBNJournal of Clinical Biochemistry and Nutrition0912-00091880-5086the Society for Free Radical Research JapanKyoto, Japanjcbn09-11210.3164/jcbn.09-112Original Article
Structure Effect on Antioxidant Activity of Catecholamines toward
Singlet Oxygen and Other Reactive Oxygen Species in vitro
Takako Shimizu1, Yuji Nakanishi1, Meiko Nakahara2, Naoki Wada1, Yoshihiko Moro-oka3,
Toru Hirano4, Tetsuya Konishi5 and Seiichi Matsugo1,*
1School of Natural System, College of Science and Engineering, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
2Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Takeda 4-3-11, Kofu, Yamanashi 400-8511, Japan
3Emeritus Prof. of Tokyo Institute of Technology
4Hamamatsu University School of Medicine, Photon Medical Research Center,
Hamamatsu, Handayama, Higashi-ku, Hamamatsu 431-3192, Japan
5Faculty of Applied Life Sciences, Niigata University of Pharmacy & Applied Life Sciences,
Higashijima 265-1, Akiha-ku, Niigata 956-2081, Japan
1120101692010473181190Received 16.11.2009; accepted 22.12.2009
*To whom correspondence should be addressed.
Tel: +81-76-264-6219 Fax: +81-76-234-4829
E-mail: matsugoh@t.kanazawa-u.ac.jp
Received 16 November, 2009; Accepted 22 December, 2009; Published online 16 September, 2010
Copyright © 2010 JCBN
Summary The reactivity of catecholamine neurotransmitters and the related metabolites
were precisely investigated toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reactive
oxygen species. Catecholamines reacted immediately with DPPH radicals, their reactivity
being stronger than that of ascorbic acid as a reference. Superoxide scavenging activities of
catecholamines determined by WST-1 and electron spin resonance (ESR) spin trapping
methods were also high. Whereas tyrosine, the dopamine precursor showed no reactivity
toward superoxide. The reactivity toward singlet oxygen was evaluated by observing specific
photon emission from singlet oxygen. The results revealed that reactivity of catecholamines
was markedly higher than that of sodium azide, and catechin as catechol reference. The
reaction of catecholamines and singlet oxygen was further studied by ESR using 55-dimethyl-
1-pyrroline N-oxide (DMPO) as a spin trapping reagent and rose bengal as photosensitizer.
DMPO-OH signal of epinephrine was significantly small compared to other catecholamines,
catechin, and 4-methylcatechol as a reference compound and was as small as that of tyrosine.
The signal formation was totally dependent on singlet oxygen, and the presence of catechol
compounds. These results indicated that epinephrine is the most potent singlet oxygen
quencher than other catecholamines, and the secondary amino group in its alkyl side chain
could play a role in unique singlet oxygen quenching property of epinephrine.
Key Words:catecholamine, neurotransmitters, ROS, chemiluminescence, singlet oxygen
Introduction
The catecholamines dopamine, norepinephrine, and epi-
nephrine function as neurotransmitters in the central nervous
system and also as hormones in the peripheral endocrine
system [1]. These compounds contain a characteristic cate-
chol chromophore, as indicated in their family name, and are
biosynthesized from tyrosine through an 3,4-dihydroxy-L-
phenylalanine (L-DOPA). Catecholamines affect the brain
regions associated with emotional activities such as hyper-
excitability and depression [2]. The catechol structure is
T. Shimizu et al.
J. Clin. Biochem. Nutr.
182
widely distributed in many naturally occurring antioxidants
and plays a role in scavenging reactive oxygen species
(ROS), typically, superoxide. Therefore, catecholamine
neurotransmitters and related metabolites are also expected
to react with ROS.
The brain is vulnerable to oxidative stress caused by high
contents of oxidizable substrates, such as polyunsaturated
fatty acid (PUFA) [3]. The brain consumes ca. 20% of
inhaled oxygen, thus, a large amount of ROS are believed to
be produced under normal metabolic and physiologic states
[4]. ROS produced in the brain have been associated with
various neurodegenerative disorders such as Parkinson’s
disease, which is characterized by the dysfunction of
dopaminergic neurons in the nigrostriatal system [5].
The external administration of antioxidant is usually not
effective to prevent brain oxidative stress because of the
presence of the blood/brain/barrier (BBB) [6]. In addition,
the activity of typical antioxidant enzymes such as super-
oxide dismutase (SOD) is lower in the brain than in other
tissues such as liver [7]. Hence, we focused our attention
on the reactivity of catecholamine neurotransmitters toward
ROS and radical including 1,1-diphenyl-2-picrylhydrazyl
(DPPH), superoxide, and singlet oxygen (Fig. 1). Superoxide
is the most abundant ROS generated under physiological
conditions and it is also generated by the auto-oxidation of
dopamine [8]. Singlet oxygen, on the other hand, has been
paid little attention on the oxidative stress compared with
other ROS, although it causes damages DNA [9], PUFA
[10], and amino acid [11] at the locus of generation. It is
produced by a photosensitized reaction (photodynamic
action) and also by enzyme-catalyzed reactions such as
myeloperoxidase-hydrogen peroxide-chloride reaction
in vivo
system [12]. It also plays a role in cellular signaling and
apoptosis [13–15]. Therefore, it is necessary to study the
reactivity of singlet oxygen toward neurotransmitter mole-
cules. Although several fragmented studies have been
reported on the reactions of catecholamines toward ROS,
we have re-evaluated the scavenging activity of a series of
catecholamine neurotransmitters towards ROS, with special
focus on their reactivity toward singlet oxygen.
Materials and Methods
Reagents
Dopamine hydrochloride, 3,4-dihydroxy-L-phenylalanine
(L-DOPA), L-noradrenaline (norepinephrine), L-adrenaline
(epinephrine), tyrosine, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), 4-methyl catechol, hypoxanthine, and xanthine
oxidase (XOD) were purchased from Nacalai Tesque Co.
(Kyoto, Japan). Ascorbic acid and rose bengal were
purchased from Sigma-Aldrich Co. Japan (Tokyo, Japan).
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate sodium salt (WST-1) and 55-Dimethyl-
1-pyrroline N-oxide (DMPO) were purchased from Dojindo
Co. (Kumamoto, Japan). (+)-Catechin hydrate was purchased
from Spectrum Chemical Mfg. Corp. (Gardena, CA). Sodium
azide was purchased from Wako Co. (Osaka, Japan). DL-
Thioctic acid (lipoic acid) was purchased from Kanto chem-
ical Co. (Tokyo, Japan). Other chemicals were of the highest
grade. All reagents were used without further purification.
Water was prepared using Advantec PWU-100. For the
singlet oxygen photon counting method, water was prepared
using Yamato Scientific WG202.
UV-Vis absorption spectra were measured by JASCO
V550 UV/VIS spectrophotometer. Singlet oxygen chemilu-
minescence was detected by NIR-II Hamamatsu Photonics
KK, Japan.
DPPH radical scavenging activity evaluated by UV-vis
spectroscopic method
Dopamine and other test samples were dissolved in
25 mM phosphate buffer (pH 7.4) containing ethanol (final
33% v/v), except epinephrine. Since the solubility of
epinephrine in phosphate buffer was low, it was dissolved
in a mixture of phosphate buffer and 0.1% acetic acid.
Then, 0.5 ml of 1 mM DPPH in ethanol was added to 4.5 mL
of catecholamine solutions of various concentrations (final
ethanol 40% v/v), and the solution was stirred for 30 s. The
UV-Vis absorption spectrum of this solution was obtained in
the range from 350 nm to 700 nm. DPPH radical scavenging
activity of catecholamines was evaluated by determining
the absorbance at 526.5 nm. The reaction was also performed
in 40% methanol/phosphate buffer (pH 7.4), and the DPPH
Fig. 1. Chemical structures of catecholamine neurotransmitters
and catechin.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
183
radical scavenging activity was calculated according to
equation 1 (eq. 1). In the equation, A and B indicate DPPH
absorbance in the absence and presence of the test sample,
respectively.
DPPH radical scavenging activity (%) = 100 ×(A–B)/A
(eq. 1)
DPPH radical scavenging activity evaluated by ESR method
The test samples were dissolved in 25 mM phosphate
buffer (pH 7.4) containing methanol (final 37.5% v/v),
except epinephrine. Epinephrine was dissolved in a mixture
of 0.4% acetic acid and phosphate buffer as described
above. Then, 40 µl of 1 mM DPPH in methanol was added
to 160 µl of test samples of defined concentrations (final
methanol 50% v/v), and the solution was stirred for 30 s.
Electron spin resonance (ESR) spectra of the solution
were obtained after 90 s, and the DPPH radical scavenging
activity was evaluated on the basis of the peak height of
DPPH radical determined after normalization with the Mn
reference peak. The conditions used for ESR measurements
(JEOL JES TE200) were as follows: temperature, 22°C;
frequency, 9.04 GHz; power, 8.00 mW; field, 341.0
±
10 mT
;
sweep time, 0.5 min; field modulation, 0.2 mT; and amplitude,
100; and time constant, 0.03.
Superoxide scavenging activity determined by WST-1 method
The test samples of various concentrations were dissolved
in 25 mM phosphate buffer (pH 7.4), and subsequently,
0.1 U/ml XOD was added to the reaction mixture (total
volume: 3 ml) containing 333 mM WST-1, 30 µM hypo-
xanthine, and phosphate buffer. Epinephrine was dissolved
1% acetic acid (60 µl) instead of phosphate buffer as
described above. The reaction was performed at room
temperature and the absorbance at 432 nm was determined
at 1 min after the addition of XOD. Superoxide scavenging
activity was evaluated by the residual amount of WST-1 as
in the following equation (eq. 2). The A and B indicate
WST-1 absorbance in the absence and presence of the test
sample, respectively.
Superoxide scavenging activity (%) = 100 ×(A–B)/A
(eq. 2)
Superoxide scavenging activity evaluated by ESR spin
trapping method
Test samples of defined concentrations were dissolved
in 25 mM phosphate buffer (pH 7.4), and subsequently,
0.1 U/ml XOD was added to the reaction mixture (total
volume: 300 µl) containing 300 mM DMPO, 533 µM
hypoxanthine, and phosphate buffer. Epinephrine was
dissolved in 1% acetic acid (30 µl) and used as described
above. The ESR spectra were obtained at 50 s after the
addition of XOD, and the superoxide scavenging activity
was evaluated from the peak height of DMPO-OOH signal
(the superoxide DMPO adduct) after normalization with
the Mn reference peak [16]. The conditions used for ESR
measurement were the same as mentioned before.
Singlet oxygen scavenging activity evaluated by photon
counting method
To evaluate the singlet oxygen scavenging activity of
catecholamines, we dissolved the test samples and rose
bengal (photosensitizer) in deionized water at the concentra-
tions of 1 mM and 30 µM, respectively. The reaction mix-
ture containing test sample and 3 ml of rose bengal was
dispensed in a quartz cuvette and irradiated by YAG laser
at 532 nm (30 Hz, 40 mW) with a silicon filter (1100 nm
cut-off). Photon emission from singlet oxygen was detected
at 1268 nm using a highly sensitive near infrared (NIR)
spectrophotometer [17]. Data correction was achieved by
keeping a 5 µs time-lag after every pulse irradiation to avoid
the effect of strong artificial background fluorescence from
rose bengal. To evaluate the dose dependant activity of
catecholamines, we added 50 µl of test sample (up to 250 µl)
to the reaction mixture in a cuvette, and determined the
absorbance of the solution at each step. Singlet oxygen
scavenging activity was evaluated based on the signal
intensity of the peaks.
Reaction of catecholamines with singlet oxygen in the
presence of DMPO
The reaction mixture containing 250 µM test sample,
225 µM DMPO, and 24.5 µM rose bengal was prepared in
25 mM phosphate buffer (pH 7.4) (total volume: 200 µl).
The dose dependent activities of sodium azide and lipoic
acid were determined in the presence of catechin (250 µM)
or dopamine (250 µM), respectively. The reaction solution
was placed at 12 cm from the halogen lamp (650 W) and
irradiated by visible light for 1 min. After 100 s, the ESR
spectrum of the mixture was obtained (JES-FR30EX).
Statistical analyses
All data presented in this study were the average of at
least three times experiments and expressed as mean ±SD
values. Data were analyzed by Student’s t test.
Results
DPPH radical scavenging activity
The DPPH radical scavenging activity of catecholamines
dissolved in aqueous ethanol revealed that the absorption
bands of DPPH radical at 526.5 nm decreased after the
addition of catecholamines in a concentration dependent
manner. The relative DPPH radical quenching ability of
catecholamines and their IC30 values (the concentrations
T. Shimizu et al.
J. Clin. Biochem. Nutr.
184
required for scavenging 30% of DPPH radicals) are
summarized in Table 1. The reactivity of catecholamines in
aqueous ethanol was in the following order: norepinephrine
> dopamine > epinephrine > L-DOPA > ascorbic acid.
Tyrosine did not scavenge DPPH radical in the same concen-
tration range as that of catecholamines.
The DPPH scavenging activity was also determined in
aqueous methanol. The IC30 values obtained in aqueous
methanol were significantly smaller than those obtained in
aqueous ethanol. However, in both solutions, all the examined
catecholamines showed stronger scavenging activity than
ascorbic acid.
The DPPH radical scavenging activity of catecholamines
was further determined by ESR. Since this method afforded
a clear dose response up to 80% inhibition level, the
scavenging activity of catecholamines was compared on the
basis of the IC50 values (the concentration scavenging 50%
of the original DPPH); these are presented in Table 2. The
DPPH radical scavenging activities in aqueous methanol
system obtained by ESR were similar to those determined by
UV-Vis spectroscopy as described above. The scavenging
activity of epinephrine was lower than that of other
catecholamines, as determined by both these methods. The
reactivity of catecholamines was in the following order:
dopamine > norepinephrine > L-DOPA > epinephrine >
ascorbic acid. Tyrosine did not affect the peak height of
the DPPH radical signal.
Superoxide scavenging activity
Superoxide scavenging activity of catecholamines was
determined by the WST-1 method, using tyrosine and
ascorbic acid as reference agents. The dose dependant
reactivity of catecholamines is shown in Fig. 2, and the IC30
values obtained from the reaction profile are summarized in
Table 3. The superoxide scavenging activity was in the
following order: dopamine > L-DOPA > norepinephrine >
ascorbic acid > epinephrine. Tyrosine showed no reactivity
Table 1. DPPH radical scavenging activity determined by UV-vis spectroscopy
*p<0.05, **p<0.01 compared with the indicated group using student t test.
sample ethanol methanol
IC30 (µM) IC30 (µM)
L-DOPA 14.9 ±1.0 7.75 ±1.3
dopamine 12.8 ±2.9 7.14 ±1.5
norepinephrine 12.2 ±1.0 7.69 ±1.2
epinephrine 13.3 ±1.9 10.9 ±0.7
ascorbic acid 26.3 ±0.7 15.9 ±1.0
tyrosine no reaction no reaction
*
** ** ** **
** ** ** ** ** ** **
Table 2. DPPH radical scavenging activity determined by ESR
spectroscopy
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample methanol
IC50 (µM)
L-DOPA 7.51 ±1.6
dopamine 6.33 ±0.8
norepinephrine 7.10 ±1.1
epinephrine 11.8 ±1.0
ascorbic acid 15.9 ±2.3
tyrosine no reaction
*** ** ** ***
Fig. 2. Superoxide scavenging activity determined by WST-1
method. The decrease in the absorbance at 432 nm due
to superoxide-induced formazan formation by WST-1
reduction was measured by UV-Vis spectrometer in the
presence and absence of catecholamine. Superoxide was
generated in the hypoxanthine-xanthine oxidase system.
(a) dopamine, (b) norepinephrine, (c) epinephrine, (d)
L-DOPA, (e) tyrosine, (f) ascorbic acid.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
185
toward superoxide.
The superoxide scavenging activity was further evaluated
by ESR using spin trapping method. The typical ESR spectra
of DMPO-OOH signal in the presence of different concen-
trations of dopamine are shown in Fig. 3. The DMPO-OOH
signal intensity decreased in a concentration dependent
manner.
The IC50 values of different catecholamines determined
by the ESR method are listed in Table 4. The relative
activities of catecholamines determined by ESR method
were similar to those obtained by WST-1 method. The
scavenging activity of dopamine, norepinephrine, and L-
DOPA was higher than that of ascorbic acid, while the
scavenging activity of epinephrine was lower than that of
other catecholamines and ascorbic acid. The relative
activity was in the following order: dopamine > L-DOPA >
norepinephrine > ascorbic acid > epinephrine.
Singlet oxygen quenching activity
When the singlet oxygen is discharged to the ground state
oxygen, photon emission is observed at approximately
1268 nm [18]. Therefore, the photon counting of this
emission is a direct evidence of singlet oxygen generation.
In the present study, we determined the singlet oxygen
scavenging potential of catecholamines by observing this
emission using a highly sensitive NIR detection system.
The peak height of the emission spectra of singlet oxygen
decreased in the presence of dopamine in a concentration
dependent manner as shown in Fig. 4. The dose dependence
of the quenching profile shown in Fig. 5 revealed that both
Table 3. Superoxide scavenging activity determined by WST-1
reduction
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample IC30 (µM)
L-DOPA 16.8 ±3.8
dopamine 11.6 ±0.9
norepinephrine 18.1 ±1.7
epinephrine 70.9 ±7.3
ascorbic acid 26.5 ±1.5
tyrosine no reaction
** ** *** **
**
**
**
**
Fig. 3. Dopamine dependent decrease of DMPO-OOH signal.
Superoxide was determined by ESR spin trapping method
using DMPO as the spin trap reagent and hypoxanthine-
xanthine oxidase system as superoxide generator. Precise
ESR measurement condition is given in the experimental
section.
Table 4. Superoxide scavenging activity determined by ESR
using DMPO
*p<0.05, **p<0.01 compared with the indicated group using
student t test.
sample IC50 (µM)
L-DOPA 24.3 ±1.1
dopamine 14.6 ±2.3
norepinephrine 25.5 ±4.3
epinephrine 89.7 ±4.8
ascorbic acid 41.2 ±1.6
tyrosine no reaction
** ** ** ** **
**
*
**
**
Fig. 4. Singlet oxygen quenching activity of dopamine deter-
mined by photon counting method. Rose bengal was
used as a photosensitizer to generate singlet oxygen.
Photon emission was determined by photon counter after
laser irradiation at 532 nm in the presence and absence
of test sample. Precise reaction condition is given
experimental section.
T. Shimizu et al.
J. Clin. Biochem. Nutr.
186
epinephrine and norepinephrine have stronger quenching
ability than dopamine and L-DOPA. The IC50 values of test
samples are summarized in Table 5. The scavenging
activities of catecholamines were significantly higher than
those of catechin and sodium azide as typical singlet oxygen
quencher. The reactivity was in the following order:
epinephrine > norepinephrine > L-DOPA > dopamine >>
catechin > sodium azide.
The reactivity of catecholamines to singlet oxygen was
further examined by the ESR spin trapping method using
DMPO, according to a previously reported [19]. Similar to
the findings of this study, we detected the characteristic
DMPO-OH signal after irradiation in the presence of
catecholamines or catechin (Fig. 6). Simple irradiation of
rose bengal without catecholamines or catechin did not
reveal any significant radical trapped by DMPO signal.
Irradiation of catecholamines or catechin in the absence of
rose bengal did not produce any signals. Almost same signal
Fig. 5. Dose effect of catecholamines on singlet oxygen
quenching reaction. Singlet oxygen quenching activity
was measured as in Fig. 4. Precise reaction condition
is given experimental section. (a) dopamine, (b)
norepi-
nephrine, (c) epinephrine, (d) L-DOPA, (e) sodium azide
,
(f) catechin.
Table 5. Singlet oxygen quenching activity evaluated by 1268 nm photon emission
**p<0.01 compared with the indicated group using student t test.
sample IC50 (µM)
L-DOPA 23.9 ±5.1
dopamine 29.6 ±3.2
norepinephrine 15.9 ±1.0
epinephrine 14.0 ±0.2
sodium azide 181 ±15
catechin 140 ±12
** ** ** ** **
**
**
**
**
** **
**
** **
**
Fig. 6. Singlet oxygen scavenging activity of catecholamines
evaluated by ESR spin trapping method. The reaction
mixture containing catecholamines (250 µM), rose
bengal, and DMPO was irradiated for 1 min, ESR
spectra were measured at 100 s after the irradiation.
Precise reaction condition is given experimental section.
(A) none, (B) sodium azide, (C) catechin, (D) dopamine,
(E) norepinephrine, (F) L-DOPA, (G) epinephrine.
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
187
intensity was observed for all test samples with catechol
structure including 4-methyl catechol, except that the signal
intensity was small in the case of epinephrine (Fig. 6,
Table 6). Sodium azide inhibited this catechol dependent
signal in a concentration dependent manner, thereby implying
that DMPO-OH formation was dependant on the presence of
singlet oxygen and that OH-adduct formation resulted from
the reaction of singlet oxygen and catecholamines (Fig. 7).
Further, DMPO-OH signal formation was reduced in the
presence of lipoic acid, but it was not completely inhibited
even at high concentrations (up to 1.25 mM) (Fig. 8).
Discussion
The results in the present study revealed that cate-
cholamine neurotransmitters were highly reactive to ROS
and radical species. The DPPH and superoxide scavenging
activities of catecholamines were higher than those of
ascorbic acid and comparable to those of catechin, a flavan
having a catechol B-ring [20], although the reactivity of
epinephrine was rather weaker than those of ascorbic acid.
Further, tyrosine, which has only 1 phenolic OH group, did
not exhibit superoxide scavenging activity (Tables 3 and 4).
Ohkubo et al. [21] performed laser flash photolysis and
found
that cumyl peroxide reacts with neurotransmitters
(dopamine
,
norepinephrine, and epinephrine) by abstracting
the hydrogen
from the catechol OH group. The energy differ-
ence (DHT) between the original phenols and the resultant
phenoxyl radicals was several times lesser than that in
monophenols such as serotonin and tyrosine, thus, the rate of
oxidation of catecholamines was at least 10 times faster than
that of monophenols. This finding is consistent with our
present results although the ROS is not peroxyl radical but
superoxide anion radical in our present study.
Table 6. Catechol dependent DMPO-OH formation in the hoto-
dynamic singlet oxygen generating system
*p<0.05, **p<0.01 compared with the indicated group using
student t test. DMPO-OH was determined after the irradiation of
reaction mixture containing test sample, rose bengal as photo-
sensitizer and DMPO. Precise experimental condition was given
in the experimental section.
sample relative height
L-DOPA 8.9 ±0.2
dopamine 8.7 ±0.3
norepinephrine 9.1 ±0.1
epinephrine 1.5 ±0.3
4-methyl catechol 8.8 ±0.6
catechin 9.1 ±0.6
tyrosine 2.4 ±0.3
lipoic acid 1.7 ±0.3
** **
**
** ** **
Fig. 7. Effect of sodium azide on the catechin dependent
DMPO-OH formation. DMPO-OH formation was
measured as in Fig. 6 at various sodium azide concentra-
tions.
Fig. 8. Effect of lipoic acid on the dopamine dependant DMPO-
OH formation. DMPO-OH formation was measured as
in Fig. 6 at various lipoic acid concentrations.
T. Shimizu et al.
J. Clin. Biochem. Nutr.
188
Interestingly, we noted that the reaction of catecholamines
with DPPH showed a marked solvent effect. The DPPH
scavenging activities (estimated on the basis of IC values)
of catecholamines determined in aqueous methanol were
comparable between UV-Vis spectroscopy and ESR
methods, although there was a small difference among the
catecholamines. However, the IC30 values in aqueous
ethanol were markedly different from those in aqueous
methanol (Tables 1 and 2). When the relative scavenging
activities of catecholamines were compared with those of
ascorbic acid, which was used as the reference scavenging
agent, the activities of catecholamines were found to be
similar in both the solvents. Therefore, the DPPH assay is
only adaptable for the relative comparison of free radical
scavenging activities of a series of test samples, and a
common reference is required for accurate evaluation of
scavenging activity. Further, our results indicated that the
superoxide scavenging activity of epinephrine was lower
than that of other catecholamines. The same tendency was
observed for DPPH radical scavenging activity. Among all
the catecholamines used in this study, dopamine showed
the highest DPPH and superoxide scavenging activities in
all assays.
Further, the present study revealed the unique reactivity of
catecholamines toward singlet oxygen, whose reactivity was
different from that of other ROS. It is not a free radical but a
strong oxidant since it has unique electronic configuration
and 94.1 kJ/mol higher energy than the ground state oxygen
[22]. Singlet oxygen can cause damage in a wide range of
biologically important molecules [23]. The involvement of
singlet oxygen in a reaction is usually indirectly evaluated
on the basis of the presence of characteristic products, such
as “ene”-type products, or by using specific quenchers, such
as 1,4-diazabicyclo[2.2.2]octane (DABCO) or sodium azide
[24, 25]. In the present experiment, the reaction of singlet
oxygen with catecholamines and catechin were directly
examined by the photon counting method. The obtained
results revealed that the singlet oxygen quenching activity of
catecholamines was several times stronger than that of
catechin and sodium azide (Fig. 5).
The quenching of singlet oxygen proceeds via chemical
and physical processes. Sodium azide quenches singlet
oxygen mainly by the physical process [26]. It has been
reported that catechin, the green tea polyphenol, quenches
singlet oxygen mainly by the physical process and that the
catechol structure at B-ring is responsible for the physical
quenching process [27, 28]. It was also known that singlet
oxygen interacts with certain amino acids such as tryptophan
,
histidine, and tyrosine mainly by the chemical process. For
example, tyrosine and tyrosine derivatives that bear electron-
donating groups on their aromatic rings readily react with
singlet oxygen via the chemical process [29, 30]. In our
study, the reactivity of catecholamines toward singlet
oxygen, which was determined by evaluating the quenching
activity corresponding to the 1268 nm emission peak, was
considerably higher than that of catechin and sodium azide.
Among the catecholamines examined, norepinephrine and
epinephrine exhibited stronger quenching activities than
dopamine and L-DOPA did (Table 5). Therefore it was
expected not only the catechol structure but other factor such
as alkyl side chain property are involved in the singlet
oxygen quenching probably via both physical and chemical
processes.
In the present study, the reaction of singlet oxygen and
catecholamines was further examined by ESR using DMPO
as the spin trapping reagent. It has been reported that the
hydroxyl radical adduct of DMPO (DMPO-OH) is formed in
a photosensitized singlet oxygen generating system in the
presence of phenolic antioxidants or reduced glutathione,
thereby indicating that the hydrogen donating property of
antioxidants is essential for the formation of DMPO-OH
[31, 32]. Indeed, in our experiment, catecholamines and
catechin produced large DMPO-OH signals, however lipoic
acid, which is a strong antioxidant but is not a hydrogen
donating molecule, formed only a weak DMPO-OH signal
(Table 6). Further, catechin dependent DMPO-OH forma-
tion was inhibited by sodium azide as singlet oxygen
quencher in a concentration dependent manner (Fig. 7). The
DMPO-OH formation was therefore found to be primarily
dependent on the generation of singlet oxygen, as reported
previously [19, 31, 32]. Tyrosine did not give rise to
significant signal, although it has the same alkyl side chain
structure as L-DOPA but does not have catechol structure.
However, 4-methyl catechol, which is used as the reference
for catecholamine, formed as large DMPO-OH signal as that
of catecholamines (Table 6). Therefore, a catechol structure
is the primary requisite for DMPO-OH formation, and the
contribution of alkyl side chain to this process may not be
large. The interesting finding is that the DMPO-OH signal
of epinephrine was significantly small compared to other
catecholamines including norepinephrine, and the signal
level was as small as that of tyrosine even though it has
catechol structure.
Nishizawa et al. [31] reported the mechanism of the
DMPO-OH formation that the DMPO-singlet oxygen adducts
primarily generated is transformed into DMPO-OH in the
presence of hydrogen donating antioxidant, and they also
suggested that the free hydroxyl radical produced by
catechol oxidation is involved in DMPO-OH formation.
To evaluate the contribution of free hydroxyl radical in the
DMPO-OH formation, we studied the effect of lipoic acid on
the reaction, because lipoic acid and dihydrolipoic acid are
known to effectively scavenge hydroxyl radicals generated
from metal free system [33, 34]. Increasing concentrations
of lipoic acid inhibited catecholamine dependent DMPO-
OH formation, but this inhibitory effect was not very
Reactivity of Catecholamine toward Singlet Oxygen and ROS
Vol. 47, No. 3, 2010
189
remarkable (Fig. 8). Therefore, the free hydroxyl radicals
might not be the major source of DMPO-OH. Moreover,
epinephrine inhibited the dopamine dependant DMPO-OH
signal in a concentration dependent manner (data not
shown). Taken these discussions together, DMPO-OH signal
suppressing effect of epinephrine might be due to its higher
singlet oxygen scavenging activity. This is supported from
the data in Table 5 in that epinephrine showed the highest
reactivity toward singlet oxygen among the catecholamines.
The only structural difference between epinephrine and
norepinephrine is the alkyl side chain, the former has a
secondary amino group while the latter has the primary
amino group. Therefore, it can be presumed that owing to
the presence of the secondary amino group, epinephrine
exhibits higher activity toward singlet oxygen as compared
to other catecholamines, which has a primary amino group.
The present study showed the high reactivity of catechol
neurotransmitters (dopamine, norepinephrine, or epinephrine
)
and their precursor (L-DOPA) toward ROS. Therefore,
under oxidative stress conditions, catecholamine neuro-
transmitters will readily react with ROS in the brain and
oxidize dopamine to semiquinone, quinone, and dopamino-
chrome, which exert neurotoxic effects [35–40]. Although
the critical role of singlet oxygen in brain disorders has not
been elucidated yet, the results of this study suggest that
further investigation to understand the significant contribu-
tion of singlet oxygen-catecholamine neurotransmitter reac-
tions in brain disorders is warranted in the future.
References
[1] Hauser, S.L. and Longo, D.L.: Part IV Clinical Pharmaco-
logy, in Harrison’s Principles of Internal Medicine 15th, eds.
By Braunwald, E., Fauci, A.S., Kasper, D.L., and Jameson,
J.L., McGraw-Hill Professional Publishing, New York,
pp. 427–457, 2006.
[2] McQueen, J.K.: Biochemistry of transmitter molecules, in
Transmitter molecules in the brain, eds. By Flink, G.,
McQueen, J.K., Harmar, A.J., Arbuthnott, G.W., Mitchell, R.,
and Christie, J.E., Springer, Berlin, pp. 10–16, 1987.
[3] Liu, X., Yamada, N., Maruyama, W., and Osawa, T.:
Formation of dopamine adducts derived from brain poly-
unsaturated fatty acids: mechanism for Parkinson disease. J.
Biol. Chem., 283, 34887–34895, 2008.
[4] Floyd, R.A.: Antioxidants, oxidative stress, and degenerative
neurological disorders. Proc. Soc. Exp. Biol. Med., 222, 236–
245, 1999.
[5] Serra, P.A., Esposito, G., Enrico, P., Mura, M.A., Migheli, R.,
Delogu, M.R., Miele, M., Desole, M.S., Grella, G., and
Miele, E.: Manganese increases L-DOPA auto-oxidation in
the striatum of the freely moving rat: potential implications
to L-DOPA long-term therapy of Parkinson’s disease. Br. J.
Pharmacol., 130, 937–945, 2000.
[6] Gilgun-Sherki, Y., Melamed, E., and Offen, D.: Oxidative
stress induced-neurodegenerative diseases: the need for anti-
oxidants that penetrate the blood brain barrier. Neuro-
pharmacology, 40, 959–975, 2001.
[7] Marklund, S.L., Westman, N.G., Lundgren, E., and Roos,
G.: Copper- and zinc-containing superoxide dismutase,
manganese-containing superoxide dismutase, catalase, and
glutathione peroxidase in normal and neoplastic human cell
lines and normal human tissues. Cancer Res., 42, 1955–
1961, 1982.
[8] Graham, D.G.: Oxidative pathways for catecholamines in
the genesis of neuromelanin and cytotoxic quinones. Mol.
Pharmacol., 14, 633–643, 1978.
[9] Cadet, J., Douki, T., and Ravanat, J.L.: Oxidatively generated
damage to the guanine moiety of DNA: mechanistic aspects
and formation in cells. Acc. Chem. Res., 41, 1075–1083,
2008.
[10] Schafer, F.Q. and Buettner, G.R.: Singlet oxygen toxicity is
cell line-dependent: a study of lipid peroxidation in nine
leukemia cell lines. Photochem. Photobiol., 70, 858–867,
1999.
[11] Davies, M.J. and Truscott, R.J.: Photo-oxidation of proteins
and its role in cataractogenesis. J. Photochem. Photobiol. B.,
63, 114–125, 2001.
[12] Kiryu, C., Makiuchi, M., Miyazaki, J., Fujinaga, T., and
Kakinuma, K.:
Physiological production of singlet molecular
oxygen in the myeloperoxidase-H2O2-chloride system.
FEBS
Lett.
,
443, 154–158, 1999.
[13] Reiter, R.J.: Oxidative damage in the central nervous system:
protection by melatonin. Prog. Neurobiol., 56, 359–384,
1998.
[14] Klotz, L.O.: Oxidant-induced signaling: effects of peroxy-
nitrite and singlet oxygen. Biol. Chem., 383, 443–456, 2002.
[15] Chan, W.H., Yu, J.S., and Yang, S.D.: Apoptotic signaling
cascade in photosensitized human epidermal carcinoma
A431 cells: involvement of singlet oxygen, c-Jun N-terminal
kinase, caspase-3 and p21-activated kinase 2. Biochem. J.,
351, 221–232, 2000.
[16] Clément, J.L., Ferré, N., Siri, D., Karoui, H., Rockenbauer,
A., and Tordo, P.: Assignment of the EPR spectrum of 5,5-
dimethyl-1-pyrroline N-oxide (DMPO) superoxide spin
adduct. J. Org. Chem., 70, 1198–1203, 2005.
[17] Hirano, T., Kohno, E., Gohto, Y., and Obama, A.: Singlet
oxygen generation due to ICG irradiation. Photomedicine
and Photobiology, 28, 15–16, 2006.
[18] Arakane, K., Ryu, A., Takarada, K., Masunaga, T., Shinmoto,
K., Kobayashi, R., Mashiko, S., Nagano, T., and Hirobe, M.:
Measurement of 1268 nm emission for comparison of singlet
oxygen (1 delta g) production efficiency of various dyes.
Chem. Pharm. Bull (Tokyo), 44, 1–4, 1996.
[19] Ueda, J., Takeshita, K., Matsumoto, S., Yazaki, K.,
Kawaguchi, M., and Ozawa, T.: Singlet oxygen-mediated
hydroxyl radical production in the presence of phenols:
whether DMPO-*OH formation really indicates production
of *OH? Photochem. Photobiol., 77, 165–170, 2003.
[20] Rahman, M.M., Ichiyanagi, T., Komiyama, T., Hatano, Y.,
and Konishi, T.: Superoxide radical and peroxynitrite-
scavenging activity of anthocyanins; structure-activity rela-
T. Shimizu et al.
J. Clin. Biochem. Nutr.
190
tionship and their synergism. Free Radic. Res., 40, 993–
1002, 2006.
[21] Ohkubo, K., Moro-oka, Y., and Fukuzumi, S.: Hydrogen
abstraction from neurotransmitters by active oxygen species
facilitated by intramolecular hydrogen bonding in the radical
intermediates. Org. Biomol. Chem., 4, 999–1001, 2006.
[22] Straight, R.C. and Spikes, J.D.: Photosensitized oxidation of
biomolecules. In Singlet Oxygen vol. 4, ed. By Frier, A.A.,
CRC Press, Florida, pp. 91–143, 1985.
[23] Davies, M.J.: Singlet oxygen-mediated damage to proteins
and its consequences. Biochem. Biophys. Res. Commun.,
305, 761–770, 2003.
[24] Schaefer, M. and Hardeland, R.: The melatonin metabolite
N-acetyl-5-methoxykynuramine is a potent singlet oxygen
scavenger. J. Pineal. Res., 46, 49–52, 2009.
[25] Baier, J., Maisch, T., Maier, M., Landthaler, M., and
Bäumler, W.: Direct detection of singlet oxygen generated
by UVA irradiation in human cells and skin. J. Invest.
Dermatol., 127, 1498–1506, 2007.
[26]
Hall, R.D. and Chignell, C.F.: Steady-state near-infrared
detection of singlet molecular oxygen: a Stern-Volmer quench-
ing experiment with sodium azide.
Photochem. Photobiol.,
45, 459–464, 1987.
[27] Mukai, K., Nagai, S., and Ohara, K.: Kinetic study of the
quenching reaction of singlet oxygen by tea catechins in
ethanol solution. Free Radic. Biol. Med., 39, 752–761, 2005.
[28] Nagai, S., Ohara, K., and Mukai, K.: Kinetic study of the
quenching reaction of singlet oxygen by flavonoids in
ethanol solution. J. Phys. Chem. B., 109, 4234–4240, 2005.
[29] Michaeli, A. and Feitelson, J.: Reactivity of singlet oxygen
toward amino acids and peptides. Photochem. Photobiol., 59,
284–289, 1994.
[30] Wright, A., Bubb, W.A., Hawkins, C.L., and Davies, M.J.:
Singlet oxygen-mediated protein oxidation: evidence for the
formation of reactive side chain peroxides on tyrosine
residues. Photochem. Photobiol., 76, 35–46, 2002.
[31] Nishizawa, C., Takeshita, K., Ueda, J., Mizuno, M., Suzuki,
K.T., and Ozawa, T.: Hydroxyl radical generation caused
by the reaction of singlet oxygen with a spin trap, DMPO,
increases significantly in the presence of biological reduc-
tants. Free Radic. Res., 38, 385–392, 2004.
[32] Bilski, P., Reszka, K., Bilska, M., and Chignell, C.F.:
Oxidation of the spintrap 5,5-dimethyl-1-pyrroline N-oxide
by singlet oxygen in aqueous solution. J. Am. Chem. Soc.,
118 , 1330–1338, 1996.
[33] Matsugo, S., Yan, L.J., Han, D., Tritschler, H.J., and Packer,
L.: Elucidation of antioxidant activity of alpha-lipotic acid
toward hydroxyl radical. Biochem. Biophys. Res. Commun.,
208, 161–167, 1995.
[34] Matsugo, S., Yan, L.J., Han, D., Tritschler, H.J., and Packer,
L.: Elucidation of antioxidant activity of dihydrolipoic acid
toward hydroxyl radical using a novel hydroxyl radical
generator NP-III. Biochem. Mol. Biol. Int., 37, 375–383,
1995.
[35] Smythies, J. and Galzigna, L.: The oxidative metabolism of
catecholamines in the brain: a review. Biochim. Biophys.
Acta, 10, 159–162, 1998.
[36] Terland, O., Flatmark, T., Tangerås, A., and Grønberg, M.:
Dopamine oxidation generates an oxidative stress mediated
by dopamine semiquinone and unrelated to reactive oxygen
species. J. Mol. Cell. Cardiol., 29, 1731–1738, 1997.
[37] Terland, O., Almås, B., Flatmark, T., Andersson, K.K., and
Sørlie, M.: One-electron oxidation of catecholamines
generates free radicals with an in vitro toxicity correlating
with their lifetime. Free Radic. Biol. Med., 41, 1266–1271,
2006.
[38] Hattoria, N., Wanga, M., Taka, H., Fujimura, T., Yoritaka, A.,
Kubo, S., and Mochizuki, H.: Toxic effects of dopamine
metabolism in Parkinson’s disease. Parkinsonism Relat.
Disord., 15, S35–38, 2009.
[39] Miyazaki, I. and Asanuma, M.: Dopaminergic neuron-
specific oxidative stress caused by dopamine itself. Acta
Med. Okayama, 62, 141–150, 2008.
[40] Stokes, A.H., Hastings, T.G., and Vrana, K.E.: Cytotoxic and
genotoxic potential of dopamine. J. Neurosci. Res., 15, 659–
665, 1999.
