Hydroxyl radical generation by dissociation of water molecules during 1.65?MHz frequency ultrasound irradiation under aerobic conditions

Article abstract of DOI:10.1016/j.bbrc.2016.12.171

The dissociation of water molecules by ultrasound irradiation under aerobic conditions was demonstrated experimentally. To be able to detect the dissociation of water molecules, we performed the ultrasound irradiation of ¹⁷O-labelled water (H₂¹⁷O) under aerobic conditions. The hydroxyl and hydrogen radicals generated during the ultrasound irradiation process were trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and electron spin resonance (ESR) spectroscopy was performed on the DMPO spin adducts. In the ESR spectrum, a 15-line signal attributable to the trapping of the hydroxyl radicals containing ¹⁷O (¹⁷OH radicals) by DMPO together with a 4-line signal attributable to the trapping of the hydroxyl radicals containing ¹⁶O (¹⁶OH radicals) by DMPO were observed. The generation of ¹⁷OH radicals indicated that H₂¹⁷O was dissociated by the sonolysis process under aerobic conditions. On the other hand, the ESR signal attributable to the trapping of hydrogen radicals by DMPO was not observed, suggesting that hydrogen radicals were not generated during the dissociation of water molecules.

Full text of DOI:10.1016/j.bbrc.2016.12.171

Biochemical and Biophysical Research Communications xxx (2017) 1e5
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Biochemical and Biophysical Research Communications
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Hydroxyl radical generation by dissociation of water molecules during
1.65 MHz frequency ultrasound irradiation under aerobic conditions
a
b
c
a, *
Akimitsu Miyaji , Masahiro Kohno , Yoshihiro Inoue , Toshihide Baba
a
School of Materials and Chemical Technology, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502, Japan
School of Bioscience and Biotechnology, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan
Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The dissociation of water molecules by ultrasound irradiation under aerobic conditions was demon-
strated experimentally. To be able to detect the dissociation of water molecules, we performed the ul-
trasound irradiation of O-labelled water (H O) under aerobic conditions. The hydroxyl and hydrogen
2
Received 16 December 2016
Accepted 26 December 2016
Available online xxx
17
17
radicals generated during the ultrasound irradiation process were trapped with 5,5-dimethyl-1-
pyrroline-N-oxide (DMPO), and electron spin resonance (ESR) spectroscopy was performed on the
DMPO spin adducts. In the ESR spectrum, a 15-line signal attributable to the trapping of the hydroxyl
Keywords:
High-frequency ultrasound
Hydroxyl radical
17
17
radicals containing O ( OH radicals) by DMPO together with a 4-line signal attributable to the trapping
1
6
16
17
17
of the hydroxyl radicals containing O ( OH radicals) by DMPO were observed. The generation of OH
O-labelled water
radicals indicated that H1
7
O was dissociated by the sonolysis process under aerobic conditions. On the
Oxidative stress
Water sonolysis
2
other hand, the ESR signal attributable to the trapping of hydrogen radicals by DMPO was not observed,
suggesting that hydrogen radicals were not generated during the dissociation of water molecules.
©
2016 Published by Elsevier Inc.
1. Introduction
thorough understanding of the mechanism of free-radical genera-
tion under ultrasound irradiation is essential.
Ultrasound is being used in medical and dental fields. Especially,
ultrasound with the frequency above 1 MHz is used for therapeutic
and cleaning tools [1e4]. In the therapeutic applications, the high
frequency has been chosen due to its depth of penetration into
tissue [5e7]. For ultrasound cleaning, the high frequency ultra-
sound is effective to remove particles from the surface of object
such as tooth [8,9], which is due to a strong acceleration of the
water molecules. However, there are concerns whether ultrasound
irradiation can induce oxidative damage in the human body,
because free radicals are generated when water is exposed to ul-
trasound waves with an intensity higher than a certain threshold.
These free radicals have the ability to damage biological molecules
When bulk water is subjected to ultrasound irradiation, cavi-
tation bubbles grow and collapse repeatedly [10]. During their
collapse, the local temperature and pressure can be higher than
5000 K and 500 atm, respectively [11,12]. The high temperature and
pressure are what result in the generation of free radicals [13e16].
The primary reaction responsible for the generation of free radicals
is the thermal dissociation of the water molecule into a hydroxyl
ꢀ
ꢀ
radical ( OH) and a hydrogen radical ( H), as shown below:
ꢀ
ꢀ
H
2
O / OH þ H
(1)
The reaction (1) has been confirmed in bulk water exposed by
(
e.g., enzymes, DNA, and lipids) in vitro. Thus, to prevent any acci-
ultrasound with 50 kH frequency under argon gas conditions
through electron spin resonance (ESR)-spin trapping measure-
ments; the ESR signals attributable to hydroxyl radicals and
hydrogen atoms were detected [15e17]. However, it is confirmed
only under limited measurement conditions.
dental damage resulting from the use of ultrasound waves and to
ensure the safety and reliability of ultrasound irradiation with
respect to its application in the medical and dental fields, a
Under aerobic conditions, only the signal attributable to hy-
droxyl radicals has been observed [16,18]. Therefore, the reaction
*
Corresponding author. School of Materials and Chemical Technology, Tokyo
(1) may not be the primary reaction of water sonolysis under aer-
Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-
8
502, Japan.
obic conditions. Alternatively, under aerobic conditions, oxygen
E-mail address: tbaba@chemenv.titech.ac.jp (T. Baba).
http://dx.doi.org/10.1016/j.bbrc.2016.12.171
006-291X/© 2016 Published by Elsevier Inc.
0
Please cite this article in press as: A. Miyaji, et al., Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency
ultrasound irradiation under aerobic conditions, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2016.12.171

2
A. Miyaji et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
molecules also undergo thermal dissociation in the interior of
determined on the basis of the signal height using an external
standard, TEMPOL. The concentration of OH was determined using
Digital Data Processing (JEOL) and expressed in terms of the con-
centration of DMPO-OH, a spin adduct of OH. All the tests were
performed at least in triplicate at room temperature.
ꢀ
ꢀ
collapsing bubbles, resulting in biradical oxygen atoms ( O )
19,20]:
[
ꢀ
O
2
/ 2 Oꢀ
(2)
Based on the reactions involving oxygen atoms known from
combustion chemistry, the following reactions have been proposed
to explain the generation of hydroxyl radicals:
3
. Results and discussion
2
During the ultrasound irradiation to H O in the presence of air, a
4
-line ESR signal with the intensity ratio 1:2:2:1 was observed, as
ꢀ
ꢀ
ꢀ
ꢀ
16
O þ H
2
O / OH þ OH
(3)
shown in Fig. 1. This signal was attributable to DMPO- OH, which
has a g value of 2.0064 and hyperfine constants of 1.49 (a ) and
.49 mT (a ) [24]. No signal related to hydrogen radicals was
observed, in keeping with previous reports.
N
In this reaction, only hydroxyl radicals are generated under ul-
trasound irradiation of water in the presence of oxygen molecules.
Based on this reaction mechanism, the isotopic exchange of O-
labelled oxygen molecules (
eration of hydrogen peroxide labelled with one O atom (H
1
H
16,18
This result indi-
1
8
cated that hydroxyl radicals were generated during the ultrasound
irradiation to water under aerobic conditions in the present study,
in agreement with previous studies.
18
16
2 2
O ) and water (H O), and the gen-
18
18 16
2
O O)
under ultrasound irradiation can be explained [21]. Therefore, Re-
action (2) and (3) are commonly accepted as the mechanism of
hydroxyl radical generation by ultrasound irradiation of liquid
water in the presence of air. However, to confirm that the proposed
mechanism is correct, the primary reaction involved in the sonol-
ysis of water under aerobic conditions, represented by Reaction (3),
should be validated experimentally.
The dependence of the generation of DMPO-OH on the ultra-
sound irradiation time is shown in Fig. 2. In keeping with what has
been reported previously, the DMPO-OH signal increased linearly
until an irradiation time of 30 s. For further increases, it plateaued,
owing to the degradation of DMPO-OH because of the ultrasound
waves [25]. To investigate the initial stage of the water sonolysis
process (i.e., the stage prior to the degradation of DMPO-OH), we
In this study, in order to elucidate the primary reaction involved
in the sonolysis of water under aerobic conditions, we performed
performed ultrasound irradiation for 30 s. In this case too, the ul-
17
trasound irradiation to H
2
O was performed under aerobic condi-
17
the ultrasound irradiation of O-labelled air-containing water. The
free radicals generated were detected using 5,5-dimethyl-1-
16
tions. In addition to the 4-line signal attributable to DMPO- OH,
another multiple-lines signal was observed, as shown in Fig. 3(a).
Using a simulation of the ESR measurement results, the signals
shown in Fig. 3(a) could be fitted well using a combination of the 4-
line signal attributable to DMPO- OH and a 15-line signal attrib-
utable to DMPO- OH, as shown in Fig. 3(b). The 4-line signal
17
pyrroline-N-oxide (DMPO). The nuclei spin number of O is 5/2,
while that of 16O is 0. Because of this difference in the spin
17
numbers, the ESR signal attributable to OH-trapping DMPO
16
1
7
16
(
DMPO- OH) was different from that related to OH-trapping
17
16
DMPO (DMPO- OH). This allowed the hydroxyl radicals generated
from water and those from oxygen molecules to be distinguished.
Further, ultrasound at 1.65 MHz frequency, which is used in ther-
apeutic and cleaning tools in medical and dental fields [1e4], was
used in the present study.
16
related to DMPO- OH has a g-value of 2.0064 and hyperfine con-
stants of 1.49 mT (a
signal related to DMPO- OH has a g-value of 2.0064 and hyperfine
constants of 1.49 (a ), 1.47 mT (a ), and 0.47 mT (a17O) (Fig. 3(d))
24]. This result indicates that not only OH radicals but also OH
N H
) and 1.49 mT (a ) (Fig. 3(c)), while the 15-line
17
N
H
16
17
[
radicals were generated by based on the results of the ESR mea-
surements, we concluded that hydroxyl radicals are generated by
the dissociation of water molecules when they are exposed to ul-
trasound waves under aerobic conditions.
2
. Materials and methods
DMPO was obtained from LABOTEC Co., Ltd. (Tokyo, Japan)
while 2,2,6,6-teteramethyl piperidinol (TEMPOL) was obtained
from Sigma-Aldrich (Tokyo, Japan). H O (>90 atom% O) was
2
purchased from NUKEM Isotopes GmbH (Alzenau, Germany).
A previously reported experimental device for ultrasound gen-
eration was used in this study [22,23]. In this device, the ultrasound
According to the simulation results shown in Fig. 3(c) and (d),
17
17
16
17
the ratio of the intensities of the DMPO- OH and DMPO- OH
signals was nearly 1. This ratio was almost constant until the ul-
trasonic irradiation time was at least 45 s. These results indicate
1
6
17
that the amounts of OH and OH radicals generated during water
17
generator is placed at the bottom of a water bath. First, 200
water containing 100 mM DMPO was transferred to a glass tube
15 mm in diameter and 85 mm in length). The glass tube was
ml of
2
sonolysis are almost equal when ultrasound is exposed to H O in
(
placed in the water bath and was fixed above the ultrasound
generator. The temperature of the bulk water was controlled at
ꢁ
2
0 ± 1 C by the low temperature circulator (Tokyo Rikakikai Co.,
Ltd, Japan). Next, ultrasound irradiation (power of 30 W and fre-
quency of 1.65 MHz) was performed for 30 s.
The water sample exposed to the ultrasound waves was trans-
ferred to a flat quartz cell (150 mL) for the ESR measurements. The
optical path length was 0.25 mm. The ESR spectra were recorded at
room temperature using an X-band spectrometer (JES-FA-100, JEOL
Ltd., Tokyo, Japan), which was operated at 9.43 GHz. The magnetic
field was modulated at 100 kHz. The conditions for measuring the
ESR spectra were as follows: microwave power of 4 mW; magnetic
field of 335.5 ± 5.0 mT; field modulation width of 0.1 mT; sweep
time of 2 min; and time constant of 0.1 s.
The signal intensities were normalized with respect to a MnO
marker, and the concentrations of the stable radical products were
2
Fig. 1. ESR spectrum of the spin adduct formed during the sonolysis of H O for 30 s
under aerobic conditions in the presence of 100 mM DMPO.
Please cite this article in press as: A. Miyaji, et al., Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency
ultrasound irradiation under aerobic conditions, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2016.12.171

A. Miyaji et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
3
ꢀ
16
ꢀ
17
O / ꢀ OH þ ꢀ17OH
16
O þ H
2
(5)
According to this mechanism and the purity of H
2
7O used in this
1
study, the ratio of the numbers of ꢀ OH and ꢀ OH generated by
water sonolysis should be 1.0e1.2, a value that would be consistent
with the ratio determined experimentally.
17
16
We did not detect the DMPO-H-related signal attributable to the
trapping of the hydrogen radicals by DMPO during the sonolysis of
water under aerobic conditions. Under the conditions used in this
study, had hydrogen radicals been generated, a signal attributable
to DMPO-H should have been detected, in spite of the short lifetime
of DMPO-H. The reaction rate constant of hydrogen radicals with
respect to oxygen molecules at around 298 K is approximately
1
0
ꢂ1 ꢂ1
1
2
0
M
s
[26e29], while that with respect to DMPO at around
9
ꢂ1 ꢂ1
98 K is approximately 10 M
s [30e32]. In this study, 100 mM
of DMPO was dissolved in water; this amount is approximately
2
5
ꢃ 10 times higher than the concentration of the oxygen dis-
solved in water (approximately 200 M). Thus, the hydrogen rad-
m
icals generated probably reacted with DMPO more quickly than
they did with the oxygen molecules. Given the difference in the
reaction rates, that fact that no signal attributable to DMPO-H was
detected can be interpreted as the two meanings as follows.
Fig. 2. Effect of the sonication time on the yield of DMPO-OH under aerobic conditions.
the presence of 16O
for hydroxyl radical generation by water sonolysis under aerobic
. Based on the proposed reaction mechanism
1) Hydrogen radicals are not generated during the ultrasound
irradiation of water under aerobic conditions, as suggested by
Reactions (4) and (5).
2) Hydrogen radicals are generated and are rapidly scavenged by
the oxygen molecules present in the gas bubbles when water is
2
17
conditions [14,21], hydroxyl radical generation from H
2
O by
16
sonolysis in the presence of
O
2
can be described as follows:
16O
/
ꢀ
16
ꢀ
O þ ꢀ16Oꢀ
(4)
2
Fig. 4. ESR spectra of the spin adducts formed during the sonolysis of H17
O for 30 s in
2
an argon atmosphere in the presence of 100 mM DMPO. (a) Experimental spectrum,
Fig. 3. ESR spectra of the spin adducts formed during the sonolysis of H17O for 30 s
2
17
(
b) simulated spectra of DMPO-H, (c) simulated spectrum of DMPO- OH, (d) simulated
under aerobic conditions in the presence of 100 mM DMPO. (a) Experimental spec-
16
spectrum of DMPO- OH, and (e) simulated spectrum of the DMPO-trapping carbon-
centered radical.
trum, (b) simulated spectra of both DMPO-17OH and DMPO- OH, (c) simulated
16
17
16
spectrum of DMPO- OH, and (d) simulated spectrum of DMPO- OH.
Please cite this article in press as: A. Miyaji, et al., Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency
ultrasound irradiation under aerobic conditions, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2016.12.171

4
A. Miyaji et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
subjected to ultrasound irradiation under aerobic conditions
19,35].
adducts attributable to the hydroxyl and hydroxyl radicals, which
are expected to be generated during the dissociation of the water
[
17
molecule. Based on the fact that an ESR signal attributable to OH
radicals was observed, we concluded that hydroxyl radicals are
generated by the dissociation of water molecules during ultrasound
irradiation under aerobic conditions. The dissociation of the water
molecule under anaerobic conditions had been reported previously,
wherein hydroxyl and hydrogen radicals were detected. However,
we did not detect an ESR signal attributable to hydrogen radicals
under aerobic conditions. Thus, the hydroxyl radical is probably
generated via a reaction of the water molecule with the oxygen
biradical (Reaction (5)), as proposed previously, or/and because of
the thermal dissociation of the water molecule (Reaction (6)).
If hydrogen radicals are generated by the thermal dissociation of
17
water as the mechanism 2), then OH radicals are also generated
16
from water. Further, an equivalent amount of OH radicals can be
generated by the following reaction:
1
7O / ꢀ OH þ H
17
ꢀ
H
2
(6)
(7)
ꢀH þ 16,16O
ꢀ
/
16
O þ ꢀ16OH
ꢀ
2
The oxygen biradical generated in Reaction (7) can react with a
water molecule as shown in Reaction (3). Even in this case, the ratio
of the number of 17OH radicals to that of the OH radicals would be
around 1, as observed in the experimental results described above.
Alternatively, there was a possibility that hydrogen radicals
16
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scien-
tific Research (KAKENHI) (Grant No. 25630363) from the Japan
Society for the Promotion of Science.
17
cannot be effectively trapped by DMPO in H
2
O. In the previous
work, the ESR signal due to DMPO-H was not observed in the
sonolysis of H1
signal was observed in the sonolysis of H
the generation of hydrogen radicals during the sonolysis of argon-
7
O in the presence of argon gas [17], though the
2
16
2
O. However, we detected
Transparency document
containing H1
7
O. As shown in Fig. 4, distinct ESR signals related to
2
17
16
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2016.12.171.
DMPO-H, DMPO- OH, and DMPO- OH, which are attributable to
the generation of hydrogen, OH, and 16OH radicals, respectively,
17
were observed. Further, a low-intensity signal related to DMPO-C,
16
References
which is attributable to the generation of OH radicals and
carbon-centered radicals, was also observed. The generation of
hydrogen, hydroxyl, and carbon-centered radicals was also
[1] K.G. Baker, V.J. Robertson, F.A. Duck, A review of therapeutic ultrasound:
biophysical effects, Phys. Ther. 81 (2001) 1351e1358.
1
6
observed during the sonolysis of H
2
O (data not shown). This result
[2] D. Miller, N. Smith, M. Bailey, G. Czarnota, K. Hynynen, I. Makin, American
institute of ultrasound in medicine bioeffects committee, overview of thera-
peutic ultrasound applications and safety considerations, J. Ultrasound Med.
indicated that the isotopic substitution of the oxygen atom in the
water molecule does not affect the dissociation of water molecules
under ultrasound irradiation as depicted by Reaction (1), and not
affect the ability of DMPO for trapping hydrogen radicals. No
detection of the ESR signal due to DMPO-H is probably due to the
31 (2012) 623e634.
[3] A.R. Biesbrock, L.M. Bayuk, D.S. Yates, M.V. Santana, R.D. Bartizek, The clinical
effectiveness of a novel power toothbrush and its impact on oral health,
J. Contemp. Dent. Pract. 15 (2002) 1e10.
[4] W.B.D. Forfang, B.H. You, I.-H. Song, Ultrasonic dental therapy: trends and
difference in the frequency of the ultrasound waves used or/and the
prospects, Eur. Int. J. Sci. Tech. 2 (2013) 1e6.
purity of H1
7
O also probably affect the amount of hydrogen radicals
[5] D.O. Draper, J.C. Castel, D. Castel, Rate of temperature increase in human
muscle during 1 MHz and 3 MHz continuous ultrasound, J. Orthop. Sports
Phys. Ther. 22 (1995) 142e150.
2
generated during ultrasound irradiation.
According to Reaction (1), the hydrogen and hydroxyl radicals
would be generated in equal amounts. The rate constant for the
reaction of DMPO with hydrogen radicals is almost equal to that for
the reaction of DMPO with hydroxyl radical; however, the life time
of DMPO-H is shorter than that of DMPO-OH. Therefore, the in-
tensity of the DMPO-H signal was expected to be equivalent to or
[
[
[
[
6] G.R. ter Haar, Basic physics of therapeutic ultrasound, Physiotherapy 64
(1978) 100e103.
7] R.J. Baker, G.W. Bell, The effect of therapeutic modalities on blood flow in the
human calf, J. Orthop. Sports Phys. Ther. 13 (1991) 23e27.
8] K. Shinada, L. Hashizume, K. Teraoka, N. Kurosaki, Effect of ultrasonic tooth-
brush on Streptococcus mutans, Jpn. J. Conserv. Dent. 42 (1999) 410e417.
9] M.R. Costa, V.C. Silva, M.N. Miqui, T. Sakima, D.M.P. Spolidorio, J.A. Cirelli,
Efficacy of ultrasonic, electric and manual toothbrushes in patients with fixed
orthodontic appliances, Angle Orthod. 77 (2007) 361e366.
17
smaller than that of the DMPO- OH signal. However, the intensity
of the DMPO-H signal was approximately three times higher than
[10] G. R.ter. Haar, S. Daniels, Evidence for ultrasonically induced cavitation in vivo,
Phys. Med. Biol. 26 (1981) 1145e1149.
[11] K.S. Suslick, Sonochemistry, Science 247 (1990) 1439e1445.
17
that of the DMPO- OH signal, which was also observed during the
sonolysis of H1
6
2
O [18]. When sonolysis is performed in the presence
[
12] E.B. Flint, K.S. Suslick, The temperature of cavitation, Science 253 (1991)
of argon gas, the local temperature can be as high as approximately
700 K; this is higher that the temperature for the case when
sonolysis is performed in the presence of oxygen and nitrogen
gases [33]. From combustion chemistry, it is known that the hy-
droxyl radical dissociates into the hydrogen radical and the oxygen
biradical at such high temperatures, as follows [34]:
1397e1399.
[
[
13] K.S. Suslick, The chemical effects of ultrasound, Sci. Am. 260 (1989) 80e86.
14] P. Riesz, T. Kondo, Free radical formation induced by ultrasound and its bio-
logical implications, Free Rad. Biol. Med. 13 (1992) 247e270.
1
[15] K. Makino, M.M. Mossoba, P. Riesz, Chemical effects of ultrasound on aqueous
solutions. Evidence for ꢀOH and ꢀH by spin trapping, J. Am. Chem. Soc. 104
(1982) 3537e3539.
[
[
16] K. Makino, M.M. Mossoba, P. Riesz, Chemical effects of ultrasound on aqueous
solutions. Formation of hydroxyl radicals and hydrogen atoms, J. Phys. Chem.
87 (1983) 1369e1377.
17] T. Kondo, C.M. Krishna, P. Riesz, Effect of non-volatile scavengers of hydroxyl
radicals on thymine radical formation induced by gamma-rays and ultra-
sound, Int. J. Radiat. Biol. 53 (1988) 891e899.
ꢀ
ꢀ
ꢀ
ꢀ
OH / O þ H
(8)
This dissociation reaction is probably what causes the amount of
hydrogen radicals to be higher than the amount of hydroxyl
radicals.
[18] M. Kohno, T. Mokudai, T. Ozawa, Y. Niwano, Free radical formation from
sonolysis of water in the presence of different gases, J. Clin. Biochem. Nutr. 49
(2011) 96e102.
[
[
[
19] E. Hart, A. Henglein, Free radical and free atom reactions in the sonolysis of
aqueous iodide and formate solutions, J. Phys. Chem. 89 (1985) 4342e4347.
20] M.A. Beckett, I. Hua, Elucidation of the 1,4-dioxane decomposition pathway at
discrete ultrasonic frequencies, Environ. Sci. Technol. 34 (2000) 3944e3953.
21] C.H. Fischer, E.J. Hart, A. Henglein, Ultrasonic irradiation of water in the
4
. Conclusions
In this study, we performed the sonolysis of 17O-labelled air-
presence of 1
8,
18
containing water and measured the ESR spectra of the spin
2 2 2
O : isotope exchange and isotopic distribution of H O ,
Please cite this article in press as: A. Miyaji, et al., Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency
ultrasound irradiation under aerobic conditions, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2016.12.171

A. Miyaji et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
5
J. Phys. Chem. 90 (1986) 1954e1956.
[28] J. Elliot, A pulse radiolysis study of the temperature dependence of reactions
involving H, OH and eaq. in aqueous solutions, Radiat. Phys. Chem. 34 (1989)
753e758.
[29] S. Gordon, E.J. Hart, J.K. Thomas, The ultra violet spectra of transients pro-
duced in the radiolysis of aqueous solutions, J. Phys. Chem. 68 (1964)
1262e1264.
[30] E. Finkelstein, G.M. Rosen, E.J. Rauckman, Spin trapping of superoxide and
hydroxyl radical: practical aspects, Arch. Biochem. Biophys. 200 (1980) 1e16.
[31] E. Finkelstein, G.M. Rosen, E.J. Rauckman, Spin trapping. Kinetics of the re-
action of superoxide and hydroxyl radicals with nitrones, J. Am. Chem. Soc.
102 (1980) 4994e4999.
[32] P.R. Marriott, M.J. Perkins, D. Griller, Spin trapping for hydroxyl in water: a
kinetic evaluation of two popular traps, Can. J. Chem. 58 (1980) 803e807.
[33] F.R. Young, Sonoluminescence from water containing dissolved gases,
J. Acoust. Soc. Am. 60 (1976) 100e104.
[
[
22] A. Iwasawa, K. Saito, T. Mokudai, M. Kohno, T. Ozawa, Y. Niwano, Fungicidal
action of hydroxyl radicals generated by ultrasound in water, J. Clin. Biochem.
Nutr. 45 (2009) 214e218.
23] Y. Matsumura, A. Iwasawa, T. Kobayashi, T. Kamachi, T. Ozawa, M. Kohno,
Detection of high-frequency ultrasound-induced singlet oxygen by the ESR
spin-trapping method, Chem. Lett. 42 (2013) 1291e1293.
24] G.R. Buettner, Spin trapping: ESR parameters of spin adducts, Free Radic. Biol.
Med. 3 (1987) 259e303.
25] S.N. Nam, S.K. Han, J.W. Kang, H. Choi, Kinetics and mechanisms of the
sonolytic destruction of non-volatile organic compounds: investigation of the
sonochemical reaction zone using several OH* monitoring techniques,
Ultrason. Sonochem. 10 (2003) 139e147.
[
[
[
26] G.V. Buxton, C.L. Greenstock, W.P. Helman, A.B. Ross, Critical review of rate
constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl
ꢂ
radicals (,OH/,O ) in aqueous solution, J. Phys. Chem. Data Ref. 17 (1988)
[34] J. Colussi, L.K. Weavers, M.R. Hoffmann, Chemical bubble dynamics and
quantitative sonochemistry, J. Phys. Chem. A 102 (1998) 6927e6934.
[35] M.H. Uddin, S. Hatanaka, S. Hayashi, Effects of aqueous temperature on
sonolysis of bisphenol A: rate constants increasing with temperature under
oxygen, J. Chem. Eng. Jpn. 42 (2009) 303e308.
513e886.
[
27] J. Elliot, D.R. McCracken, G.V. Buxton, N.D. Wood, Estimation of rate constants
for near-diffusion-controlled reactions in water at high temperatures, J. Chem.
Soc. Faraday Trans. 86 (1990) 1539e1547.
Please cite this article in press as: A. Miyaji, et al., Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency
ultrasound irradiation under aerobic conditions, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2016.12.171