RESEARCH ARTICLE

EPR-Spin Trapping and Flow CytometricStudies of Free Radicals Generated Using
Cold Atmospheric Argon Plasma and X-Ray
Irradiation in Aqueous Solutions andIntracellular Milieu
Hidefumi Uchiyama1, Qing-Li Zhao2, Mariame Ali Hassan2, Gabor Andocs1,
Nobuyuki Nojima1, Keigo Takeda3, Kenji Ishikawa3, Masaru Hori3, Takashi Kondo2*
1 Tateyama Machine Co., Ltd., Toyama 930–1305, Japan, 2 Department of Radiological Sciences,
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930–0194,
Japan, 3 Plasma Nanotechnology Research Center Nagoya University, Nagoya 464–8601, Japan* kondot@med.u-toyama.ac.jp
OPEN ACCESS
Citation: Uchiyama H, Zhao Q-L, Hassan MA,
Andocs G, Nojima N, Takeda K, et al. (2015) EPRSpin Trapping and Flow Cytometric Studies of Free
Radicals Generated Using Cold Atmospheric Argon
Plasma and X-Ray Irradiation in Aqueous Solutions
and Intracellular Milieu. PLoS ONE 10(8): e0136956.
doi:10.1371/journal.pone.0136956
Editor: Honglian Shi, University of Kansas, UNITEDSTATES
Received: September 29, 2014
Accepted: August 12, 2015
Published: August 28, 2015
Copyright: © 2015 Uchiyama et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information file.
Funding: This work was supported by a Grant-in-Aid
for Scientific Research on Innovative Areas, Grant
No. 25108503 from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, and a
Grant-in-Aid for Practical and Commercial Applicationof Advanced Technology (TONIO), Toyama
Prefecture. The funders had no role in study design,
data collection, analysis, decision, and preparation of
the manuscript or publishing. Tateyama Machine Co.,

Abstract
Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to
identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous
solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP
was generated using a high-voltage power supply unit with low-frequency excitation. The
characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra
measurements. Hydroxyl (OH) radicals and hydrogen (H) atoms in aqueous solutions wereidentified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAPinduced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate(DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Humanlymphoma U937 cells were used to study intracellular oxidative stress using five fluorescentprobes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of OH radicals using ArCAP compared with that by X-irradiation. Very small amounts of H atoms were detected
whereas nitric oxide was not found. The formation of OH radicals depended on the type ofrare gas used and the yield correlated inversely with ionization energy in the order of krypton> argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed
to Ar-CAP. Intracellularly, OH, H2O2, which is the recombination product of OH, and OClwere the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts ofsuperoxides were formed. Despite the superiority of Ar-CAP in forming OH radicals, theexposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous
solutions and an intracellular milieu is discussed.

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Free Redicals Generated by Cold Ar-Plasma

Ltd provided support in the form of salaries for
authors (HU, GA and NN), but did not have any
additional role in the study design, data collection andanalysis, decision to publish, or preparation of themanuscript. The specific roles of these authors arearticulated in the "author contributions" section.
Competing Interests: HU, GA and NN are
employees of Tateyama Machine Co., Ltd. This does
not alter the authors' adherence to PLOS ONE
policies on sharing data and materials.

Introduction
Plasma medicine is an emerging interdisciplinary field including the technology of low-temperature plasma [1,2]. Despite the novelty of this technology, its potential has been demonstrated in various applications such as sterilization [3,4], blood coagulation [5], wound healing[6,7], cancer treatment [8–12], dentistry [13,14], and regenerative medicine of bone [15,16].
The technology offers many advantages including high efficiency, convenience of operation interms of design and cost, being a dry procedure, applicability to temperature-sensitive materials, and absence of no hazardous residues. A physical cold plasma is a partially ionized gas generated by flowing a gas through a relatively high electric field. A series of excitations anddissociative collisions with the surrounding gas at or near atmospheric pressure takes place
producing a mixture of electrons, negative and positive ions, excited gas species, free radicals,and electromagnetic radiation without heating the surrounding gas. Hence, this type of plasma
is called cold atmospheric plasma (CAP) [1–16]. Plasma produces a cocktail of oxygen andnitrogen radicals that can promptly react with all biomolecules that come across their way
including living cells. These free radicals are the focus of emerging interests in biomedical CAP
applications. In general, free radicals are a double-edged sword that can modify a plethora of
biomolecules and cellular pathways wiring a range of functions from proliferation to cell death
depending on their composition and dose [17]. The chemical output of CAP can be tuned inorder to achieve the best results. As tunability requires knowledge, relentless research has
started to pay off in terms of the understanding of some of the mechanisms underlying plasma
effects and in defining some reactive species as key players. For example, in bacteriostatic andbactericidal applications, it has been demonstrated that plasma-generated particles and UV
photons can modify DNA constituents and induce DNA strand breaks, and inactivate proteinsin addition to damage of the cellular envelope [18–21]. In one study, dose-dependent effects
were reported to range from enhancement of cell proliferation to induction of apoptosis primarily due to the formation of intracellular reactive oxygen species (ROS). The observed DNA
damage is initiated by the production of active neutral species induced via organic peroxides ina growth medium [22]. In HeLa cells, nitrogen and air plasma jet induce apoptosis via ROS
generation and dysfunction of mitochondria, as confirmed by the use of various ROS scavengers, which mitigate apoptosis induction [23]. Hydrogen peroxide (H2O2) has been shown toplay a central role in physical plasma-induced oxidative stress in human blood cells [24]. Endoplasmic stress in response to oxidative and nitrative stresses induced by a CAP jet of helium
containing 1% oxygen has been claimed to mediate apoptosis in HepG2 cells [25]. ROS and/or
reactive nitrogen species (RNS) generated by plasmas trigger signaling pathways involving JNK
and p38, and promote mitochondrial perturbation leading to apoptosis [26]. To date, there has
been no unifying concept for the chemical make-up of the output jet and how the different species can modify cellular activities. Owing to the absence of a universal plasma set up and thehigh variability of operating parameters, extensive efforts are still required in order to gain
knowledge and hence control of CAP performance. In this study, we examined the fluxes ofreactive moieties generated by a CAP generator developed in house with argon gas flow inaqueous solutions and cultured cells using electron paramagnetic resonance (EPR) spin
trapping.
EPR-spin trapping is an analytical technique employed in the detection and identificationof short-lived free radicals. Spin trapping involves the addition of radicals to nitrone spin traps
to form a spin adduct, a nitroxide-based radical of relatively longer half-life to allow its detection using an EPR spectrometer. Depending on the radical, each spin adduct usually yields a
distinctive EPR spectrum characteristic to that radical. The spin adduct is usually identified
based on the basis of the measurement of hyperfine-coupling constants of relevant nuclei. The

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Free Redicals Generated by Cold Ar-Plasma

relative amount of spin adducts can also be obtained by comparing their spectra with that of astable nitroxide. In this study, EPR-spin trapping using a number nitrone spin traps was utilized to identify and quantify free radicals in aqueous solutions exposed to Ar-CAP. The
obtained EPR spectra were compared with the spectra from radiolysis of air-saturated water.
Other radicals that might arise following exposure to Ar-CAP were detected using the nitrone
spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) [27,28]. DBNBS is a useful agent
for detecting pyrolysis radicals which are formed in high temperature interfacial regions produced by ultrasonic cavitation, e.g., methyl radicals in sodium acetate aqueous solutions [29].
Furthermore, the intracellular oxidative stress induced by Ar-CAP was evaluated by flow
cytometry using a number of fluorescent probes to identify intracellular reactive species.

Materials and Methods
Chemicals
The inert gases helium (He), neon (Ne), argon (Ar), krypton (Kr), and xenon (Xe) were boughtfrom Hokusan Co., Ltd. (Toyama, Japan). All gases were of pure grade ( 99.9%). The spin traps
5,5-dimethyl-1-pyrroline N-oxide (DMPO; Labotec Co., Ltd., Tokyo, Japan), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO; Sigma Aldrich Chem. Co., MO), phenyl N-t-butylnitrone(PBN; Sigma Aldrich Chem.), 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS; Wako Pure
Chemical Industries, Ltd., Osaka, Japan), and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; Dojindo, Kumamoto, Japan) were used to detect the presence of different free radical species in ultrapure water following exposure to CAP by EPR spin trapping.
Thymine, thymidine, uracil, uridine, sodium acetate, and L-alanine were obtained from
Wako Pure Chemical Industries, Ltd. Hydroxyphenyl fluorescein (HPF), aminophenyl fluorescein (APF), and diaminofluorescein-2 (DAF-2DA) were from Sekisui Medical Co., Ltd.
(Tokyo, Japan). Hydroethidine (HE) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) werefrom Molecular Probes Inc. (Eugene, OR).

Cells
Human myelomonocytic lymphoma U937 cells were obtained from Human Sciences Research
Resource Bank (Japan Human Sciences Foundation, Tokyo, Japan). They were maintained in
RPMI-1640 medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum(FBS) at 37°C in humidified air with 5% CO2.

Cold atmospheric argon plasma irradiation system
A jet of Ar-plasma was produced in a quartz tube of 2.0 mm inner diameter and about 150 mm
length. Two electrodes connected to a high-voltage power supply unit with low frequency (18kV peak-to-peak, 20 kHz) were fixed on the quartz tube surface. Discharge voltage was measured with a high-voltage probe (P6015A; Tektronik, Inc., Tokyo, Japan). The Ar-gas flow rate
was controlled with a gas flow controller (MODEL8500 series; KOFLOC Co., Ltd., Kyoto
Japan). The Ar gas flow was at 2 L/min. The length of the plasma jet was approximately 10
mm. The operational parameters for typical experiments were the irradiation distance between
the tip of the quartz tube and the solution surface, and irradiation time. The system and theexperimental setup are illustrated in Fig 1.

Temperature measurement of the medium during Ar-CAP irradiation
The temperature changes of the medium during the Ar-CAP irradiation was carried out by afour channel fluoroptic temperature measurement system. (Luxtron m3300 Biomedical Lab

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Free Redicals Generated by Cold Ar-PlasmaFig 1. Block diagram of cold atmospheric plasma (CAP) exposure system. Al; aluminum, Ar; argon.
doi:10.1371/journal.pone.0136956.g001

Kit, Lumasense Technologies, Santa Clara, CA). The temperature measurement probe is a 0.5
mm in diameter optical fiber, totally insensitive for electromagnetic field. The sample to bemeasured was 1ml tap water in a well of a 24 well plate. All the four probes of the temperature
measurement system were immersed into the water to be irradiated with Ar-Cap, on a bottomof the well, exactly in the way of the plasma plume.

Optical emissions from Ar-CAP
Optical emissions Ar-CAP were collected by an optic fiber and a lens directed to a position at 8mm in the remote region of Ar-CAP. The emission were observed using a spectrometer (Shamrock SR-561-B1; grating, 600 l/mm-1; ANDOR, Belfast, UK) and an intensified charge-coupled-device (ICCD) camera (iStar DH734-25F-03; ANDOR).

Atomic radicals generated by Ar-CAP in gas phase
The absolute densities of O (3Pj) and N (4So) atoms were measured by vacuum ultraviolet
absorption spectroscopy (VUVAS) with a micro discharge hollow cathode lamp (MHCL) as alight source operated at a fixed pressure of 1 atm [30]. The transition lines used for measuring
O atoms were 3s 3So-2p4 3P2 at 130.217 nm, 3s 3So-2p4 3P1 at 130.487 nm, and 3s 3So-2p4 3P0 at
130.604 nm. Those used for measuring N atoms were 4P5/2-4So3/2 at 119.96 nm, 4P3/2 -4So3/2 at120.02 nm, and 4P1/2-4So3/2 at 120.07 nm. The VUV light emitted from the MHCL was transmitted through a measured region with an absorption length of 7 mm set using two MgF2 windows attached to the light-guide pipes made of ceramics. The transmitted VUV light wasdispersed by a VUV monochromator and detected by a photomultiplier tube. The total

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Free Redicals Generated by Cold Ar-Plasma

absorption intensity for three sublevels of O and N atoms was measured. Ar-CAP was located
8 mm above the measured region. To remove background absorption due to atmosphericgases, N2 gas was used as a purge gas to maintain the pressure of the chamber at atmosphericpressure. The absorption intensity of the O2 gas feed was measured using the transition lines of
H2 molecules emitted from MHCL at approximately 128 nm [31]. All optical parts were evacuated at high vacuum using a turbo molecular pump. The slit width of the VUV monochromator was set to 100 μm, and the wavelength resolution was within 0.4 nm.

Chemical dosimeter
The chemical effects of Ar-CAP were measured by a ferrous-ferric ion (Fricke) dosimeter.
Changes in absorbance of the chemical system with exposure time were determined with aspectrophotometer at 304 nm.

X-ray irradiation
X-ray irradiation was carried out at room temperature using an X-ray generator (MBR-1520R3, Hitachi Medico Technology, Kashiwa, Japan) operated at 150 kV and 20 mA with a 1 mm Al
filter at a dose rate of 5 Gy/min determined by Fricke’s chemical dosimetry. The air-saturated
sample solutions (1 ml) in plastic tubes were irradiated on a rotating table at 6 rpm. Immediately after X-irradiation, samples were measured with EPR and UV-spectrophotometer. Finaldoses were 5, 7.5 and 10 Gy.

Free radical detection by EPR spin trapping
The detection of OH radicals and H atoms induced by Ar-CAP was carried out using the spin
traps DMPO, PBN, and M4PO. An aqueous solution containing a spin trap at a concentrationof 10 mM was treated with CAP at increasing durations and distances. Immediately after irradiation, the samples were transferred to a glass capillary tube (VC-HO75P, Terumo, Tokyo,
Japan) and inserted into a special quartz tube in an EPR spectrometer (RFR-30, Radical
Research Inc., Tokyo, Japan). In general, EPR setting were microwave power; 4 mW, frequency;
9.425 GHz, center magnetic field; 329.5 mT, and modulation width; 0.1 mT. The nitroso spin
trap DBNBS was used at a concentration of 5 mM for the detection of radicals derived fromthymine (20 mM), thymidine (100 mM), uracil (20 mM), uridine (20 mM), L-alanine (1 M),
and sodium acetate (3 M) aqueous solutions. The EPR spectra of the treated samples wererecorded at room temperature. The yields of spin adducts were determined using the stable
nitroxide radical 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrroline-1-oxide as a standard. The peakheights of EPR signals were expressed in relative units compared with that of the Mn2+ internal
standard, one unit was calculated to approximately equal 7.7 x 106 M nitroxide radicals.

Intracellular oxidative stress measured by flow cytometry
Intracellular oxidative stress following CAP treatment was assessed flow cytometrically using a
number of fluorescent probes with different affinities to detect a wide range of free radical species. One million cells were preloaded with a probe in phosphate-buffered saline (PBS) for 15
min at 37°C and then exposed to Ar-CAP. Immediately after the exposure, the cells were
injected into the flow cytometer to analyze at least 10,000 events. The probes included were
DCFH-DA (10 μM) for overall oxidative stress, HE (10 μM) for superoxide anion (O2-), HPF(2.5 μM) for OH, and peroxy nitrite (ONOO-); APF (2.5 μM) for OH, ONOO-and hypochlorite (OCl-), DAF-2 DA (10 μM) for nitric oxide (NO).

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Free Redicals Generated by Cold Ar-Plasma

Apoptosis measurement by Annexin V-FITC/ PI staining
Apoptosis induction following exposure to Ar-CAP and X-rays for different periods and doses,
respectively, was investigated flow cytometrically. The irradiated cells were incubated at 37°C
for 6 h before they were collected, washed with cold PBS, and suspended in the binding bufferof the Apoptosis Detection Kit (Immunotech, Marseille, France). The cells were then stainedwith fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) at 4°C for
20 min in the dark according to the manufacturer’s instructions. Finally, samples were injected
into the flow cytometer where at least 10,000 events were counted. The dot plots display thepercentages of cells externalizing phosphatidylserine bound to FITC-annexin V (early apoptosis), cells with damaged membranes internalizing PI (necrosis), or both (secondary necrosis).
The total apoptosis fraction is expressed as the sum of early apoptotic and secondary necrotic
fractions.

Measurement of nitrate/nitrite concentration in aqueous solutions
Briefly, 1 ml milli-Q water was added to 24-well tissue culture plate, and Ar-CAP irradiation
was performed for 1 min. After treatment, 80 μL sample was taken out to 96-well plate, and a
NO2/NO3 assay was performed using a NO2/NO3 Assay Kit-CII (Dojindo, Kumamoto, Japan),the optical density was measured at 570 nm by an automatic microplate reader (Bio-Rad, Hercules, CA).

Statistical analysis
Experiments were performed in independent triplicates. All results are shown asmean ± standard deviation (SD). Tests of significance were performed using unpaired t-student
test (two-tailed) with p < 0.05 considered to indicate a statistically significant difference. Pearson’s correlation coefficient was used to test the correlation among obtained data. All tests
were performed using GraphPad Prism 5 (GraphPad Software; San Diego, CA).

Results and Discussion
1. Characterization of Ar-CAP
Optical emission spectra were measured to obtain the spectral lines emitted from excited-state
species in Ar-CAP. Fig 2A and 2B show typical optical emission spectra from Ar-CAP ranging
from 300 to 900 nm at distances of 8 and 18 mm. Because only Ar gas was fed into the plasma
generator, most of the emission lines and bands observed between 700 and 850 nm weremainly attributed to the excited states of Ar. Typically, the emission lines from excited Ar (3p5
4s!3p5 4p) were observed at 696.5, 706.7, 727.3, 738.4, 750.4, 751.5, 763.5, 772.4, 794.8, 800.6,
801.5, 810.4, 811.5, 826.5, 840.8, 842.5, and 852.1 nm. In addition, between 300 and 450 nm,
the emission bands from excited N2 were observed at approximately 316, 337, 358, 381, 406,
and 416 nm for the N2 2nd positive system (C 3Pu!B 3Pg). The emission band signals for theN2+ 1st negative system (B 2Su+!X 2Sg+) appeared relatively weak in the range between 391
and 428 nm. An emission band from OH (A 2Sg! X 2P) occurred with a relatively low signal
intensity at approximately 308 nm. Because the emission bands of N2 and OH were also
detected, these findings indicate that the effluent Ar gas mixed with atmospheric air at remoteregions beyond the exit of the glass tube. Optical emission spectroscopy is essentially more sensitive for measuring electron temperature than for measuring the densities of the observed species. Thus, the optical emission data do not enable the quantitative estimation of the density of
any species.

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Free Redicals Generated by Cold Ar-PlasmaFig 2. Optical emission and atomic radicals generated by Ar-CAP. Emission spectra (λ: 300–900 nm) of Ar-CAP at distances of 8 and 18 mm. (a) (λ:300–450 nm), emission spectra from excited N2 and OH. (b) (λ: 700–850 nm), emission spectra from excited states of Ar.
doi:10.1371/journal.pone.0136956.g002

Furthermore, the absolute densities of atomic species were measured by VUVAS. The average numbers of O atoms were 6.3×1013 cm-3 at 8 mm distance, 4.5×1013 cm-3 at 13 mm, and2.3×1013 cm-3 at 18 mm. The density of O atoms thus correlates indirectly with the distancefrom the plasma tube nozzle and the O atoms fed into the samples at remote regions can becalculated using the first-order kinetics within the distance range tested. Assuming that the gas
is at equilibrium at room temperature, then the O atom fluxes would be in the order of 1017
cm-2 s-1, and the total O atom dose for 60s would be approximately 6×1018 cm-2 in a 6 ml-volume sample, i.e., 1018 atoms/ml.

2. Free radicals generated using CAP in aqueous solutions
Plasma irradiation produces immense quantities of free radicals. The chemical activity of ArCAP was confirmed by Fricke dosimeter where the oxidation of ferrous ions to ferric ionsincreased linearly with increasing exposure time (Fig 3). However, the exact species produced
by Ar-CAP are still unidentified. EPR spin trapping was employed for the detection of free radicals generated by Ar-CAP irradiation for 2 min in aqueous solutions. Ar-CAP was set at 8 mm
distance between the glass tube nozzle and the surface of the sample solution. A number ofspin traps with showing different characteristic spectra were used namely DMPO, M4PO, andFig 3. Fricke dosimetery. The effect of different Ar-CAP exposure time on the oxidation of ferrous ions toferric ions. The oxidation increased linearly with increasing plasma irradiation time.
doi:10.1371/journal.pone.0136956.g003

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Free Redicals Generated by Cold Ar-Plasma

PBN. To measure temperature rise, Ar-CAP irradiation in aqueous solution was performed
under continuous real time temperature data recording. The temperature rise after 2 and 5 min
exposure was 1.7°C ± 0.2°C (n = 4) and 2.3 °C ± 0.2°C (n = 4), respectively. When 1 ml of
10 mM DMPO aqueous solution was exposed to Ar-CAP for 2 min, an EPR spectrum consisting of a 1:2:2:1 quartet as well as other small peaks was observed. The equal nitrogen andhydrogen hyperfine coupling constants (hfcc; aN = aβH = 1.49 mT) are characteristic of thehydroxyl spin adduct of DMPO (DMPO-OH) as shown in Fig 4A. The small peaks corresponded to DMPO-H adducts showing a primary triplet (aN = 1.65 mT) that is further split by
two secondary hydrogens (aβH = 2.25 mT). An EPR spectrum of DMPO-OH and DMPO-H
generated by X-irradiation is shown in Fig 4B.
The formation of OH radicals and H atoms by Ar-CAP was further confirmed using M4PO
and PBN. For M4PO, the resulting EPR spectra consisted of a primary triplet due to nitrogen(aN = 1.53 mT), with each line split into a doublet by a secondary proton (aH = 1.69 mT). Thesehyperfine coupling constants are in agreement with those reported previously for M4PO-OH[32,33] (Fig 4C). Other lines were analyzed as primary nitrogen triplets (aN = 1.65 mT) further
split by two secondary protons (aβH = 2.17 mT), consistent with the M4PO spin adduct of H
atoms. The EPR spectrum obtained from PBN solution (10mM) showed a primary nitrogen
triplet (aN = 1.67 mT) further split by two secondary protons (aβH = 1.06 mT), consistent with
the PBN spin adduct of H atoms [34] (Fig 4D). All the hyperfine coupling constants of ArCAP- generated radicals detected by nitrone spin trapping are shown in Table 1.
Moreover, the use of ethanol as OH radical scavenger further confirmed the formation ofOH by Ar-CAP irradiation. As shown in Fig 5, the signal of DMPO-OH decreased withincreasing the ethanol concentration and the signal nearly disappeared at a concentration of
0.5%. Simultaneously, the EPR signal of the DMPO-CH3CHOH adduct increased with ethanol
concentration.
Compared with 5 Gy X-ray irradiation, Ar-CAP irradiation yielded huge amounts of OH
radicals but smaller amounts of H atoms (Fig 4A and 4B). The amount of DMPO-OH adducts
generated by Ar-CAP irradiation was also compared with that generated by UV photolysis(λ = 254 nm; at 0.45 mW/cm2) of hydrogen peroxide (H2O2) solution (40 mM), and the decay
in the EPR spectra of DMPO-OH adducts was investigated. The half-lives of the first-orderdecay of DMPO-OH adducts induced by Ar-CAP irradiation and UV-photolysis of H2O2 were
calculated as 6.1 ± 0.7 min and 19.2 ± 2.6 min, respectively. The rapid decay of the DMPO-OH
adduct formed after Ar-CAP irradiation indicates a change in pH and/or the generation of
nitric acid in the plasma-irradiated solution [35–37].
When the extent of OH radical production was assessed at graded distances from theplasma tube nozzle, the amount of DMPO-OH adducts was found to decrease with increasing
distance (Fig 6A). With the yield at 8 mm taken as 100%, 91.2 ± 12.9% yield was obtained at
10 mm, 78.2 ± 4.8% at 12 mm, 45.9 ± 4.8% at 14 mm, and 9.1 ± 3.9% at 16 mm. With respect
to Ar-CAP irradiation time, Fig 6B shows that the amount of DMPO-OH adducts increased
with increasing irradiation time. The yield at 30s was about 1.6 times that at 15s, and the yield
at 60s was about 2.3 times that at 15s.
The effect of different rare gases on CAP-induced OH radical production was investigated.
The yield of DMPO-OH adducts was in the order Kr > Ar = Ne > He. No plasma discharge
was observed when Xe was used. When the yield of DMPO-OH adducts from different rare gas
plasmas under similar conditions was plotted against the ionization energies of the rare gases
used, an inverse correlation was observed (Fig 7). The NO probe cPTIO at a concentration of
200 μM was used to examine NO generated by Ar-CAP irradiation. No specific signal of cPTIO-NO was observed in the generated EPR spectrum after a 2 min exposure. A reaction-diffusion simulation study by Ikuse et al. showed that NO generation occurs at the interface

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Free Redicals Generated by Cold Ar-Plasma

Fig 4. EPR spectra of spin adducts in aqueous solutions irradiated with Ar-CAP for 2 min at 8 mm distance. (a) EPR spectrum of DMPO showing a
1:2:2:1 quartet of DMPO-OH and several other small peaks due to DMPO-H. (b) DMPO aqueous solution irradiated with 5 Gy X-rays showing typical
DMPO-OH and DMPO-H peaks. (c) EPR spectrum of M4PO-OH and M4PO-H adducts. (d) EPR spectrum of PBN-H adducts. Arrows indicate internal
standard lines.
doi:10.1371/journal.pone.0136956.g004

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Free Redicals Generated by Cold Ar-Plasma

Table 1. Hyperfine coupling constants of Ar-CAP-induced radicals detected by nitrone spin traps.
Spin Trap

RadicalaN (mT)aβH (mT)

DMPO

OH

1.49

1.49

H

1.66

2.25 (2H)C2H5O

1.58

22.80

OH

1.53

1.69

H

1.65

2.17 (2H)

H

1.67

1.06

M4PO
PBN
doi:10.1371/journal.pone.0136956.t001

between the irradiated solution and plasma jet where it rapidly reacts with other moieties producing a variety of species [38]. Therefore, NO generation might be below the detection limit
in our setup. When the nitrate/nitrite generation due to Ar-CAP irradiation was examined, theconcentration in aqueous solutions was 77.3 ± 2.0 μM. It is thus concluded that Ar-CAP irradiation in water produced mainly large amounts of OH radicals and subsequently H2O2, owing
to the rapid recombination of OH radicals, small amounts of H atoms, and nitrate/nitrite werealso detected.

3. Effects of CAP on pyrimidine derivatives, L-alanine, and sodium
acetate aqueous solutions
The water-soluble, non-volatile, aromatic nitroso spin trap DBNBS was used for the detectionof intermediate radicals formed from DNA constituents, sodium acetate, or L-alanine at highconcentrations to detect specific radicals following Ar-CAP irradiation. Solutions containing
thymine (20 mM) and DBNBS (5 mM) irradiated with Ar-CAP for 2 min showed an EPR spectrum of a triplet (aN = 1.30 mT) which can be attributed to the spin-trapped radicals at the C-5
position most likely formed by OH addition (and H addition) to the C-6 position of the thymine ring (Fig 8A). The same triplet (aN = 1.30 mT) was also observed for the similarly irradiated thymidine aqueous solution (100 mM) (Fig 8B). Analysis of the EPR spectra of the spin
adducts indicated that there were no magnetic nuclei at the β-position of the spin adducts. In
the case of uracil (20 mM) and uridine (20 mM) aqueous solutions, the EPR spectra showedsecondary doublets due to a β -proton which exhibited a different hyperfine splitting, and secondary triplets due to a β-nitrogen (Fig 8C and 8D). It is speculated that the major spin adducts
originated from the 6-yl radical formed by OH addition (and H addition) to the C-5 positionof the base moiety [29,39].
Sodium acetate and L-alanine of high concentrations were irradiated with Ar-CAP to detect
whether methyl radicals are formed due to pyrolysis or direct degradation, namely dissociationof C-O or C-C bond. The EPR spectrum for the sodium acetate aqueous solution at concentrations of 3 M and 5 mM DBNBS showed a primary nitrogen triplet (aN = 1.37 mT) further splitby two secondary protons (aβH = 1.0 mT) (Fig 8E). The spectrum is consistent with that of thespin adduct of the acetate (CH2COO-) radical. Unlike the case of sonication of 3.3 M sodium
acetate solution for 10 min in the presence of Ar, there was no evidence of methyl (CH3) radicals in Ar-CAP [29]. L-alanine aqueous solution (1 M) irradiated with Ar-CAP showed an EPR
spectrum with a primary nitrogen triplet (aN = 1.36 mT) further split by two secondary protons(aβH = 1.36 mT) which indicates CH2-CH(NH3+)-COO- radical formation as a result of H
abstraction from the methyl group of alanine. A primary nitrogen triplet (aN = 1.37 mT) further split by a secondary proton (aβH = 0.65 mT) was also observed (Fig 8F). This is consistent
with CH3-CH-COO- radical formation through the deamination of the alanine molecule [29].

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Free Redicals Generated by Cold Ar-Plasma

Fig 5. Effect of ethanol on OH formation. DMPO-OH adduct signal intensity decreasing with ethanol
concentration with simultaneous increase in DMPO-CH3CHOH adduct signal intensity.
doi:10.1371/journal.pone.0136956.g005

No evidence of CH3 radicals was observed. All the hyperfine coupling constants of the ArCAP-generated radicals detected by a nitroso spin trap are shown in Table 2.

Fig 6. Effect of distance and exposure time on OH formation. (a) DMPO-OH adducts signal intensity decreases with increasing the distance from theplasma tube nozzle to sample. The yields at different distances are given as percentage from that at 8 mm considered as 100% (b) DMPO-OH adducts signal
intensity increases with increasing Ar-CAP irradiation time.
doi:10.1371/journal.pone.0136956.g006

PLOS ONE | DOI:10.1371/journal.pone.0136956 August 28, 201512 / 19

Free Redicals Generated by Cold Ar-PlasmaFig 7. Amount of OH radicals produced by different rare gas—plasmas. The four rare gases krypton(Kr), argon (Ar), neon (Ne), and helium (He) were used to generate cold atmospheric plasma. The amountof.OH radicals formed (indicated by DMPO-OH adduct EPR signal) correlated inversely with the ionizationenergy of the gas. Represented data are the mean ± SD of three independent replicates.
doi:10.1371/journal.pone.0136956.g007

4. Intracellular ROS formation induced by Ar-CAP irradiation
Ar-CAP irradiation has been shown to produce immense amounts of free radicals in aqueous
solutions. In this part, the formation of free radicals within cells was investigated using fivefluorescent probes, namely, HPF for the detection of OH and ONOO-; APF for OH, ONOO-,
and OCl-; HE for O2-; and DAF-2 for NO. When HPF and APF were utilized to measure theintracellular oxidative stress due to Ar-CAP irradiation, the shift of distribution of fluorescenceintensity was observed (Fig 9A and 9B). The quantitative data are shown in Fig 9C and 9D. A
non specific fluorescent probe DCFH-DA was also used for confirming the total intracellularoxidative stress [40]. Compared with X-ray irradiation at 10 Gy, Ar-CAP irradiation for 1 min
induced greater intracellular oxidative stress (Fig 9E). The increase in DCF(H) fluorescenceintensity after Ar-CAP and X-ray irradiation could be explained in terms of the enhanced formation of OH radical and OCl- as well as H2O2 resulting from the combination of two OHradicals. This result is in part consistent with the superiority of Ar-CAP in the formation ofOH radicals shown by EPR spectrometry as discussed earlier. Since the increase in HPF fluorescence intensity could be mainly attributed to intracellular OH radicals while APF fluorescence could be attributed to both OH radicals and OCl-, then X-rays seem to generate more
OCl- (Fig 9C and 9D). The exact mechanism by which OH radicals are formed inside cells is
still unclarified; however, there are three possibilities: 1) OH radicals may form directly inside
the cells, 2) a certain potion of the enormous amounts of extracellular OH radicals can traverse
the cell membrane, or 3) OH radicals may form via the Fenton reaction of H2O2.
No increase in HE fluorescence intensity was observed in the X-irradiated cells whereas
very little increase was observed in cells treated with Ar-CAP for 1 min indicating the formation of small amounts of O2- by Ar-CAP (Fig 9F). The intracellular formation of ONOO-

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Free Redicals Generated by Cold Ar-Plasma

Fig 8. EPR spectra of DBNBS-spin adducts. (a) thymine, (b) thymidine, (c) uracil, (d) uridine, (e) sodium acetate, and (f) L-alanine. Arrows indicate internal
standard lines.
doi:10.1371/journal.pone.0136956.g008

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Free Redicals Generated by Cold Ar-Plasma

Table 2. Hyperfine coupling constants of Ar-CAP-induced radicals detected by the nitroso spin trapDBNBS
aβN (mT)aβH (mT)

1.35

0.28

0.44

1.30

0.36

0.36

RadicalaN (mT)

Thymine1.30

Thymidine

1.30

UracilUridineSodium acetate
CH2COO-

1.37

1.00 (2H)

1.36

1.36 (2H)

1.37

0.65 (H)

L-Alanine
CH2CH(NH3+)-COO.

-

CH3- CHCOO

doi:10.1371/journal.pone.0136956.t002

following either treatment was unlikely because the formation of NO was excluded based on
DAF-2 data (Fig 9G).

Fig 9. Intracellular ROS formation induced by Ar-CAP irradiation. Intracellular oxidative stress was investigated flow cytometrically using five different
fluorescent probes of different affinities to a number of free radicals. U937 cells were exposed to Ar-CAP for 1 min or X-rays (10 Gy). Control samples were
sham-exposed and handled exactly as irradiated cells were. The typical histograms showing the increase in fluorescence intensity following Ar-CAP (2 min)in presence of (a) HPF and (b) APF. All percentages acquired from three independent replicates are plotted in bar diagrams as mean ± SD for (c) HPF, (d)APF, (e) DCF(H), (f) HE, and (g) DAF-2. RFI, Relative Fluorescence Intensity
doi:10.1371/journal.pone.0136956.g009

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Free Redicals Generated by Cold Ar-PlasmaFig 10. Comparison of Ar-CAP and X-irradiation with respect to apoptosis induction. The total apoptotic cell fractions in U937 cells exposed to Ar-CAP
and X-irradiation investigated at 6 h post treatment by flow cytometry using annexin V-FITC and PI double staining.
doi:10.1371/journal.pone.0136956.g010

5. Intracellular ROS formation at isoapoptotic doses of Ar-CAP and Xray irradiation
Apoptosis has been associated with enhanced intracellular oxidative stress under many conditions [41,42]. Here, the total apoptosis fraction in U937 cells exposed to Ar-CAP was investigated at 6 h post treatment by annexin V-FITC and PI double staining with flow cytometry.
The fractions were 5.2 ± 1.1% after 30 s irradiation, 13.3 ± 0.9% after 60 s irradiation, and28.3 ± 9.7% after 120 s irradiation compared with 2.6 ± 0.5% in the control. In the case of Xray irradiation, the percentages of the total apoptosis were 4.6 ± 1.2% at 5 Gy, 13.3 ± 0.2% at
7.5 Gy, and 19.9 ± 4.2% at 10 Gy compared with 2.5 ± 0.4% in the control (Fig 10). Thus, thetotal apoptosis fraction induced by 1 min-Ar-CAP was almost similar to that induced by 7.5
Gy X-irradiation. When the amount of DMPO-OH adducts generated by X-irradiation wasmeasured at increasing radiation doses, the slope value was 0.03 relative unit /Gy. Consequently, if Ar-CAP treatment for 1 min produced about 6.75 relative value of DMPO-OH
adducts, this would correspond to that obtained at 225 Gy of X-irradiation, i.e., Ar-CAP would
generate approximately 30 times the amount of OH radicals produced by X-ray irradiation.
Despite this striking comparison, X-ray irradiation remains more lethal than Ar-CAP irradiation and the intriguing question about ROS and their lethality still awaits answers.

Conclusions
In this study, we have shown that Ar-CAP irradiation can generate enormous amounts of OH
radicals and H2O2—the combination product of OH radicals in the aqueous phase. A theoretical calculation reveals that Ar-CAP irradiation for 1 min would be sufficient for producing
OH radicals in aqueous solutions located at 8 mm away from the plasma tube nozzle equivalent to X-ray exposure at a dose of approximately 225 Gy. However, apoptosis level induced by
Ar-CAP (1min) was almost similar to that of X-irradiation at a dose of 7.5 Gy. Thus, although
Ar-CAP (1min) would create about 30 times more OH radicals than that formed by X-

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Free Redicals Generated by Cold Ar-Plasma

irradiation (7.5 Gy), the apoptosis-inducing efficacy of both treatments was similar reflecting
the superiority of X-rays in inducing cell killing.
Our EPR-spin trapping study revealed that no pyrolysis or direct degradation radicals wereformed in aqueous solutions containing non volatile solutes. Intracellularly, OH radicals andOCl- were detected in large amounts. The latter may have resulted from the reaction betweenOH radicals and chloride anions (Cl-) that are abundant in cells. Unlike OCl-, it is not clear
whether OH radicals were formed intracellularly or traversed from the extracellular milieu
through the cell membrane. The elucidation of the mechanism by which reactive oxygen andnitrogen species are formed, and the consequent effects on cellular responses remain for futureplasma research to unveil. All data are available in S1 File.

Supporting Information
S1 File. The biological effects and formation of free radicals by Ar-CAP compared to Xirradiation. Biological effects were determined based on apoptosis induction in U937 cells
assessed flow cytometrically. Formation of free radicals was traced by different methods. In
extracellular milieu, different spin adducts were detected by EPR spin trapping method. In
presence of cells, various intracellular fluorescent probes were used with flow cytometry. Moreover, the overall chemical activity of Ar-CAP was detected with Fricke dosimeter. Data are
given as mean ± SD.
(XLSX)

Author Contributions
Conceived and designed the experiments: HU QLZ TK. Performed the experiments: HU QLZ
MAH GA TK. Analyzed the data: HU QLZ TK NN KT. Contributed reagents/materials/analysis tools: NN KT KI MH. Wrote the paper: HU MAH QLZ TK.

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