Accepted Manuscript

Comparison of free radicals formation induced by cold atmospheric plasma,

ultrasound, and ionizing radiation

Mati Ur Rehman, Paras Jawaid, Hidefumi Uchiyama, Takashi Kondo

PII: S0003-9861(16)30115-1
DOI: 10.1016/j.abb.2016.04.005
Reference: YABBI 7260

To appear in: Archives of Biochemistry and Biophysics

Received Date: 31 December 2015

Revised Date: 10 April 2016
Accepted Date: 11 April 2016

Please cite this article as: M.U. Rehman, P. Jawaid, H. Uchiyama, T. Kondo, Comparison of free
radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation, Archives of
Biochemistry and Biophysics (2016), doi: 10.1016/j.abb.2016.04.005.

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 ACCEPTED MANUSCRIPT

Comparison of free radicals formation induced by cold atmospheric

plasma, ultrasound, and ionizing radiation
Mati Ur Rehmana*, Paras Jawaida, Hidefumi Uchiyamab, Takashi Kondoa*
a
Department of Radiological Sciences, Graduate School of Medicine and

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Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194,

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Japan
b
Tateyama Machine Co., Ltd., Business Promotion Department, 30 Shimonoban,

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Toyama 930–1305, Japan.

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*Correspondence: Dr. Mati Ur Rehman and Prof. Dr. Takashi Kondo, Department of

Radiological Sciences, Graduate School of Medicine and Pharmaceutical Science,

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University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Tel: +81-76-434-7267;

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Fax: +81-76-434-5190.

E-mail: rehman.mu84@yahoo.com, and kondot@med.u-toyama.ac.jp

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Abstract
Plasma medicine is increasingly recognized interdisciplinary field combining

engineering, physics, biochemistry and life sciences. Plasma is classified into two

categories based on the temperature applied, namely “thermal” and “non-thermal” (i.e.,

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cold atmospheric plasma). Non-thermal or cold atmospheric plasma (CAP) is produced

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by applying high voltage electric field at low pressures and power. The chemical effects

of cold atmospheric plasma in aqueous solution are attributed to high voltage discharge

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and gas flow, which is transported rapidly on the liquid surface. The argon-cold

atmospheric plasma (Ar-CAP) induces efficient reactive oxygen species (ROS) in

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aqueous solutions without thermal decomposition. Their formation has been confirmed
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by electron paramagnetic resonance (EPR) spin trapping, which is reviewed here. The
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similarities and differences between the plasma chemistry, sonochemistry, and radiation

chemistry are explained. Further, the evidence for free radical formation in the liquid
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phase and their role in the biological effects induced by cold atmospheric plasma,
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ultrasound and ionizing radiation are discussed.

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Keywords: Cold atmospheric plasma, Ionizing radiation, Ultrasound, Free radicals,

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Reactive oxygen species, Platinum nanoparticles, Cell killing, EPR spin trapping
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1. Introduction
In recent decades, several physical factors have been utilized in the diagnosis

and treatment of different medical conditions such as cold atmospheric plasma (CAP),

ultrasound and X-irradiation. Plasma, which is often regarded as the “fourth state of

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matter”, is a partially ionized gas, which contains a mixture of electrons, photons, atoms,

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radicals and various excited and non-excited molecules. Plasma medicine is an

emerging interdisciplinary field, found to be highly effective for biological and medical

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purposes. Recently, remarkable efforts have been made in the development of low

temperature atmospheric plasma technology and its potential has been demonstrated in

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different applications such as disinfection and sterilization [1, 2], haemostatis [3],
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dentistry [4,5], regeneration of bones [6], wound healing and cancer treatment [7-9].
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Ideally, plasma can be generated by using any gas. However, the most commonly used

gases are argon and helium because these are chemically inert and results in the
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production of stable plasma [10]. The CAP has been well known to induce a variety of
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reactive oxygen species (ROS) in the liquid phase and inside the cells.

Ultrasound has also been widely applied in the diagnostics, however, recent
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progress on the biological, chemical and physical effects of ultrasound, revealed

promising results, and its therapeutic applications are well documented such as in
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hyperthermia for cancer therapy, tissue ablation using high-intensity focused ultrasound
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(HIFU), and the use of microbubbles to improve the ultrasonic imaging techniques. In

addition, low intensity pulsed ultrasound (LIPUS) is used in bone fractures to accelerate

bone healing and in elastography for diagnosis. The expected future clinical application

of ultrasound includes its efficiency for DDS (drug delivery system) and gene transfer

due to its sonoporation properties. The effects of ultrasound are dependent on the

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acoustic cavitation and can be observed only above certain threshold intensity, resulting

in the generation of ultrasound-induced ROS in the liquid phase [11].

X-ray is an electromagnetic wave which is essential for medical diagnosis and

treatment; similarly, other types of radiation include heavy charged particles, protons,

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neutrons, etc, which also showed remarkable potential for cancer therapy. This review

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especially focuses on the argon-cold atmospheric plasma, ultrasound, and ionizing

radiation induced free radical generation and its role in the mechanism of action. These

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three physical factors possess difference in energy, reaction time, pressure, etc. (Fig. 1).

However, they share very similar behavior for generation of ROS, particularly in the

liquid phase.
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2. Free radical generation induced by ultrasound and ionizing
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radiation
2.1. Three regions of sonochemistry
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The chemical effects of ultrasound are attributed to the acoustic cavitation,

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which is the formation and collapse of small gas bubbles. The sonochemical reaction

can take place in three different regions. The first one is the high temperature region of
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a collapsing gas bubble. The second one is the interfacial region between the

surrounding liquid and a hot gas phase. In this region, the relative efficiencies of
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non-volatile solutes to thermally decomposition depend on their hydrophobicity.

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Increased hydrophobicity of the solute results in the greater formation of thermal

decomposition products. The third region is the bulk of solution at ambient temperature,

where the free radicals, formed in the cavitation bubbles and not scavenged in the

interfacial region, react with organic solutes giving rise to products similar to those

observed in the radiation chemistry (Fig. 2) [12].

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2.2. Initial process and free radical formation of ionizing radiation

In the case of ionizing radiation, when it passes through matters, energy

is emitted through the track. However, the track in Electromagnetic radiation, such as

X-rays, is formed by electrons due to photoelectric effects and Compton effects. These

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electrons cause excitation and ionization in the track. Primary electrons formed here

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deposit energy (about 60~80 eV) to secondary electrons within about 2 nm diameter and

produce ion pairs so called “spur”. Since, 80% of our body is consists of water, which

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absorbs radiation energy to cause excitation and ionization (Fig. 3). Initially, OH, H,

aqueous electrons and H3O+ form in spur, and subsequently the recombination occurs to

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produce H2O2 and H2. Although the reaction is discontinuous microscopically, overall
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events appear to be homogeneous and continuous because these reactions happen in a

very short time like within 10-7s [13].

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3. Free radical formation induced by cold atmospheric plasma

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3.1. Set-up and specification

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3.1.1. Argon cold atmospheric plasma (Ar-CAP) irradiation system

A jet of argon plasma was produced in a quartz tube of 2.0 mm inner diameter
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and about 150 mm length. The conditions applied for the argon plasma are (voltage:

peak-to-peak 18 kV, frequency: 20 KHz and a gas flow rate: 2 L/min). Since, plasma
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chemical effects are inversely proportional to the distance; therefore the main
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experiments were conducted at an 8 mm irradiation distance between the tip of the quartz

tube and the solution surface [14].

3.1.2. Helium cold atmospheric plasma (He-CAP) irradiation system

A cold atmospheric plasma system (PN-120TPG, NU Global, Japan) consisted

of a gas flow controller, a voltage power supply and a hand-piece of the plasma jet,

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constructing an inner micro-hollow-type electrode and an outer dielectric barrier

electrode. The inner and outer diameter of dielectric tube was 1 and 2 mm, respectively.

A high voltage power with a frequency of 60 Hz and a peak-to-peak voltage of 7 kV

was supplied to the two electrodes. Helium gas with a gas flow rate of 2L/min was

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applied for the generation of a plasma jet. The line-averaged electron density in the

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plasma source is approximately 2 x 1015 cm-3. The length of the plasma jet was

approximately 20 mm in atmospheric ambient. The gas temperature of the plasma jet

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was below 350 K.

3.2. Free radicals detected by EPR-spin trapping

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The formation of free radicals by Ar-CAP has been confirmed by electron
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paramagnetic resonance (EPR) spin trapping. EPR spin trapping, using several spin
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traps with different characteristics spectra such as 5, 5-dimethyl-1-pyrroline N-oxide

(DMPO) and N-tert-Butyl-α-phenylnitrone (PBN) are regarded as a powerful tool for

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detecting and identifying free radicals. Exposure of DMPO aqueous solution to Ar-CAP
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showed conclusive evidence for the formation of OH radicals and H atoms by spin

trapping, and the spin adducts, DMPO-OH and DMPO-H were obtained. The
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confirmation of Ar-CAP induced formation of OH radicals and H atoms

using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) and PBN resulted in the EPR

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spectrum, which is in agreement with the spin adducts, M4PO-OH, M4PO-H and the
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PBN spin adduct of H atoms PBN-H.

When the aqueous DMPO solution containing various concentrations of

ethanol as OH radical scavenger was irradiated with Ar-CAP, a decrease in the signal

intensity of DMPO-OH adduct and a simultaneous increase in the EPR spectrum of

DMPO-CH3CHOH adduct was observed as the concentration of ethanol increased. This

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suggests the competitive scavenging of OH by DMPO [14]. These findings strongly

indicate that Ar-CAP irradiation in water induces a huge amount of OH radicals, which

subsequently results in the formation of H2O2 [15]. In addition, small amounts of H

atoms and nitrate/nitrite are also formed [14], (Fig. 4).

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H2O → OH + H

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OH + OH → H2O2

The spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), which is a

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water soluble, non-volatile, aromatic nitroso compound was used to examine the

possibility of detecting intermediate radicals specifically formed from DNA constituents

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following Ar-CAP irradiation. When a solution containing thymidine and DBNBS is
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exposed to Ar-CAP irradiation, an EPR spectrum showed three main lines by resonance
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absorption in nitrogen (Fig. 5). The same spectrum was also observed for the other

nucleic acid base thymine, which is mainly attributed to the spin-trapped radicals
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formed by the reaction of OH. To elucidate the role of free radicals ( OH) induced by
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Ar-CAP irradiation, thymidine solutions were irradiated in the presence of different

concentrations of sodium formate as a OH scavenger. The EPR signal intensity of

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thymidine spin adducts begins to decrease at 2 mM concentration and decreased to

approximately 15% at 1 M concentration of sodium formate (Fig. 5). This result

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suggests that about 85 % thymidine spin adduct is due to free radical reactions
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(mainly OH) generated by Ar-CAP irradiation. These results obtained from Ar-CAP

irradiation are consistent with the findings of ultrasonic irradiation, which showed

that OH is the main active species in the liquid phase.

Similarly, the identification of radical species generated in sodium acetate and

L-alanine was investigated. To examine the formation of pyrolysis radicals, such as

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methyl radicals, spin trapping was done with high solute concentration. In Ar-CAP

exposed solution the formation of acetate ( CH2COO-) radical, but not of methyl ( CH3),

was observed. In L-alanine solution, only the radicals formed by abstraction of a

hydrogen atom were detected [14]. However, in ultrasonic irradiation, at higher solute

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concentrations of both L-alanine and sodium acetate, generation of new radicals

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(typically methyl radical) has been reported due to the thermal decomposition of both

molecules. This is related to the high-temperature interfacial regions by ultrasonic

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cavitation [12].

Taken together, in Ar-CAP irradiation, the efficient production of ROS has

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been demonstrated, however, thermal decomposition is not involved in the mechanism.
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In Ar-CAP, ROS are generated due to high voltage discharge and gas flow, which is
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considered as a rapid transportation mean on the liquid surface [16], (Fig. 4). Therefore,

the distance from the interface is important for the chemical activity of Ar-CAP [17]. It
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has been observed that the amount of OH radical production and DMPO-OH adducts
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decrease with increasing distance.

3.3. Intracellular ROS formation at iso-apoptotic doses of X-ray irradiation

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Comparison of intracellular ROS formation at iso-apoptotic doses of Ar-CAP

and X-ray irradiation showed that the total apoptosis fraction induced by 1 min of
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Ar-CAP was similar to that is induced by 7.5 Gy X-ray irradiation. Consequently

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Ar-CAP irradiation for 1 min produced sufficient OH approximately 5.8 x 10-5 M (in

terms of nitroxide), which is equivalent to X-ray irradiation exposure at a dose of

approximately 225 Gy, i.e., Ar-CAP would generate approximately 30 times the amount

of OH radicals produced by X-ray irradiation [14]. Despite this, X-ray irradiation

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remains more lethal than Ar-CAP irradiation and the intriguing question about ROS and

their lethality still need to be answered.

4. Impact of platinum nanoparticles (nano-Pts) on the biological effects

In past few years, extensive research has been focused on the nanoscience and

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nanotechnology. The concept of nanoscale materials has been widely applied and it

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holds promising features for technical, biological, industrial and biomedical applications.

This field is rapidly growing due to the increased interest of researchers from academic,

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industry and federal sector.

Recently, platinum nanoparticles (nano-Pts) protected by polyacrylic acid (PAA)

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were manufactured through reduction with ethanol. These nano-Pts have gained much
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attention due to the fact that they may use as an antioxidants to scavenge ROS
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persistently and catalytically in living organisms. In fact, these nano-Pts can scavenge

superoxide anions (O2.-) and peroxides (H2O2), indicating that they can act as
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superoxide dismutase (SOD)/catalase mimetics [18]. Previously findings from our

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group showed that these nano-Pts showed anti-inflammatory effects against

LPS-induced inflammatory response [19], and ultraviolet-induced skin inflammation

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[20], as well as inhibiting hyperthermia-induced apoptosis [21]. This inhibition is

attributed mainly due to the protection against intracellular oxidative stress induced by
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these physicochemical stresses.

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4.1. Effects of nano-Pts on biological effects of ultrasound

Ultrasound can induce variety of cell lesions including cell membrane damage

which disrupts cellular homeostasis reversibly or irreversibly, and may cause DNA

damage, apoptosis and/or cell lysis [22]. Interestingly, when the effect of these nano-Pts

was investigated in combination with ultrasound, the antioxidant properties of nano-Pts

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were questioned as a possible mechanism of protection against apoptotic cell death

based on the reported involvement of ROS generation in ultrasound-induced apoptosis.

When U937 cells were sonicated at 0.4 W/cm2 for 2 min, a considerable increased in

DNA fragmentation was observed, and pre-treatment with nano-Pts significantly

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suppressed the ultrasound-induced extent of DNA fragmentation. However, nano-Pts

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does not show any protective effects on ultrasound-induced DNA damage, which

excludes any protective role of nano-Pts on genomic DNA. Interestingly, pre-treatment

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with nano-Pts suppress the activation of the apoptotic pathway by restoring the

ultrasound-induced loss of MMP and caspase-3, t-Bid expression. Furthermore, it was

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found that these nano-Pts inhibit the ultrasound-induced autophagy, which operates as
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pro-survival pathway, and its blockade resulted in the enhanced cell killing [23]. As for
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the underlying mechanism, it was suggested that these nano-Pts may interfere with

apoptosis and consequently block autophagy induction through the suppression of

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caspase-8-mediated Atg3 cleavage as a link between apoptosis and autophagy inhibition,

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and the enhancement of cell killing in combination treatment is not dependent on the

scavenging effects of nano-Pts, rather it depends on the mechanical stress induced by

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ultrasound in the presence of metal nanoparticles.

4.2. Effects of nano-Pts on biological effects of ionizing radiation

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The typical biological effects of ionizing radiation are mostly supervened by

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indirect action of radiation, and free radicals such as hydroxyl radicals which are

produced due to the ionization of water molecules; react with DNA and other critical

targets in the cells [24]. It has been believed that scavengers of hydroxyl radicals can

suppress radiation-induced cell death as estimated by colony formation assay. In

contrast, when the effects of antioxidants on radiation-induced apoptosis in human

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lymphoma U937 cells were examined, the correlation between the concentration of

antioxidants to inhibit DNA fragmentation by 50% and the reaction rate constant of

antioxidants for hydroxyl radical was not observed [25]. It suggests that the prevention

of radiation-induced cell death by antioxidants is not solely dependent on their ability to

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scavenge hydroxyl radical. However, the involvement of hydrogen peroxide in the

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radiation-induced cell death has been well known. When human lymphoma U937 cells

were pre-treated with nano-Pts, radiation-induced cell death was suppressed. This is

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mainly due to the scavenging effects of nano-Pts on radiation-induced hydrogen

peroxide production. As for the underlying mechanism concerned, in the presence of

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nano-Pts, restoration of both intrinsic and extrinsic pathway of apoptosis was observed.
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Nano-Pts suppressed radiation-induced cell death mainly by scavenging
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radiation-induced ROS, and prevented ROS-mediated FAS receptor activation so that

the activation of downstream cell death signaling pathway was suppressed [26].
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4.3. Effects of nano-Pts on biological effects of helium cold atmospheric plasma

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The effects of CAP have been attributed to the generation of ROS and helium cold

atmospheric plasma (He-CAP) was found to induce apoptosis in human lymphoma

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U937 cells over a certain dose. Therefore, it is worthy to investigate the effects of

nano-Pts on the He-CAP-induced apoptosis, to identify the role of intracellular

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oxidative stress. Two sensitive fluorescent probes were used for measuring the
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intracellular reactive oxygen species induced by He-CAP, namely dichlorofluorescein

diacetate (DCFH-DA) a total oxidative stress index but more specific to H2O2 and

hydroethidine (HE) specific to O2.-.

A marked increase in the production of ROS was observed immediately after

He-CAP treatment in the cells. However, nano-Pts suppress the helium plasma induced

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ROS generation and ultimately results in the reversal of apoptosis through suppression

of all the involved micro-molecular pathways. These findings suggest the crucial role of

ROS in He-CAP induced biological effects particularly OH and its recombination

product H2O2, as more profound effects of nano-Pts were observed on He-CAP induced

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H2O2 generation.

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5. Concluding remarks with a perspective of ROS induced by ionizing
radiation, ultrasound and cold atmospheric plasma

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Ionizing radiation, ultrasound, and the cold atmospheric plasma are three

physical factors which generate free radicals in the liquid phase or ultimately produce

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ROS in the cells for their biological action, as summarized in (Fig. 6). Radiation can
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induce ROS production in the liquid phase and intracellularly. Ultrasound also induces
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ROS in the liquid phase, but this is attributed to the occurrence of cavitation that means

more than a specified amount of ultrasound strength (sound pressure) will require.
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Subsequently, ROS generation can be observed only above certain threshold intensity. In
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addition, this threshold in the liquid phase depends on the purity of liquid and on the

history of its temperature and pressure exposure. Cavitation thresholds increase with
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increasing frequency, ambient pressure, and liquid viscosity but decrease with increasing

gas content and temperature of the liquid. Therefore, the possibility of ROS generation
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directly into the cells is very low. In case of cold atmospheric plasma, a relatively large
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amount of ROS is generated in the liquid phase, but it is also necessary to consider the

amount of ROS generated directly into the cells. The huge amount of extracellular ROS

produce by cold atmospheric plasma seems to be not completely recognized

intracellularly.

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Considering the biological effects the ROS generated extracellularly acts on the

cell membrane and the intracellular ROS interacts with DNA and protein activity, which

causes an initial response. In particular, changes in the mitochondria to induce secondary

functional disruption, intracellularly, are important. For example, the mitochondrial

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damage in intact remaining cells by ultrasound-induced mechanical shock wave is

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possible, and generation of intracellular ROS from mitochondria rather than

extracellular ROS (which were directly produced by inertial cavitation in the medium),

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are involved in the regulation of ultrasound-induced biological effects [27]. In case of

cold atmospheric pressure plasma ROS which is generated in the liquid phase, diffuse

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through the plasma membrane or react with plasma membrane to generate intracellular
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ROS through lipid peroxidation and cause damage to intracellular components, promote,
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or inhibit intracellular signaling pathways [28,29]. The biological effects of ionizing

radiation are mostly mediated through indirect action of radiation. Radiation interacts
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with water molecules to produce free radicals such as OH radical which has been
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regarded as the cause of cell death induced by radiation. However, the studies

performed in our laboratory showed that role of OH radical in the radiation-induced

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apoptosis is relatively small, and it was found that the long lived hydrogen peroxide

which is formed by OH radical recombination reaction appear to be important for

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radiation-induced apoptosis [24,25].

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Taken together it is suggested, although the effects of these physical stresses

are mainly dependent on the ROS production especially in the liquid phase, yet when

considering the future therapeutic application of these physical factors, the elucidation

of the precise molecular mechanism of generating intracellular ROS remains crucial.

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Acknowledgements

This work was in part supported by Grant-in-Aid for Scientific Research on Innovative

Areas (no.25108503 and no.15H00892).

The authors would like to thank Dr. Alaa Eldin Tawfik Ismail Refaat Department of

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Cancer Cell Biology, University of Toyama for valuable comments.

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Figure Legends

Fig.1. Characteristics of atmospheric plasma chemistry, radiation chemistry,

sonochmistry and thermochemistry.

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The figure shows features of different chemical effects depending on energy, pressure,

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and reaction time induced by atmospheric plasma, ultrasound, heat and ionizing

radiation. The image was taken and modified from original Suslick, K. S. "The

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Chemical Effects of Ultrasound”, Scientific American 1989 (2) 260, 80-86.

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Fig. 2. Mechanism of ultrasound-induced ROS or free radicals
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The generation of free radicals or ROS induced by ultrasound observed only over a
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certain amount of ultrasound intensity. The chemical effects of ultrasound in aqueous

solution are attributed to acoustic cavitation, which refers to the formation, growth and
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collapse of small gas bubbles in liquids. The very high temperature and pressure
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resulting from a collapsing gas bubble lead to thermal dissociation of water. In case of

ultrasonic intensity below cavitation threshold ROS is not generated.

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Fig. 3. Mechanism of ionizing radiation-induced free radicals or ultimate ROS

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Ionizing radiation induced generation of free radicals and other ROS are due to initial
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excitation and ionization of water molecules. The recombination of resulting ions and

free radicals take place which give rise to generation of more stable ROS species. In the

presence of (O2), aqueous electrons and hydrogen atoms produce superoxide anion

radicals. Biological actions of ionizing radiation are classified as "direct action" and

"indirect action". The indirect action is mainly due to free radicals formed in water and

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plays an important role (approximately 70%) in radiation-induced cell death as

estimated by clonogenic survival assay.

Fig. 4. Mechanism of cold atmospheric plasma-induced free radicals or ROS.

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Generation of cold atmospheric pressure plasma-induced free radicals or ROS species

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are dependent on ionization-excitation of the gas utilized and also on the distance from

the generation source. For biological activities of plasma it is necessary to consider the

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electric field ion as well as the influence of ultraviolet rays. *Shows the excited state of

molecules.

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Fig.5. Effects of argon-cold atmospheric plasma on EPR spectrum of thymidine
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solution.
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Thymidine solution 20 mM was exposed to Ar-CAP irradiation 1 min in the presence of

DBNBS as the spin trap. Spin trapping agent, DBNBS concentration is 5 mM. To
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elucidate the role of free radicals induced by Ar-CAP, thymidine solution was irradiated
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in the presence of various concentrations of sodium formate, which act as OH radical

scavenger. The figure on the right panel shows the reaction of OH radical with
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thymidine.
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Fig.6. Radiation, ultrasound, and cold atmospheric plasma (CAP) induced free radicals

or ultimately ROS in the liquid phase and intracellularly, and subsequent biological

effects

Radiation generates similar amount of ROS in both cases, ultrasound induces only in the

liquid phase under conditions to create acoustic cavitation. CAP produces marked

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amount of ROS in the liquid phase compared with inside the cells. If biological effects

induced by these three physical stresses are similar, the quantities, qualities and special

distribution of ROS are largely different.

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Highlights:

1. Argon-cold atmospheric plasma induced free radicals in an aqueous solution.

2. Argon-cold atmospheric plasma induced intracellular ROS formation.
3. Free radicals formation induced by Ar-CAP, ultrasound and ionizing radiation was

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explained.

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