Singlet Oxygen-mediated Hydroxyl Radical Production in the Presence of Phenols: Whether DMPO-.OH Formation Really Indicates Production of .OH?

Article abstract of DOI:10.1562/0031-8655(2003)077<0165:SOMHRP>2.0.CO;2

The reaction of singlet oxygen (¹O₂) generated by ultraviolet-A (UVA)-visible light (λ > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-^.OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-^.OH decreased in the presence of either a ^.OH scavenger or a quencher of ¹O₂ and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both ¹O₂- and DMPO-mediated formation of free ^.OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that ¹O ₂, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-^.OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of ^.OH. These observations indicate that ¹O₂ reacts first with DMPO, and the resulting DMPO-¹O₂ intermediate is immediately decomposed/reduced to give ^.OH. Phenolic compounds would participate in this reaction as electron donors but would not more, DEPMPO did not cause the spin-trapping agent-mediated generation of ^.OH like DMPO did.

Full text of DOI:10.1562/0031-8655(2003)077<0165:SOMHRP>2.0.CO;2

Singlet Oxygen–mediated Hydroxyl Radical Production in the Presence of Phenols:
Whether DMPO–·OH Formation Really Indicates Production of ·OH?
Author(s): Jun-ichi Ueda, Keizo Takeshita, Shigenobu Matsumoto, Kinya Yazaki, Mitsuru Kawaguchi,
and Toshihiko Ozawa
Source: Photochemistry and Photobiology, 77(2):165-170.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2003)077<0165:SOMHRP>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282003%29077%3C0165%3ASOMHRP
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2003, 77(2): 165–170
Singlet Oxygen–mediated Hydroxyl Radical Production
in the Presence of Phenols: Whether DMPO–ꢀOH Formation
ꢀ
Really Indicates Production of OH?{
Jun-ichi Ueda*1, Keizo Takeshita1, Shigenobu Matsumoto2, Kinya Yazaki3, Mitsuru Kawaguchi3
and Toshihiko Ozawa1
1National Institute of Radiological Sciences, Inage-ku, Chiba-shi, Japan;
2Laboratory of Biochemistry and Isotopes, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan and
3Department of Pharmacology, Tokyo Dental College, Mihama-ku, Chiba-shi, Japan
Received 8 August 2002; accepted 11 November 2002
ABSTRACT
more, DEPMPO did not cause the spin-trapping agent–
mediated generation of OH like DMPO did.
ꢀ
The reaction of singlet oxygen (1O2) generated by ultraviolet-A
(UVA)–visible light (k > 330 nm) irradiation of air-saturated
solutions of hematoporphyrin with phenolic compounds in the
presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO), gave an electron spin resonance (ESR) spectrum
characteristic of the DMPO–hydroxyl radical spin adduct
(DMPO–ꢀOH). In contrast, the ESR signal of 5,5-dimethyl-2-
pyrrolidone-N-oxyl, an oxidative product of DMPO, was
observed in the absence of phenolic compounds. The ESR
signal of DMPO–ꢀOH decreased in the presence of either
INTRODUCTION
Reactive oxygen species (ROS) such as superoxide (O2 2), the
ꢀ
hydroxyl radical (ꢀOH) and singlet oxygen (1O2) have been
implicated both in the aging process and in degenerative disease
2
ꢀ
ꢀ
(1–3). O2 and OH are oxygen radicals produced by electron
donation to molecular oxygen, whereas 1O2 is generated from
ꢀ
molecular oxygen by energy transfer. As well as OH, the most
1
reactive oxygen radical, O2 oxidizes many biological substances
1
ꢀ
a OH scavenger or a quencher of O2 and under anaerobic
conditions, whereas it increased depending on the concentra-
such as amino acids, polyunsaturated fatty acids and nucleosides
(4,5), thereby causing cell death and mutations (6).
1
tion of DMPO. These results indicate both O2- and DMPO-
mediated formation of free OH during the reaction. When
The interaction of 1O2 with some compounds may generally lead
to the generation of molecular oxygen by energy transfer reactions
ꢀ
DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-
pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen
radical species was obtained. This suggests that 1O2, as an
oxidizing agent, reacts little with DEPMPO, in which a strong
electron-withdrawing phosphoryl group increases the oxida-
tion potential of DEPMPO compared with DMPO. A linear
correlation between the amounts of DMPO–ꢀOH generated
and the oxidation potentials of phenolic compounds was
observed, suggesting that the electron-donating properties of
phenolic compounds contribute to the appearance of ꢀOH.
2
or to other ROS including O2ꢀ by electron transfer reactions (or
both) (7). Concerning the latter case, Saito et al. (8) reported that
organic compounds with oxidation potentials of less than 0.5 V (vs
SCE) were capable of undergoing single-electron transfer to 1O2 to
2
ꢀ
generate O2 preferentially in aqueous solvents. On the other
hand, the possibility of direct conversion of 1O2 to ꢀOH by
biological reductants such as reduced glutathione and reduced
nicotinamide adenine dinucleotide phosphate (NADPH) has been
suggested (9,10).
Skin is a defensive barrier and is constantly exposed to certain
kinds of oxidative stress such as ultraviolet and ionizing irradiation
1
(11). O2 is produced through the interaction of the ultraviolet-A
1
These observations indicate that O2 reacts first with DMPO,
and the resulting DMPO–1O2 intermediate is immediately
ꢀ
decomposed/reduced to give OH. Phenolic compounds would
component (UVA) of sunlight with endogenous photosensitizers
such as porphyrins and flavins in the skin (6,12). Because phenolic
compounds are widely used in the production of pharmaceutical as
well as cosmetic and food-flavoring products, the skin is inevitably
exposed to phenolic compounds (13). Therefore, it is considered
that a phenolic compound, which is as potent an electron donor as
reduced glutathione or NADPH, may also cause the conversion of
1O2 to another ROS. Then, we attempted to elucidate whether
oxygen radicals are produced from 1O2 in the presence of phenolic
compounds.
participate in this reaction as electron donors but would not
contribute to the direct conversion of 1O2 to OH. Further-
ꢀ
{Posted on the website on 25 November 2002.
*To whom correspondence should be addressed at: National Institute of
Radiological Sciences, 9-1 Anagawa 4-chome, Inage-ku, Chiba-shi 263-
8555, Japan. Fax: 81-43-255-6819; e-mail: ueda@nirs.go.jp
Abbreviations: DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-
oxide; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DMPOX, 5,5-dimeth-
yl-2-pyrrolidone-N-oxyl; ESR, electron spin resonance; HP, hematopor-
phyrin; NADPH, reduced nicotinamide adenine dinucleotide phosphate;
1O2, singlet oxygen; O2ꢀ2, superoxide; ꢀOH, hydroxyl radical; ROS,
reactive oxygen species; TEMPD, 2,2,6,6-tetramethyl-4-piperidone;
TEMPON, 2,2,6,6-tetramethyl-4-piperidone-1-oxyl; UVA, ultraviolet-A.
1
In this study, O2 was generated by irradiation of hematopor-
phyrin (HP), a model compound for endogenous porphyrins in the
skin (14), with UVA–visible light (k . 330 nm), and the
assignment of oxygen radicals was performed by electron spin
ꢀ 2003 American Society for Photobiology 0031-8655/01
$5.0010.00
165

166 Jun-ichi Ueda et al.
Measurements of oxidation potentials. To characterize the chemical
structure of phenolic compounds, we determined oxidation potentials in
phosphate buffer (pH 7.4) using a cyclic voltammetric method. Using an
ALS 630A Electrochemical Analyzer (BAS Co., Tokyo, Japan), cyclic
voltammetric measurements were made using a three-electrode system
consisting of a glass carbon electrode, a platinum counter electrode and an
Ag–AgCl electrode as a reference. To remove dissolved oxygen, nitrogen
gas was passed through the sample solution for 15 min. Cyclic voltammo-
grams were recorded at a scan rate of 100 mV/s. Cyclic voltammetry
indicated that the oxidation of phenols was not reversible. Therefore, the
experimental half-peak oxidation potentials were used as the oxidation
potential Epa and further calculated with the equation E0 (NHE) 5 Epa
1
0.212 (V).
ESR measurements. ESR measurements were made on a JEOL FE-2X
spectrometer (X-band) with 100 kHz field modulation. ESR spectra were
recorded at room temperature.
Statistical analysis. Linear regression analysis was performed using
Statview-4.5J (Abacus Concepts, Berkeley, CA) and used to determine the
relation between the signal intensities of DMPO adducts and the oxidation
potential of phenolic compounds.
RESULTS
1O2-mediated DMPO–ꢀOH formation in the presence
of phenolic compounds
A four-line ESR signal was observed 2 s after UVA–visible light
(k . 330 nm) irradiation of an air-saturated solution containing
100 mM DMPO and 25 lM HP at pH 7.4 in the presence of
100 lM dopamine (Fig. 2b) but not in the absence of dopamine
(Fig. 2a). The hyperfine splitting constants (aN 5 aH 5 14.9 G) of
this signal corresponded to those of DMPO–ꢀOH (18). No signal
from DMPO–ꢀOH was seen without HP, suggesting that DMPO–
ꢀOH is derived from the photochemistry dependent on the
sensitizer (Fig. 2c). Furthermore, DMPO–ꢀOH was vastly di-
minished either under anaerobic conditions (Fig. 2d) or with a 1O2
quencher, NaN3 (Fig. 2e), indicating that its formation is mediated
Figure 1. Chemical structures of the phenolic compounds used.
resonance (ESR) using as a spin trap, 5,5-dimethyl-1-pyrroline-N-
oxide (DMPO). DMPO is useful for identifying oxygen-centered
radicals because the resultant spin adducts have characteristic ESR
parameters and can be readily distinguished from other spin
adducts. The chemical structures of nine phenolic compounds
examined in this study are shown in Fig. 1. We observed the
ꢀ
production of the DMPO adduct of OH (DMPO–ꢀOH) derived
1
by O2. No signal for the DMPO adduct of the azide radical (ꢀN3)
from 1O2 in the presence of phenolic compounds and exam-
ined whether the presence of the adduct really reflects the
was observed.
To elucidate whether the DMPO–ꢀOH adduct was derived either
1
ꢀ
generation of free OH during the reaction of O2 with phenolic
compounds.
2
ꢀ
from the decomposition of the DMPO adduct of O2 (DMPO–
2
ꢀ
O2 ) or from the reaction of free ꢀOH with DMPO, the photoreac-
ꢀ
tion was performed in the presence of OH scavengers such as
MATERIALS AND METHODS
ethanol (5%) and sodium formate (0.25 M). The irradiation of the
respective solutions gave a new six-line ESR signal corresponding
to the DMPO adduct of the a-hydroxyethyl radical (ꢀCH(CH3)OH)
(aN 5 15.8 G, aH 5 22.8 G) (18) (Fig. 2f) or to that of the carbon
dioxide anion radical (ꢀCOO2) (aN 5 15.6 G, aH 5 18.7 G) (18)
(Fig. 2g) with a reduction in the signal intensity of the DMPO–ꢀOH
Materials. Trolox C, isoeugenol, p-hydroxybenzoic acid, 2,2,6,6-tetra-
methyl-4-piperidone (TEMPD) and 2,2,6,6-tetramethyl-4-piperidone-1-
oxyl (TEMPON) were purchased from Aldrich Chemical Co. (Milwaukee,
WI). p-Eugenol, p-methoxyphenol, phenol, 2,6-dimethoxyphenol and
guaiacol were obtained from Wako Pure Chemical Co. (Osaka, Japan).
HP and dopamine were purchased from Nakarai Chemical Co. (Kyoto,
Japan). DMPO was provided by Labotec (Tokyo, Japan) and used without
further purification. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide
(DEPMPO) was purchased from Oxis International Inc. (Portland, OR) and
also used without further purification. Chelex 100 resin (sodium form) was
obtained from Bio-Rad Co. (Hercules, CA).
1
ꢀ
adduct, indicating the O2-mediated formation of free OH during
the photoreaction of HP in the presence of dopamine. Catalase had
little effect on the signal intensity of DMPO–ꢀOH (data not
ꢀ
shown), indicating that the formation of OH is not mediated by
hydrogen peroxide.
Generation and reaction with phenolic compounds of 1O2. 1O2 was
generated by irradiating HP with UVA–visible light as described previously
(15) and was detected as an oxidative product, TEMPON, of TEMPD with
1O2 (16,17). Furthermore, the occurrence of 1O2 was confirmed from the
inhibition of TEMPON formation by histidine or sodium azide (NaN3),
The effect of a substituent group of phenol
on DMPO–ꢀOH formation
1
known O2 quenchers. UVA–visible light was irradiated through a UV-33
The time course of the signal intensity of the DMPO–ꢀOH adduct
was examined in the presence of various concentrations of
dopamine (Fig. 3). The signal intensity of DMPO–ꢀOH increased
upon irradiation, reached a maximal level at around 2 s and
decreased thereafter. This suggests the production of DMPO–ꢀOH
adduct at an initial stage and subsequent decomposition. Because
a similar decline in DMPO–ꢀOH signal was observed in most
phenolic compounds after irradiation for longer than 2 s, the ESR
filter (cut-off: 330 nm, Toshiba Glass Co., Tokyo, Japan) using Supercure-
203S with a 200 W Mercury–Xenon lamp (Radical Research Inc., Tokyo,
Japan) connected to the ESR cavity by a quartz fiber. The dose was 142
mW/cm2 at 365 nm on the surface of the flat cell (JEOL, Tokyo, Japan).
One-hundred microliters of 100 lM HP, various concentrations of phenolic
compounds and 100 lL of 400 mM DMPO or DEPMPO were added to 0.1
M phosphate buffer (total volume, 400 lL). After a quick stir, the reaction
mixture was transferred into a flat quartz cell. ESR spectra were measured
immediately after the appropriate irradiation time.

Photochemistry and Photobiology, 2003, 77(2) 167
Figure 3. Time course of DMPO–ꢀOH adduct formation in the presence of
various concentrations of dopamine. h: 20 lM, ): 100 lM, s: 250 lM,
Á: 500 lM and ,: 1 mM. Data are the average of three experiments.
Figure 2. ESR spectra due to DMPO adduct detected in a reaction mixture
containing NaN3, ethanol or HCOONa. (A) Complete system containing
25 lM HP and 100 mM DMPO in 100 mM phosphate buffer, pH 7.4. (B) as
in (A) but with addition of 100 lM dopamine. (C) as in (B) but without HP.
(D) as in (B) except that sample was purged with N2 gas for 15 min before
irradiation. (E) as in (B) but with addition of 10 mM NaN3. (F) as in (B) but
with 5% ethanol. (G) as in (B) but with 1 M HCOONa. s: DMPO adduct
of ꢀOH; ): DMPO adduct of ꢀCH(OH)CH3; Á: DMPO adduct of
ꢀCO22ꢀ. Conditions: microwave power, 10 mW; modulation amplitude,
0.79 G; scan range, 100 G; amplitude, 790 (1000 in the case of A); time
constant, 0.03 s; scan time, 2 min.
reach a point of saturation until at least 100 mM DMPO (Fig. 6). In
contrast, the increase in the signal intensity of the DMPO–ꢀOH
adduct produced from the reaction of DMPO with ꢀOH, which was
generated by the Fenton reaction, was saturated at 50 mM DMPO.
This suggests the participation of DMPO in the 1O2-mediated
ꢀ
formation of free OH.
To ascertain the role of DMPO in the conversion of 1O2 to ꢀOH,
a spin-trapping experiment was undertaken using DEPMPO
instead of DMPO. The irradiation of a solution containing 25
lM HP and 100 mM DEPMPO with UVA–visible light did not
produce any spin adduct irrespective of the presence or absence of
100 lM dopamine (Fig. 7a,b), although the reaction rate constant
signal intensities of the DMPO–ꢀOH adduct in the presence of
various phenolic compounds were compared from 2 s after
irradiation.
The relative ESR signal intensities of DMPO–ꢀOH were plotted
against the concentrations of phenolic compounds including
dopamine at a constant concentration of DMPO as shown in Fig. 4.
Phenolic compounds, except for phenol and p-hydroxybenzoic
acid, increased the amount of DMPO–ꢀOH with an increase in
concentration at relatively low levels (less than 250 lM). As
shown in Fig. 5, there existed a linear relationship (correlation
9
21
for DEPMPO with OH (7.1 3 10 M s21) is slightly higher
than that for DMPO (3.4 3 109 M21 s21) (19). When DMPO was
added to the reaction mixture described above (Fig. 7c), however,
an eight-line ESR signal appeared in addition to the four-line signal
due to DMPO–ꢀOH. The hyperfine splitting constants (aP 5 46.8
G, aN 5 13.8 G, aH 5 13.7 G) of this eight-line ESR signal
corresponded to those of the DEPMPO adduct of hydroxyl radical
(DEPMPO–ꢀOH) (19). These results strongly indicate that the
ꢀ
ꢀ
formation of OH is dependent on DMPO.
coefficient:
r 5 0.918) between the signal intensity of
DMPO–ꢀOH in 100 lM phenolic compounds and oxidation
potential. A similar relationship was obtained at concentrations
below 250 lM. This suggests that the electron-donating
properties of phenolic compounds contribute to the formation
of ꢀOH. A further increase in the concentration of phenolic
compounds except for Trolox C and isoeugenol led to a plateau
in the signal intensity of DMPO–ꢀOH, whereas an abrupt
decrease in the signal intensity of DMPO–ꢀOH was obtained
at a relatively high concentration of Trolox C and isoeugenol
(Fig. 4).
The formation of 5,5-dimethyl-2-pyrrolidone-N-oxyl
in the absence of phenolic compounds
The oxidative product of DMPO, 5,5-dimethyl-2-pyrrolidone-N-
oxyl (DMPOX) (aN 5 7.2 G, a(2)H 5 4.1 G) (20), was obtained 20 s
after UVA irradiation of an air-saturated solution of 25 lM HP and
20 mM DMPO in the absence of phenolic compounds (Fig. 8a).
No signal for DMPO–ꢀOH was found, although it has been
1
reported that DMPO reacts with O2 generated by the reaction of
excited photosensitizers with visible light to yield DMPO–ꢀOH
(21–25). The signal intensity of DMPOX increased with time,
reached a maximum and subsequently decreased as shown in Fig. 8b.
The increase in DMPO concentration accelerated the initial rate
of production of DMPOX but hastened the reduction of
DMPOX signal. In contrast, no DMPOX signal was observed
ꢀ
Involvement of DMPO in the formation of OH
When the ESR signal intensity of the DMPO–ꢀOH adduct was
measured in the presence of 100 lM dopamine, it was found to
increase with the concentration of DMPO, and the increase did not

168 Jun-ichi Ueda et al.
Figure 5. Correlation between signal intensities of DMPO–ꢀOH adducts
and oxidation potential of phenolic compounds at a concentration of 100
lM. Numbers correspond to those in Fig. 1.
Figure 4. Effect of varying the concentration of phenolic compounds on
the ESR signal intensity of DMPO adduct of ꢀOH radical in the presence of
100 mM DMPO. h: Trolox C, ): isoeugenol, s: p-eugenol, Á: p-
methoxyphenol, ,: guaiacol, : dopamine, ¤: 2,6-dimethoxyphenol, f:
n
phenol and m: p-hydroxybenzoic acid. Data are the average of two or
three experiments.
than that of the phosphorylated form (Ep[a] 5 1.60 V vs SCE) (26).
Then, 1O2 may hardly react with DEPMPO. This may explain why
no DEPMPO–ꢀOH was found in the absence of DMPO. The
reaction of DMPO with 1O2 should form a nitronium-like
compound with C-hydroperoxide (–CH(OO2)–N1(‚O)–) (in-
termediate I) as shown in Scheme 1.
in the presence of phenolic compounds at any DMPO
concentration (data not shown).
Phenolic compounds affected the formation of the DMPO–ꢀOH
adduct, depending on their substituent group. Because the
concentration of DMPO is larger than that of phenolic compounds,
1O2 reacts preferentially with DMPO to produce intermediate (I).
Therefore, phenolic compounds may react either with photo-
activated HP (triplet state) or with intermediate (I) as an electron
donor. The reduction of photoactivated HP by phenolic compounds
may not contribute to the formation of DMPO–ꢀOH because it
should decrease the generation of DMPO–ꢀOH by suppressing 1O2
production. Phenolic compounds, except for phenol and p-
hydroxybenzoic acid, increased the intensity of DMPO–ꢀOH in
a dose-dependent manner at relatively low concentrations of
phenolic compounds. In addition, a linear relationship between the
signal intensity of DMPO–ꢀOH in the presence of phenolic
compounds and the oxidation potential of the corresponding
compounds was obtained. Thus, these results suggest that phenolic
DISCUSSION
The exposure of an air-saturated solution containing dopamine,
DMPO and HP to UVA–visible light (k . 330 nm) led to the
formation of DMPO–ꢀOH adduct. No signal for DMPO–ꢀOH was
obtained without HP, suggesting that DMPO–ꢀOH is from the
photosensitizer-dependent reaction of DMPO and not from the
direct photochemical processes including the photoionization of
1
DMPO. A competitive scavenging experiment using both an O2
quencher, NaN3, and ꢀOH scavengers, such as ethanol and sodium
formate, clearly indicated that the DMPO trapped free ꢀOH, which
was formed through a 1O2-mediated pathway in the presence of
dopamine.
Although the displacement of DMPO with DEPMPO, in which
one methyl group at position 5 of DMPO is substituted with
a diethoxyphosphoryl group, did not give any ESR signal for its
ꢀOH adduct, DEPMPO–ꢀOH, a remarkable signal for the
DEPMPO–ꢀOH adduct was observed on addition of DMPO to
the reaction mixture, strongly suggesting that the formation of ꢀOH
was mediated by DMPO. It has been reported that DMPO itself
reacted with 1O2 (k 5 1.8 3 107 M21 s21) (22) to yield
compounds act as electron donors, probably decomposing in-
1
termediate (I) leading to the O2-mediated formation of OH as
ꢀ
shown in Scheme 1. No phenoxyl radical was observed in the
presence of MgCl2, a stabilizer of the semiquinone radical,
probably because of its very short life span (data not shown).
The rapid reaction of 1O2 with a wide range of phenolic
compounds was reported to produce endoperoxides and then
hydroperoxides, and the decomposition of the hydroperoxides by
DMPO–ꢀOH and free OH via the decomposition of the reaction
ꢀ
intermediate of DMPO with 1O2 (21–25). Bilski et al. (25) reported
1
that the reaction is initiated by the electrophilic reaction of O2 at
ꢀ
UV may produce OH because it was reported that UV irradiation
ꢀ
position 2 of DMPO. The existence of a strong electron-
withdrawing phosphoryl group in DEPMPO may reduce the
electron density of the C–N bond in DEPMPO compared with that
in DMPO. This suggestion is supported by the report that the
oxidation potential of a nonphosphorylated compound, a-phenyl-N-
tert-butylnitrone (Ep[a] 5 1.47 V vs SCE in acetonitrile) was lower
of phthalimide hydroperoxide produces OH (27). In the present
case, however, the contribution of this reaction to the formation of
ꢀ
ꢀOH would be minor for the following reasons: (1) no OH was
observed without DMPO in the spin-trapping with DEPMPO,
ꢀ
suggesting that the formation of OH is mediated by DMPO and
(2) a preferential reaction of 1O2 with DMPO is presumed because

Photochemistry and Photobiology, 2003, 77(2) 169
Figure 6. Dependence of DMPO–ꢀOH signal amplitude obtained from
both photochemical and Fenton reactions on DMPO concentrations.
DMPO–ꢀOH was obtained by UVA–visible light (k . 330 nm) irradiation
of an air-saturated solution containing 25 lM HP and 100 lM dopamine
(s) and from a reaction mixture containing 12.5 lM FeSO4, 100 lM DTPA
and 250 lM H2O2 ()). Data are the average of two or three
experiments.
Figure 8. (a) Spectrum of DMPOX and (b) time course of signal intensity
of DMPOX in the presence of various concentrations of DMPO. (A)
Spectrum measured 20 s after UVA–visible light irradiation of a reaction
mixture containing 25 lM HP and 20 mM DMPO. Conditions: microwave
power, 10 mW; modulation amplitude, 0.79 G; scan range, 100 G;
amplitude, 790; time constant, 0.03 s; scan time, 2 min. (B) [DMPO] 5 h:
10 mM; ): 20 mM; s: 40 mM and Á: 100 mM. Data are the average
of two experiments.
the concentration of DMPO is much higher than that of phenolic
compounds, as discussed above. The decrease of DMPO–ꢀOH
signal in the presence of excess Trolox C and isoeugenol may be
ꢀ
due to their strong OH scavenging activity or abnormal reducing
ability (or both). Trolox C and isoeugenol markedly enhanced the
spin elimination of a nitroxyl radical TEMPON during the HP-
photoreaction but other phenolic compounds did not (data not
shown).
from the oxidation of DMPO by intermediate (I). The de-
composition of intermediate (I) by phenolic compounds, if it
occurs, may inhibit the formation of DMPOX and enhance the
formation of DMPO–ꢀOH (Scheme 1). This hypothesis is
In the absence of phenolic compounds, DMPOX instead of
DMPO–ꢀOH was observed. Bilski et al. (25) described the
formation of DMPOX in a photodynamic reaction of micellar
rose bengal in the presence of DMPO at pH 2 and proposed that
intermediate (I) oxidized DMPO–ꢀOH to yield DMPOX. DMPOX
is usually formed during the oxidation of DMPO by strong
oxidizers (20,28–30). Thus, it is likely that DMPOX is produced
Figure 7. ESR spectra due to DMPO adduct detected in a reaction mixture
containing DEPMPO and dopamine. (A) Complete system containing 25 lM
HP and 100 mM DEPMPO in 100 mM phosphate buffer, pH 7.4. (B) as in
(A) but with addition of 100 lM dopamine. (C) as in (B) but with 20 mM
DMPO. s: DMPO adduct of ꢀOH; Á: DEPMPO adduct of ꢀOH.
Conditions: microwave power, 10 mW; modulation amplitude, 0.79 G;
scan range, 100 G; amplitude, 790; time constant, 0.03 s; scan time, 2 min.
Scheme 1. Sequence for the formation of DMPO–ꢀOH or DMPOX by the
reaction of 1O2 with DMPO in the presence or absence of phenolic
compounds.

170 Jun-ichi Ueda et al.
12. Ito, K. and S. Kawanishi (1997) Site-specific DNA damage induced by
UVA radiation in the presence of endogenous photosensitizer. Biol.
Chem. 378, 1307–1312.
supported by the disappearance of DMPOX and the appearance of
DMPO–ꢀOH in the presence of phenolic compounds.
Prolonged irradiation caused the signal intensity of DMPO–ꢀOH
to decrease. The decrease may be explained as follows. The excess
reaction caused by the long irradiation produced a large amount of
DMPO–ꢀOH accompanied by the consumption of oxygen and the
subsequent formation of HP anion radical, which was generated via
a Type-I mechanism under anaerobic conditions, resulting in the
spin elimination of DMPO–ꢀOH by HP anion radical (21). A
similar reduction was observed in the signal for DMPOX after
prolonged irradiation. The presence of phenolic compounds
prevented a weakening of the DMPO–ꢀOH signal (Fig. 4). This
suggests that inhibition of the loss of DMPO–ꢀOH signal is another
possible mechanism for the appearance of DMPO–ꢀOH only in the
presence of phenolic compounds.
13. Shvedova, A. A., C. Kommineni, B. A. Jeffries, V. Castranova, Y. Y.
Tyrina, V. A. Tyurin, E. A. Serbinova, J. P. Fabisiak and V. E. Kagan
(2000) Redox cycling of phenol induces oxidative stress in human
epidermal keratinocytes. J. Investig. Dermatol. 114, 354–364.
14. Ryu, A., E. Naru, K. Arakane, T. Masunaga, K. Shinmoto, T. Nagano,
M. Hirobe and S. Mashiko (1997) Cross-linking of collagen by singlet
oxygen generated with UV-A, Chem. Pharm. Bull. 45, 1243–1247.
15. Osada, M., Y. Ogura, H. Yasui and H. Sakurai (1999) Involvement of
singlet oxygen in cytochrome P450-dependent substrate oxidations.
Biochem. Biophys. Res. Commun. 263, 392–397.
16. Moan, J. and E. Wold (1979) Detection of singlet oxygen production by
ESR. Nature 279, 450–451.
17. Lion, Y., M. Delmelle and A. van de Vorst (1976) New method of
detecting singlet oxygen production. Nature 263, 442–443.
18. Buettner, G. R. (1987) Spin trapping: ESR parameters of spin adducts.
Free Radic. Biol. Med. 3, 259–303.
19. Frejaville, C., H. Karoui, B. Tuccio, F. Le Moigne, M. Culcasi,
S. Pietri, R. Lauricella and P. Torde (1995) 5-(Diethoxyphosphoryl)-
5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone
for the in vitro and in vivo spin trapping of oxygen-centered radicals.
J. Med. Chem. 38, 258–265.
20. Ozawa, T., Y. Miura and J. Ueda (1996) Oxidation of spin-traps by
cholorine dioxide (ClO2) radical in aqueous solutions: first ESR
evidence of formation of new nitroxide radicals. Free Radic. Biol. Med.
20, 837–841.
21. Zhang, S.-P., J. Xie, J.-P. Zhang, J.-Q. Zhao and L.-J. Jiang (1999)
Electron spin resonance studies on photosensitized formation of
hydroxyl radical by c-phycocyanin from Spirulina plantensis. Biochim.
Biophys. Acta 1426, 205–211.
22. Harbour, J. R., S. L. Issler and M. L. Hair (1980) Singlet oxygen and
spin trapping with nitrones. J. Am. Chem. Soc. 102, 7778–7779.
23. Hoebeke, M., H. J. Schuitmaker, L. E. Jannink, T. M. A. R.
Dubbelman, A. Jakobs and A. van de Vorst (1997) Electron spin
resonance of the generation of superoxide anion, hydroxyl radical and
singlet oxygen during the photohemolysis of human erythrocytes with
bacteriochlorin a. Photochem. Photobiol. 66, 502–508.
24. Feix, J. B. and B. Kalyanaraman (1991) Production of singlet oxygen-
derived hydroxyl radical adducts during merocyanine-540-mediated
photosensitization: analysis by ESR-spin trapping and HPLC with
electrochemical detection. Arch. Biochem. Biophys. 291, 43–51.
25. Bilski, P., K. Reszka, M. Bilska and C. F. Chignell (1996) Oxidation of
the spin trap 5,5-dimethyl-1-pyrroline N-oxide by singlet oxygen in
aqueous solution. J. Am. Chem. Soc. 118, 1330–1338.
26. Tuccio, B., P. Bianco, J. C. Bouteiller and P. Tordo (1999) Electro-
chemical characterisation of b-phosphorylated nitrone spin traps.
Electrochim. Acta 44, 4631–4634.
27. Saito, I., M. Takayama, T. Matsuura, S. Matsugo and S. Kawanishi
(1990) Phthalimide hydroperoxides as efficient photochemical hydroxy
radical generators. A novel DNA-cleaving agent. J. Am. Chem. Soc.
112, 883–884.
In conclusion, the 1O2-mediated formation of ꢀOH in the
presence of phenolic compounds was obviously initiated by the
reaction of 1O2 with DMPO used as a spin trap. Phenolic
compounds would participate in the production of ꢀOH as electron
donors but not in the direct conversion of 1O2 to ꢀOH. Furthermore,
ꢀ
DEPMPO did not cause the generation of OH.
Acknowledgements—This work was supported by Grants-in-aid for Scien-
tific Research (12672168 and 10357021) from the Ministry of Education,
Science, and Culture, Japan, and in part by a Grant from AOA Japan Re-
search Foundation.
REFERENCES
1. Ames, B. N. (1983) Dietary carcinogens and anticarcinogens. Science
221, 1256–1264.
2. Cerutti, P. A. (1985) Prooxidant states and tumor promotion. Science
227, 375–381.
3. Halliwell, B. and J. M. C. Gutteridge (1999) In Free Radicals in
Biology and Medicine, 3rd ed., Oxford University Press, New York,
NY.
4. Decuyper-Debergh, D., J. Piette, C. Laurent and A. van de Vorst (1989)
Cytotoxic and genotoxic effects of extracellular generated singlet
oxygen in human lymphocytes in vitro. Mutat. Res. 225, 11–14.
5. Ravanat, J.-L., P. Di Mascio, G. R. Martuez, M. H. G. Medeiros and
J. Cadet (2000) Singlet oxygen induces oxidation of cellular DNA.
J. Biol. Chem. 275, 40601–40604.
6. Ryter, S. W. and R. M. Tyrrell (1998) Singlet molecular oxygen (1O2):
a possible effector of eukaryotic gene expression. Free Radic. Biol.
Med. 24, 1520–1534.
7. Foote, C. S. (1976) ‘‘Photosensitized oxidation and singlet oxygen:
consequences in biological systems. In Free Radicals in Biology, Vol. II
(Edited by W. A. Pryor), pp. 85–133. Academic Press, New York.
8. Saito, I., T. Matsuura and K. Inoue (1983) Formation of superoxide ion
via one-electron transfer from electron donors to singlet oxygen. J. Am.
Chem. Soc. 105, 3200–3206.
9. Buettner, G. R. (1985) Thiyl free radical production with hematopor-
phyrin derivative, cysteine and light: a spin-trapping study. FEBS Lett.
177, 295–299.
10. Takeshita, K., C. A. Olea-Azar, M. Mizuno and T. Ozawa (2000)
Singlet oxygen-dependent hydroxyl radical formation during uropor-
phyrin-mediated photosensitization in the presence of NADPH. Antiox.
Redox Signal. 2, 355–362.
28. Rosen, G. M. and E. J. Rauckman (1980) Spin trapping of the primary
radical involved in the activation of the carcinogen N-hydroxy-2-
acetylaminofluorene by cumene hydroperoxide-hematin. Mol. Pharma-
col. 17, 233–238.
29. Mao, G. D., P. D. Thomas and M. Poznansky (1994) Oxidation of spin
trap 5,5-dimethyl-1-pyrroline-1-oxide in an electron paramagnetic
resonance study of the reaction of methemoglobin with hydrogen
peroxide. Free Radic. Biol. Med. 16, 493–500.
30. Gunther, M. R., R. A. Tschirret-Guth, H. E. Witkowska, Y. C. Fann,
D. P. Barr, P. R. Ortiz de Montellano and R. P. Mason (1998) Site-
specific spin trapping of tyrosine radicals in the oxidation of
metmyoglobin by hydrogen peroxide. Biochem. J. 330, 1293–1299.
11. Fuchs, J., N. Groth, T. Herrling and G. Zimmer (1997) Electron
paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-
4-piperidin-1-oxyl (TEMPO) redox reactions in human skin. Free
Radic. Biol. Med. 22, 967–976.