Effects of Non-Thermal Plasma on Mammalian Cells
Sameer Kalghatgi1, Crystal M. Kelly2, Ekaterina Cerchar3, Behzad Torabi2, Oleg Alekseev2, Alexander
Fridman4, Gary Friedman1, Jane Azizkhan-Clifford2*
1 Department of Electrical and Computer Engineering, Drexel University, Philadelphia, Pennsylvania, United States of America, 2 Department of Biochemistry and
Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America, 3 Department of Surgery, Drexel University College of
Medicine, Philadelphia, Pennsylvania, United States of America, 4 Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, Pennsylvania,
United States of America
Abstract
Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in
contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical
applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding
of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here
that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from
increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of
intracellular reactive oxygen species (ROS). We have utilized c-H2AX to detect DNA damage induced by non-thermal plasma
and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides
in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM
independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks;
however, plasma does not lead to formation of bulky adducts/thymine dimers.
Citation: Kalghatgi S, Kelly CM, Cerchar E, Torabi B, Alekseev O, et al. (2011) Effects of Non-Thermal Plasma on Mammalian Cells. PLoS ONE 6(1): e16270.
doi:10.1371/journal.pone.0016270
Editor: Sotirios Koutsopoulos, Massachusetts Institute of Technology, United States of America
Received October 10, 2010; Accepted December 9, 2010; Published January 21, 2011
Copyright: ß 2011 Kalghatgi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Drexel University Major Research Initiative. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jc19072@gmail.com
blood coagulation without significant heating [11,14]. Nonthermal plasma treatment has also been shown to promote cell
proliferation [15], enhance cell transfection [16,17], sterilize root
canals [18,19,20] and possibly increase wound healing [21]. The
simplicity and flexibility of devices required to generate nonthermal plasma and apply it to tissues is particularly appealing.
However, an understanding of mechanisms by which non-thermal
plasma interacts with living cells and tissues is required to fully
develop its clinical applications.
Several different methods of non-thermal plasma generation at
atmospheric pressure are known [22]. The type of non-thermal
plasma employed in this study is called Dielectric Barrier
Discharge (DBD) [23], which occurs at atmospheric pressure in
air when high voltage of time-varying waveform is applied
between two electrodes, with at least one electrode being insulated
[24], that prevents current build-up, creating electrically safe
plasma without substantial gas heating (Figure 1.). This approach
allows direct treatment living tissues without thermal damage [1].
Plasma is an ionized gas composed of charged particles (electrons,
ions), electronically excited atoms and molecules, radicals, and UV
photons. Plasma treatment exposes cells or tissue surface to active
short and long lived neutral atoms and molecules, including ozone
(O3), NO, OH radicals, and singlet oxygen (O2 1Dg), and a
significant flux of charged particles, including both electrons and
positive and negative ions like super oxide radicals [22,25,26].
Non-thermal plasma density, temperature, and composition can
be changed to control plasma products.
Introduction
The term plasma in physics refers to a partially ionized medium,
usually gas. Importantly, plasma not only produces electrons and
various ions, but also neutral (uncharged) atoms and molecules,
such as free radicals and electronically excited atoms having high
chemical reactivity and the capability to emit UV. The
temperature and components of the gas, as well as the strength
and pulse duration of the electric field determine the exact
composition of plasma. In man-made systems, plasma is usually
generated by electrical discharges and can be generally classified
according to its gas temperature. In thermal plasma, gas
temperature can reach several thousand degrees Kelvin. Devices,
such as argon plasma coagulators, which are used clinically to
cauterize living tissues, typically generate plasmas at temperatures
far exceeding room temperature. The effects of such thermal
plasmas on tissues are non-selective and difficult to control because
they occur primarily through transfer of intense heat [1]. In
contrast, in non-thermal plasmas, gas can be maintained close to
room temperature. Although electrical discharges that generate
non-thermal plasma have been known for a long time, their
clinical potential has been largely ignored and until recently,
applications have been confined to sterilization of inert surfaces
[2,3,4,5,6,7,8,9,10,11] or modulation of cell attachment [12,13]
through surface modification. It has recently been demonstrated
that non-thermal atmospheric pressure plasma can be applied
directly to living cells and tissues [11], killing bacteria and inducing
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure 1. Dose-dependent effects of non-thermal atmospheric pressure dielectric barrier discharge (DBD) plasma on MCF10A cells.
(A) Photograph of DBD plasma treatment of cells. (B) 104 MCF10A cells plated on glass cover slips were treated with the indicated dose of DBD
plasma as described. Cells were counted 24 and 72 hours after treatment. Data are plotted relative to the # of cells in the untreated plate at 24 hours
relative to the # at 72 hours, which was set at 1.0. (C) Cells were treated with the indicated dose of DBD plasma; and colony survival assays were
performed as described. Data are expressed relative to the # of colonies in the untreated control. (D) Three days after treatment with the indicated
dose of DBD plasma, cells were harvested and stained with Annexin V/propidium iodide (PI) and analyzed by Guava.
doi:10.1371/journal.pone.0016270.g001
Prior studies have focused mainly on bactericidal effects of
plasma [27], which require the presence of oxygen [10,28],
consistent with the suggestions in the literature that oxidative stress
(among other factors) may be mediating the interaction between
non-thermal plasma and living organisms [4,5,13]; however, to
date there are no data to indicate that plasma treatment induces
oxidative stress in cells. Since plasma does not produce sufficiently
energetic particles or photons to penetrate cells, it had been
assumed that cellular genetic material would not be affected;
however, formation of intracellular ROS is the major mechanism
by which ionizing radiation produces DNA damage [29]. DNA
damage resulting from ROS includes small or bulky modifications
to bases, interstrand and intrastrand cross-links, as well as singlestrand and double-strand breaks (DSB) [30]. DNA damage
induces a cascade of signals through activation of cellular kinases,
including the phosphatidylinositol 3-kinase–related kinases
(PIKK), specifically ataxia-telangiectasia mutated (ATM), DNA
dependent protein kinase (DNA-PK), and ATM and Rad3-related
(ATR), which phosphorylate the histone variant H2AX in the
vicinity of damage [31]. We demonstrate that non-thermal plasma
induces a variety of effects on mammalian cells, ranging from
increased cell proliferation to apoptosis, and it leads to DNA
damage through formation of intracellular ROS. The DNA
damage signaling cascade activated by non-thermal plasma
treatment is different from that associated with ionizing radiation
(IR), ultraviolet (UV) or H2O2 [29].
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Materials and Methods
Cell Culture
Mammalian breast epithelial cells (MCF10A) were maintained
in high glucose Dulbecco’s Modified Eagle’s Medium/Ham’s F12
50:50 mixture (Cellgro, Mediatech, VA, USA) supplemented with
5% horse serum, Epidermal Growth Factor (100 mg/ml),
Hydrocortisone (1 mg/ml), Cholera Toxin (1 mg/ml), Insulin
(10 mg/ml) and Penicillin/Streptomycin (500 ml, 10000 U/ml
penicillin and 10 mg/ml streptomycin), which were all purchased
from Sigma, St Louis, MO. For plasma treatment, cells were
washed with phosphate buffered saline (PBS), detached with
0.25% Trypsin (GIBCO, Invitrogen, CA, USA), and seeded near
confluence (46105 cells/well) on 22622 mm square glass cover
slips (VWR, PA, USA) in 6-well plates (Greiner Bio One, NC,
USA). Cells were cultured in complete medium in a 37uC, 5%
CO2 incubator for 24 hours prior to plasma treatment.
Amino acids serine, methionine, cysteine, arginine, leucine,
lysine, isoleucine, valine, proline, glutamate and glutamine
(100 mM, Sigma, St Louis, MO) were used to treat cells directly
and separately. N-Acetyl L-cysteine (NAC, 4 mM, Sigma, St Louis,
MO), was used as an intracellular reactive oxygen species scavenger.
Plasma Treatment
DBD plasma was produced using an experimental setup shown in
Figure 1A and schematically illustrated in Figure S1 [11]. Plasma
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Effects of Non-Thermal Plasma on Mammalian Cells
conditions employed here was much higher than 1-ppm level
concentration of nitric oxide (NO), which is entirely consistent with
other published reports [4,5,34]. Concentration of ozone produced
by DBD plasma in the gas phase was measured using an optical
ozone meter MedOzon-245/5 (MedOzone, Russia) and the
concentration of NO was measured using a NO/NOx chemiluminescent analyzer Model 600 CLD (California Analytical Instruments, USA).
MCF10A cells on glass cover slips were exposed to plasma at
various doses (0.13–7.8 J/cm2). Briefly, each cover slip was removed
from the 6-well plate, drained, and placed on a microscope slide.
100 ml of complete medium or defined medium was added to the
glass cover slip before plasma treatment to prevent sample drying.
After treatment, cells were held in the treated medium for one
minute (unless indicated otherwise) and then the cover slip was
placed in a new 6-well plate containing 2 ml of complete medium,
and the samples were returned to the incubator for one hour before
analysis by immunofluorescence or western blot.
In some experiments, medium (complete or defined) or PBS was
treated separately from the cells, designated ‘separated treatment’.
In these cases, 100 ml of medium was placed on a cover slip and
treated with plasma as described for the cells. The treated medium
was then transferred to a cover slip on which cells had been plated
as described above.
During plasma treatment with or without cells present, the
surface of the medium is exposed to plasma and charged species
(electrons and ions) and uncharged gas species [including some
with relatively short half-life (OH, O, for example)] reach the
surface. A grounded mesh was placed between the high voltage
copper electrode and the surface of the medium to block electrons
and ions and allow only uncharged gas species to reach the
medium surface. Similarly, a magnesium fluoride (MgF2) glass
which is practically transparent to UV, but not to any gas species,
charged or uncharged, was placed over the top of the medium
surface to test for possible effects of UV.
Table 1. Typical relative concentrations of various charged
and neutral species generated by non-thermal DBD plasma in
gas phase [25,33,41].
Plasma Generated Species
Density (cm23)
Superoxide (O2N2)
1010–1012
Hydroxyl (OHN)
1015–1017
Hydrogen Peroxide (H2O2)
1014–1016
Singlet Oxygen ( O2)
1014–1016
Ozone (O3)
1015–1017
1
Nitric Oxide (NO)
1013–1014
109–1011
2
Electrons (e )
+
Positive Ions (M )
1010–1012
doi:10.1371/journal.pone.0016270.t001
was generated by applying alternating polarity pulsed (500 Hz –
1.5 kHz) voltage of 20 kV magnitude (peak to peak), 1.65 ms pulse
width and a rise time of 5 V/ns between the high voltage electrodes
using a variable voltage and variable frequency power supply
(Quinta, Russia). One mm thick quartz glass was used as an
insulating dielectric barrier covering the 1-inch diameter copper
electrode. The discharge gap between the bottom of the quartz and
the treated sample surface was fixed at 2 mm. Discharge power
density was measured to be 0.13 Watts/cm2 (at 500 Hz) and 0.31
Watts/cm2 (at 1.5 kHz) [32]. Plasma treatment dose in J/cm2 was
calculated by multiplying the plasma discharge power density by the
plasma treatment duration. For example, plasma treatment at a
power density of 0.13 W/cm2 for 15 s would correspond to a dose
of 1.95 J/cm2. Non-thermal DBD plasma produces various ROS in
gas phase whose typical concentrations are provided in Table 1
[22,25,33]. The dependence of ROS concentration on plasma
power density is complex [24,34]. DBD plasma has a g-factor
(number of ROS generated per electron volt or eV) between 0.3 and
0.5 [35]. For a plasma dose of 3.9 Joules/cm2, about 561016 ROS
are generated. Characterization of DBD plasma employed here has
been presented elsewhere [11,32]. Importantly, ozone (O3)
concentration of about 100 ppm obtained at typical operating
Cell Growth Assay
MCF10A cell proliferation was measured through cell counts on
directly treated cells. MCF10A cells (16104) were seeded on
22622 mm cover slips in 6-well plates one day before plasma
Figure 2. Induction of DNA damage by DBD plasma. (A) MCF10A cells were treated with the indicated dose of plasma. After one-hour
incubation, lysates were prepared and resolved by SDS-PAGE and representative immunoblots with antibody to c-H2AX (top) or a-tubulin (bottom)
are shown. (B) Indirect immunofluorescence was performed utilizing an antibody to c-H2AX one hour after treatment of MCF10A cells with 1.55
J/cm2 DBD plasma.
doi:10.1371/journal.pone.0016270.g002
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Effects of Non-Thermal Plasma on Mammalian Cells
identified as Annexin V-positive and negative for the vital dye
propidium iodide. Floating and trypsin-released cells were
collected and centrifuged, washed thoroughly, resuspended in
Annexin binding buffer, and labeled with Annexin V-fluorescein
and propidium iodide as per manufacturer instructions (BD
Pharmingen, San Jose, CA). Samples were analyzed immediately
by flow cytometry (Guava EasyCyte Plus, Millipore, MA, USA).
Immunofluorescence
MCF10A cells were plated 24 h before treatment. One hour after
plasma treatment, cells were subjected to in situ cell fractionation as
described [36], by incubation in pre-extraction buffer (1X PBS
+0.2% Triton-X +1:50 PMSF) for 5 min at 4C, followed by one
wash with PBS and incubation in fixation solution (3% paraformaldehyde +2% Sucrose in PBS) for 10 min at room temperature.
Cells were then washed in PBS and incubated in permeabilization
buffer (1X PBS +0.5% Triton-X) for 5 min a 4uC. Cells were
washed twice with NaN3+ PBST at room temperature and
incubated overnight at 4C in primary antibody (mouse monoclonal
c-H2AX antibody, serine 139, Upstate Biotechnology, 1:1000).
After three washes in NaN3+ PBS, cells were incubated for 1 h in
the dark in secondary antibody (AlexaFlour594 donkey anti-mouse
antibody, diluted 1:1,000) followed by incubation of slides in 1 ml
DAPI + PBST + NaN3, three washes in NaN3 + PBST and
mounting using DAPI-free mounting medium (Vector Labs) on
glass microscope slides overnight. The slides were then frozen at
220uC for one day prior to imaging them on an upright
fluorescence enabled microscope.
Figure 3. Effects of DBD plasma are mediated by neutral
species and not UV generated by plasma in gas phase. (A) Cells
were subjected to plasma as described earlier (direct, D) or a grounded
mesh that filters charged particles was placed between the electrode
and the medium (indirect, I). Representative immunoblots with c-H2AX
(upper panel) or a-tubulin (lower panel) are shown. The graphs below
the immunoblots show quantification from three independent experiments using the Odyssey Infrared Imaging System (LI-COR Biosciences,
Lincoln, NE, USA). The c-H2AX signal was normalized to the amount of
a-tubulin and data are expressed relative to lowest dose that was set at
1.0. (B) UV produced in DBD plasma does not induce the observed DNA
damage. Cells overlaid with 100 ml of medium were treated with plasma
at 1.55 J/cm2 and 4.65 J/cm2 with (+) and without (2) placing
magnesium fluoride (MgF2) glass over the cells during treatment.
MgF2 glass blocks all plasma species except UV from reaching the
surface of the medium covering the cells during treatment. Representative immunoblot with c-H2AX (upper panel) or a-tubulin (lower
panel) is shown.
doi:10.1371/journal.pone.0016270.g003
Western Blot
treatment. Cells were plasma-treated as described and incubated
for an additional 3 days with a medium change on day 2. Cell
number was quantified on days 1 and 3 by counting trypsindetached cells using a Cell Viability Assay (Guava EasyCyte Plus,
Millipore, MA, USA). Fold growth was determined by taking the
ratio of the number of attached cells on day three to day one.
Protein expression and modification were analyzed by immunoblot. Total cell lysates were prepared by direct lysis of washed
cells in 2X SDS sample buffer containing 5% b-mercaptoethanol.
Samples were electrophoresed at 150 V in Tris-glycine SDS
running buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS
(pH 8.3)). Following electrophoresis, proteins were transferred to
PVDF membrane ((Millipore, MA, USA) for two hours in Trisglycine transfer buffer (10% SDS, Deionized Water, Tris-Glycine
and Methanol (VWR, PA USA)). Immunoblotting was done by
blocking membranes in 1% nonfat dried milk (Carnation) in PBS
with 0.1% Tween 20 (PBST) for a-tubulin or 5% bovine serum
albumin (BSA, Fraction V, Fisher Scientific) in PBST for c-H2AX,
followed by incubation with primary antibodies overnight for 10 to
12 h at 4uC with rocking. Primary antibodies used for immunoblot
included mouse monoclonal antibodies specific for c-H2AX
[phospho-histone H2AX (serine 139), clone JBW301; Upstate]
and a-tubulin (Santa Cruz Biotechnology). The primary antibodies were detected with fluorescently tagged goat anti-mouse Alexa
and Fluor 488 (Santa Cruz Biotechnology). Immunoblot was
developed using Odyssey Infrared Gel Imaging system (LI-COR
Biosciences, NE, USA).
Colony Survival Assay
Detection of Intracellular Reactive Oxygen Species
MCF10A cells (46105) were seeded on 22622 mm cover slips
in 6-well plates one day before plasma treatment. One day after
treatment with plasma or H2O2 (positive control), 300 cells were
seeded onto 60-mm dishes. Eleven days after plating, cells were
fixed and stained with a crystal violet solution (0.5% in 20%
ethanol), and colonies were counted.
The intracellular generation of reactive oxygen species after
plasma treatment was detected using the fluorescent probe 5-(and6)-chlroromethyl-29, 79-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, Molecular Probes). Untreated cells, cells
treated with 100 mM H2O2 and plasma treated cells were analyzed
for changes in fluorescence. MCF10A cells (46105) were plated
one day before plasma treatment. On the day of the experiment,
the cells were washed 2X with PBS, and then preincubated with
10 mM CM-H2DCFDA at 37uC for 30 min in the dark. After
30 min, the excess dye was washed off with PBS and cells were
incubated in complete medium at 37uC in the dark for 30 min,
Apoptosis
Apoptosis was measured via Annexin V/propidium iodide
labeling. Annexin V binds phosphatidylserine translocated from
the inner to the outer cell membrane. Cells in early apoptosis are
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure 4. ROS mediate induction of DNA damage by DBD plasma. (A) CM-H2DCFDA was preloaded into MCF10A cells for 30 min and then
the cells were allowed to recover for another 30 min at 37uC. After recovery, MCF10A cells were treated with the indicated dose of DBD plasma. One
hour after plasma treatment, intracellular ROS were detected using a fluorescence enabled inverted microscope. (B) MCF10A cells were incubated for
2 hours with 4 mM N-acetyl cysteine (NAC) (+), followed by treatment with the indicated dose of DBD plasma. c-H2AX (top) or a-tubulin (bottom)
was detected by immunoblot of cell lysates prepared one hour after plasma treatment.
doi:10.1371/journal.pone.0016270.g004
during which time, the acetate groups on CM-H2DCFDA are
removed by intracellular esterases, trapping the probe inside the
cells. Production of ROS was measured by detecting changes in
the fluorescence of dichlorofluorescein 5, 30, and 60 min after
plasma treatment.
37uC, and the acid was neutralized by washing five times with
PBS. Non-specific binding was blocked with 20% BSA in WB for
30 min at 37uC while shaking. Cells were incubated with the
TDM-2 primary antibody (Kindly provided by Dr. Toshio Mori at
the Nara Medical University, Nara, Japan) against cyclobutane
pyrimidine dimers at a 1:150 dilution in 1% BSA in WB for 1 h at
37uC. After five rinses with 1% BSA in WB, cells were incubated
with the Alexa Fluor 594 anti-mouse secondary antibody
(Invitrogen, Carlsbad, CA) at a 1:400 dilution in 1% BSA in WB
for 30 min at 37uC. Nuclei were counterstained with 10 mg/ml
Hoechst 33258 for 15 min at 37uC. Cells were rinsed five times
with PBS before mounting with Vectashield mounting medium
(Vector Laboratories, Burlingame, CA). All slides were visualized
with Olympus AX70 compound epifluorescence microscope
equipped with Spot RT Slider camera.
Lentivirus Production and Cell Transduction
Lentivirus was prepared from MISSION shRNA (Sigma
Aldrich, St Louis, MO, USA) following the manufacturer’s
instructions
for
targeting
ATM
(NM_000051.2-9380;
NM_000051.2-2990) and ATR (NM_001184.2-231); pLKONon-targeting (shC002) and pLKO-GFP (shC104) were used as
controls. The shRNA plasmids were then transfected into 293T
cells with VSVG, RRE and RSV-Rev packaging vectors to
generate corresponding pseudoviruses. Virus was collected 48 h
post transfection. For stable knockdown, 72 h after transduction,
MCF10A cells were selected in 1 mg/ml puromycin for 48 h.
Statistical analysis
All experimental data points were from triplicate samples and
are expressed and/or plotted as the mean 6 S.E.M. Data were
analyzed by Student’s t-test to establish significance between data
points.
Formation of Thymine Dimers
The published protocols [37,38] were used to measure
formation of thymine dimers after being modified as follows.
Monolayers were rinsed twice with PBS and then fixed with 3%
paraformaldehyde in washing buffer (WB: 0.1% Triton X-100 in
PBS) for 20 min on ice. Cells were rinsed twice in PBS,
permeabilized with WB for 15 min on ice, and rinsed two more
times with PBS. DNA was denatured with 2 N HCl for 5 min at
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Results
In order to test the effects of plasma treatment on mammalian
cells, DBD plasma was applied to human breast epithelial
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure 5. The effects of plasma on cells are dependent on ROS
concentration. (A) Cells on cover slips overlaid with 100 ml cell culture
media were treated with plasma (1.55 J/cm2), followed by dilution in
2 ml of media at the indicated holding time after treatment. Cell lysates
were collected 1 hour after dilution in 2 ml of media. Immunoblots with
c-H2AX or a-tubulin are shown. (B) Medium was separately treated with
plasma and then diluted immediately after treatment as indicated. Cells
were then exposed to the treated and diluted medium for 1 min,
followed by 1-hour incubation in 2 ml of fresh medium. Immunoblots
with c-H2AX or a-tubulin are shown.
doi:10.1371/journal.pone.0016270.g005
Figure 6. Effects of DBD Plasma are mediated by neutral
species generated in the medium. (A) Cells were subjected to
direct treatment with plasma (D) or to medium (100 ml) that was
exposed to plasma and then transferred to the cells (separate, S). (A, B)
Representative immunoblots with c-H2AX (top) or a-tubulin (bottom)
are shown. The graphs below the immunoblots show quantification
using Odyssey. The c-H2AX signal was normalized to the amount of atubulin. Data are expressed relative to lowest dose, which was set at 1.0.
(B) Medium (100 ml separated treatment) was subjected to plasma and
transferred to cells after holding for 1 to 60 min. After 1-minute
incubation with cells, cover slips with treated medium and cells were
transferred to a dish with 2 ml of medium.
doi:10.1371/journal.pone.0016270.g006
(MCF10A) cells (Figure 1A and Figure S1). Plasma is delivered
from a probe placed at a fixed distance over the media being
treated; plasma is released as an electrical discharge as a result of
electrical breakdown of the air in the gap between the high voltage
electrode and the substrate being treated. The initial experiment
involved establishing the dose-dependent effects of plasma
treatment on cell proliferation and survival by direct cell count
and colony formation. At low doses, the rate of cell proliferation
increased; the cell number between days 1 and 3 in cells treated at
0.19 J/cm2 was twice that in the untreated control, and at
intermediate doses (0.26–0.65 J/cm2), there was no significant
effect on proliferation as compared to untreated cells (Figure 1B).
At doses between 1.3 and 1.95 J/cm2, cell number decreased.
There was some correlation between growth inhibition and
survival, at a dose of 1.55 or 3.9 J/cm2—survival was inhibited
by about 40% (Figure 1C). Annexin V/PI staining of cells treated
with plasma at doses ranging from 0.65 to 3.9 J/cm2 revealed that
apoptosis was induced at higher doses (1.95 J/cm2 and higher)
where decreased survival was also observed (Figure 1D).
One possible mechanism underlying these dose-dependent effects
is generation of intracellular ROS, which at low levels is known to
increase cell proliferation and at high levels induces cell death
through DNA damage [30]. To determine whether DBD plasma
treatment of cells could induce DNA damage, we looked at
phosphorylation of H2AX, a histone variant that is phosphorylated
in response to DNA damage [39]. Western blot with an antibody
that detects H2AX phosphorylated at Ser139 (c-H2AX) revealed
that plasma treatment of cells induces a dose-dependent increase in
c-H2AX (Figure 2A). Indirect immunofluorescence also revealed
foci of c-H2AX (Figure 2B), which increased in number at higher
doses. These data demonstrate that DBD plasma treatment of cells
induces a dose-dependent increase in DNA damage.
Plasma-induced DNA damage may be initiated by charged
and/or neutral species produced by plasma in gas phase. Nonthermal atmospheric pressure DBD plasmas produce relatively
long-lived (O3, NO, HO2, H2O2) and short-lived (OH, O,
electronically excited oxygen O) neutral molecules as well as
various charged particles, including ions and electrons [40]. The
typical relative concentrations of the various species generated by
non-thermal plasma at atmospheric pressure are given in Table 1
[25,33,41]. The active species are extremely short-lived at neutral
pH and they tend to recombine in the absence of organic
substrates with which to react. To determine whether the effects of
plasma were due to charged or neutral species, a grounded mesh
was used to exclude charged particles (ions and electrons);
insertion of a grounded mesh between the plasma source and
the media interface (referred to as indirect treatment) did not
significantly affect H2AX phosphorylation (Figure 3A), indicating
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure 7. Amino acid hydroperoxides produced by plasma treatment of organic medium induce DNA damage. (A) Cells were treated
with 100 ml of medium or PBS that was separately exposed to 1.55 J/cm2 plasma. (B) 100 ml of PBS, medium without serum, or PBS with 100 mg/ml
BSA were treated with plasma (1.55 J/cm2) and immediately added to cells on a coverslip (separated, S). Cells overlaid with 100 ml of the indicated
solution were treated with plasma (1.55 J/cm2) (direct, D). (C) Solutions containing the indicated amino acid (100 mM) were separately treated with
plasma and then added to MCF10A cells. (A, B, C) After 1-minute incubation, cells on cover slips were diluted in 2 ml medium, followed by lysis and
Western blot for c-H2AX and a-tubulin. (D) Peroxidation efficiency of various amino acid components of cell culture medium when treated with IR
[43]. For each amino acid, the amount of DNA damage induced is proportional to the peroxidation efficiency.
doi:10.1371/journal.pone.0016270.g007
To further establish that the effects of DBD plasma are due to
modification of the cell medium by the plasma treatment as
opposed to a direct effect on the cells, medium was treated
separately and added to cells. Medium (100 ml) on a coverslip
(without cells) was treated with DBD plasma and then transferred
to a fresh coverslip with cells, which we have termed ‘separated
treatment’. The effect of medium separately treated with DBD
plasma and added to cells was not significantly different from the
effect of direct treatment of cells overlaid with medium (Figure 6A).
To begin addressing which component of the medium is affected
by the treatment, we assessed the stability of the separately treated
medium. Separately treated medium was held for increasing times
before being added to cells. Induction of DNA damage by the
treated medium was not reduced by holding the medium up to one
hour prior to adding it to cells (Figure 6B), suggesting that the
active species formed in the medium are relatively stable. To
identify the active components, we compared the effect of
separated treatment of complete medium vs. inorganic phosphate
buffered saline (PBS). We observed no DNA damage in cells
exposed to separately treated PBS (Figure 7A), whereas separately
treated medium induced DNA damage as anticipated. This
suggests that stable organic components in the medium, such as
organic peroxides [43] mediate the observed effects.
Cell culture medium is composed of amino acids, glucose,
vitamins, growth factors and inorganic salts, as well as serum.
Gebicki et al. have shown that c-radiation (IR) induces formation
of amino acid and protein hydroperoxides in aqueous solutions
containing BSA or individual amino acids [43]. Equivalent levels
of H2AX phosphorylation were induced in cells subjected to
separately treated serum-containing medium, serum-free medium
or PBS with BSA, but not PBS alone (Figure 7B), suggesting that
amino acid peroxidation may be involved. Peroxidation efficiency
is widely variable among different amino acids [43]. To determine
whether the observed results were related to the peroxidation
that ions and electrons do not play a significant role and that
neutral species produced in the gas phase are responsible for the
observed effects. DBD plasma is known to produce UV that is too
weak to produce significant effects on living organisms [8,27,42].
Blocking all plasma species, except UV, by inserting magnesium
fluoride glass during treatment completely blocked the phosphorylation of H2AX after plasma treatment (Figure 3B), further
demonstrating that UV does not play a role in plasma-induced
DNA damage in mammalian cells.
We next sought to directly test whether the DNA damage
induced by DBD plasma is due to the generation of intracellular
ROS. We used a common ROS detection dye, CM-H2DCFDA,
to monitor intracellular ROS levels following plasma treatment.
After 60 min incubation, we detected significantly elevated ROS
levels in plasma treated MCF10A cells, as compared to the
untreated control (Figure 4A). To determine whether DNA
damage is induced by the intracellular ROS, we pre-treated cells
with the ROS scavenger N-acetyl cysteine (NAC). NAC blocked
the induction of c-H2AX, even at high doses of DBD plasma
(Figure 4B), suggesting that the accumulation of DNA damage, as
measured by c-H2AX, is mediated by intracellular ROS.
Consistent with the result that intracellular ROS mediated
plasma induced DNA damage, we found that longer incubation in
the original 100 ml of medium, in which cells were treated (before
dilution into 2 ml of medium), resulted in higher levels of c-H2AX
(Figure 5A). Additionally, when the treated medium was subjected
to different dilutions one minute after treatment, the amount of
damage correlated with the dilution, i.e. damage was greater at the
lowest dilution (Figure 5B, 5C). These data suggest that the
generation of intracellular ROS and the induction of DNA
damage are the results of plasma’s interaction with the
extracellular medium, and that the effects of plasma depend on
the concentration of active species in the medium as well as the
length of exposure of cells to these active species.
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Effects of Non-Thermal Plasma on Mammalian Cells
phosphorylates H2AX in response to DBD plasma treatment. As
discussed, ATM, ATR and DNA-PK can phosphorylate H2AX
on Ser139. Phosphorylation of H2AX in response to plasma (as
well as H2O2) was markedly reduced in cells pretreated with
100 mM Wortmannin (Figure 8A), which at 100 mM inhibits
ATM, ATR, and DNA-PK [44]. In contrast, one hour
pretreatment with 10 mM KU55933, an ATM specific inhibitor
[45], did not significantly reduce the phosphorylation of H2AX in
response to plasma treatment, whereas it significantly reduced it in
response to H2O2 (Figure 8A). These findings indicate that ATR
and/or DNA-PK is required for the phosphorylation of H2AX at
Ser139 in response to non-thermal plasma, although they do not
rule out that other kinases may be activated.
To more directly assess the role of ATM and/or ATR, shRNAs
were utilized. ATM shRNA effectively reduced levels of ATM and
blocked phosphorylation of H2AX in response to hydrogen
peroxide, but did not significantly affect the phosphorylation of
H2AX induced by plasma treatment (1.95 J/cm2) as compared to
non-targeting shRNA (Figure 8B). These findings confirm the
results with inhibitors and indicate that ATM is not the primary
mediator of H2AX phosphorylation in response to plasma
treatment. Depletion of ATR by shRNA reduced phosphorylation
of H2AX in response to plasma treatment by 92% relative to nontargeting shRNA and 40% in response to hydrogen peroxide
(Figure 8C). Taken together, our findings demonstrate that nonthermal plasma treatment activates ATR.
Activation of ATR by UV is through the formation of thymine
dimers resulting in replication fork collapse [46]. To determine
whether plasma treatment resulted in formation of thymine
dimers, immunofluorescence with an antibody that detects
cyclobutane pyrimidine dimers (TDM-2) was performed on cells
after treatment with UV or plasma. As shown in Figure 9A, all of
the cells treated with UV showed the presence of cyclobutane
pyrimidine dimers, whereas there was no evidence of thymine
dimers in cells treated with plasma. Pretreatment of cells with
NAC did not prevent formation of bulky adducts/thymine dimers
in UV treated cells (Figure 9B), further supporting that the effects
of plasma are different from UV.
Figure 8. ATR dependence of non-thermal plasma induced
phosphorylation of H2AX. (A) Immunoblot of c-H2AX (top), and
a-tubulin (bottom) from MCF10As exposed to non-thermal plasma at a
dose of 1.95 J/cm2 or 200 mM H2O2 in the presence (+) or absence (2) of
100 mmol/L Wortmannin (Wort.) or 10 mmol/L KU55933 (KU). (B) MCF10As
were depleted of endogenous ATM by shRNA for 72 hours (bottom,
immunoblot of ATM after ATM or non-targeting (NT) shRNA). Cells were
then plated on glass cover slips and exposed to DBD plasma at a dose of
1.95 J/cm2 or 200 mM H2O2. (C) MCF10As depleted of endogenous ATR by
shRNA for 72 hours (bottom, immunoblot of ATR after ATR or nontargeting (NT) shRNA). (B,C) After knockdown, cells were plated on glass
cover slips for 24 h followed by exposure to non-thermal plasma at a dose
of 1.95 J/cm2 or 200 mM H2O2. After one-hour incubation, lysates were
prepared and resolved by SDS-PAGE and representative immunoblots with
antibody to c-H2AX (top) or a-tubulin (bottom) are shown.
doi:10.1371/journal.pone.0016270.g008
Discussion
Based on the myriad of potential clinical applications of nonthermal plasma [26] and the lack of information about the
molecular basis of the effects of plasma on mammalian cells, this
study has addressed the interaction of plasma with cells at a
molecular level. The present study demonstrates that non-thermal
plasma produces dose-dependent effects that range from increased
cell proliferation to apoptosis; these effects are the result of the
production of intracellular ROS.
Cell death in response to non-thermal plasma treatment in the
dose range examined is primarily though induction of apoptosis,
which is an important therapeutic consideration. Apoptotic cells
are broken up into apoptotic bodies, which are engulfed by
neighboring cells, leading to cell death without significant
inflammatory response [47,48]. Controlled delivery of nonthermal plasma may provide a means to kill benign and malignant
lesions in a defined area, without significant necrosis and
subsequent inflammation. Delivery can be achieved by direct
treatment of tissue surfaces or application of defined medium
treated with plasma.
Plasma-induced DNA damage is likely initiated by neutral
species and not charged species produced by plasma in gas phase.
Use of a grounded mesh to exclude charged particles (ions and
electrons) did not significantly affect H2AX phosphorylation.
efficiency of organic components in cell culture medium, 11
different amino acids with a range of peroxidation efficiencies were
dissolved individually in PBS and separately treated with plasma
and then added to cells. As shown in Figure 7C, phosphorylation
of H2AX was directly proportional to the peroxidation efficiency
of the amino acids, with valine producing the most significant level
of damage and serine and methionine producing no detectable
DNA damage (Figure 7D). There is a direct correlation between
the peroxidation efficiency of 11 different amino acids and the
level of DNA damage, providing strong support for the hypothesis
that organic peroxides are produced in the plasma treated medium
and are responsible for the observed effects on DNA.
To characterize the DNA damage pathway activated by DBD
plasma treatment, we next sought to identify the kinase that
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure 9. Plasma treatment does not induce the formation of thymine dimers. MCF10A cells grown on glass coverslips were incubated with
(A) or without (B) 4 mM NAC for 2 h, followed by treatment with direct plasma (2.3 J/cm2), UV (10 J/m2), H2O2 (200 mM), or a no treatment (NT)
control. Cells were allowed to recover for 1 h, then fixed and immunostained with TDM-2 primary antibody (kindly provided by Dr. Toshio Mori at the
Nara Medical University, Kashihara, Nara, Japan) and Alexa Fluor 594 anti-mouse secondary antibody to detect cyclobutane pyrimidine dimers.
doi:10.1371/journal.pone.0016270.g009
Further, blocking of all plasma species, except UV (by a
magnesium fluoride glass placed over the cells during treatment)
completely blocked the phosphorylation of H2AX after plasma
treatment, further demonstrating that UV does not play a role in
plasma-induced DNA damage in mammalian cells. Moreover, the
results of the experiments comparing direct treatment with
separated plasma treatment further rule out any effect of plasma
resulting from UV, temperature or electric field.
Mammalian cells undergo DNA damage as a result of ROS
generated from endogenous and exogenous sources. The intracellular ROS scavenger, NAC, completely blocked phosphorylation of H2AX after non-thermal plasma treatment of MCF10A
cells, indicating that the induction of DNA damage is mediated
through the formation of intracellular ROS. The role of ROS is
further supported by direct measurement of intracellular ROS and
by the formation of lipid peroxidation products (Figure S2A,
Methods S1), specifically malondialdehyde (MDA); however,
plasma-induced lipid peroxidation does not lead to phosphorylation of H2AX (Figure S2B).
We have provided several lines of evidence that the interaction
of plasma with organic components of cells and media is
responsible for the induction of DNA damage. First, cells exposed
to PBS that was separately treated with plasma did not exhibit
DNA damage, whereas cells exposed to medium or a solution of
specific amino acids did undergo DNA damage and the media
remains active over extended periods of time, which would not be
the case with inorganic peroxides (e.g. H2O2), and is consistent
with published reports on organic peroxides [43]. The formation
of intracellular ROS as a result of plasma treatment can result
from formation of organic peroxides in the medium; in support of
this notion, there is a direct correlation between the peroxidation
efficiency of 11 different amino acids and the amount of DNA
damage induced in cells exposed to these amino acid solutions
PLoS ONE | www.plosone.org
after plasma treatment. Taken together, these data strongly
support the conclusion that the effects of DBD plasma are
mediated by the organic peroxides formed in the medium,
although formation of organic peroxides in plasma-treated
medium should be directly measured. It should be noted that
cells in PBS directly treated with plasma exhibit DNA damage, in
contrast to cells exposed to separately treated PBS which do not.
We presume that the damage in cells after treatment in PBS is the
result of short-lived species interacting directly with oxidizable
organic substrates (including DNA) in cells. That the separated
treatment of PBS does not induce damage suggests that in absence
of organic substrates, the ROS that are generated are short-lived
probably due to their recombination and consequently are no
longer active when added to cells after the separated treatment.
The only possible explanation for the observed effects of direct
plasma treatment in PBS is that ROS coming directly from plasma
interact with organic substrates in cells producing stable ROS.
This is supported by our data demonstrating that the effects of
separated or direct treatment are blocked by an ROS scavenger.
Complex molecular networks that rapidly sense and repair
DNA damage have evolved to maintain genomic stability and
ensure cell survival [49]. ATM, ATR and DNA-PK [44] can
phosphorylate H2AX on Ser139 over a large region of chromatin
surrounding a DSB or a stalled replication fork [50,51]. In this
study, we have shown that phosphorylation of H2AX after plasma
treatment of MCF10A cells is primarily through ATR, in contrast
to the ATM-dependent phosphorylation of H2AX after treatment
of cells with IR or hydrogen peroxide. These results suggested that
plasma treatment may lead to formation of bulky adducts;
however, as shown, plasma treatment does not lead to formation
of bulky adducts/thymine dimers in MCF10A cells. Unlike IR or
H2O2, non-thermal plasma treatment may lead to formation of
stalled replication forks [52] to activate ATR; however, unlike UV,
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Effects of Non-Thermal Plasma on Mammalian Cells
Figure S2 DBD plasma-induced lipid peroxidation is not
the activation of ATR by plasma does not involve formation of
thymine dimers and it is mediated by formation of ROS. Further
studies are needed to determine if plasma induces formation of
regions of single stranded DNA or interstrand cross-links which
would activate ATR.
Understanding the mechanism underlying the effects of plasma
is essential in applying it to clinical use. We have shown here that
DBD plasma generates active oxygen species in gas phase, which
can react with organic components, such as serum or amino acids,
to produce long-lived reactive species, mostly likely amino acid
and protein hydroperoxides. The amount of intracellular ROS
produced by plasma can be controlled by varying the frequency
and voltage waveform, allowing for the fine-tuning of the
therapeutic effect, from stimulating cell proliferation to inducing
apoptosis. Generation of organic peroxides in solution may
provide an alternative means of administration. Future work will
involve investigating the mechanism by which peroxidized amino
acids are processed by the cell. Current studies are directed at
establishing whether the effects of plasma are through the uptake
of amino acid hydroperoxides by active transport mechanisms in
cells or through ROS signaling and determining the nature of the
DNA damage. Devices that deliver DBD plasma can be designed
to accommodate a wide range of clinical applications, from surface
to endoscopic administration. Possible clinical applications of
DBD plasma include wound sterilization, potential enhancement
of healing, controlled ablation of tissue, and selective targeting of
benign or malignant lesions.
responsible for the observed DNA damage. (A) Cells overlaid
with 100 ml of medium were treated with plasma at 1.55 J/cm2
with (+) and without (-) pre-incubating for 2 h with diphenyl
phenyl enediamine (DPPD, Sigma-Aldrich, St. Louis, MO, USA)
a lipophilic antioxidant which blocks lipid peroxidation. (B) Cells
overlaid with 100 ml of medium were treated with plasma (1.55 J/
cm2) with (+) and without (2) pre-incubating with DPPD. 1 h after
plasma treatment cells were lysed and immunoblots were prepared
to detect DNA damage by looking at c-H2AX signal. Representative immunoblot for c-H2AX (upper panel) or a-tubulin (lower
panel) with quantification below it is shown. The c-H2AX signal
was normalized to the amount of a-tubulin. Data are expressed
relative to the amount of c-H2AX in plasma-treated sample
without DPPD, which was set to 1.0.
(TIF)
Methods S1 Supplementary methods.
(DOC)
Acknowledgments
We would like to thank Dr. Gregory Fridman for providing the plasma
treatment setup. We would also like to thank Dr. Toshio Mori at the Nara
Medical University (Kashihara, Nara, Japan) for generously providing us
with the TDM-2 antibody.
Author Contributions
Supporting Information
Conceived and designed the experiments: SK CMK AF GF JAZ.
Performed the experiments: SK CMK EC OA. Analyzed the data: SK
CMK BZ OA AF GF JAZ. Wrote the paper: SK OA GF JAZ.
Figure S1 Schematic of the plasma treatment setup.
(TIF)
References
15. Kalghatgi S, Friedman G, Fridman A, Clyne AM (2010) Endothelial Cell
Proliferation is Enhanced by Low Dose Non-Thermal Plasma Through
Fibroblast Growth Factor-2 Release. Ann Biomed Eng 38: 748–757.
16. Coulombe S, Léveillé V, Yonson S, Leask RL (2006) Miniature atmospheric
pressure glow discharge torch (APGD-t) for local biomedical applications. Pure
and Applied Chemistry 78: 1147–1156.
17. Leduc M, Guay D, Leask RL, Coulombe S (2009) Cell permeabilization using a
non-thermal plasma. New Journal of Physics 11: 115021.
18. Jiang C, Vernier PT, Chen MT, Wu YH, Wang LL, et al. (2008) Low Energy
Nanosecond Pulsed Plasma Sterilization for Endodontic Applications . pp 77–79.
19. Xinpei L, Yinguang C, Ping Y, Qing X, Zilan X, et al. (2009) An RC Plasma
Device for Sterilization of Root Canal of Teeth. Plasma Science, IEEE
Transactions on 37: 668–673.
20. Sladek REJ, Stoffels E, Walraven R, Tielbeek PJA, Koolhoven RA (2004)
Plasma treatment of dental cavities: a feasibility study. Plasma Science, IEEE
Transactions on 32: 1540–1543.
21. Shekhter AB, Serezhenkov VA, Rudenko TG, Pekshev AV (2005) Beneficial
effect of gaseous nitric oxide on the healing of skin wounds. Nitric Oxide:
Biology and Chemistry 12: 210–219.
22. Fridman A, Chirokov A, Gutsol A (2005) Non-thermal atmospheric pressure
discharges. Journal of Physics D: Applied Physics 38: R1–R24.
23. Siemens CW (1862) On the Electrical Tests Employed During the Construction
of the Malta and Alexandria Telegraph, and on Insulating and Protecting
Submarine Cables Journal of the Franklin Institute 74: 166–170.
24. Eliasson B, Egli W, Kogelschatz U (1994) Modelling of dielectric barrier
discharge chemistry. Pure and Applied Chemistry 66: 1275–1286.
25. Fridman A, Kennedy LA (2004) Plasma Physics and Engineering: Routledge,
USA. 853.
26. Fridman G, Friedman G, Gutsol A, Shekhter AB, Vasilets VN, et al. (2008)
Applied Plasma Medicine. Plasma Processes and Polymers 5: 503–533.
27. Fridman G, Brooks A, Balasubramanian, Fridman A, Gutsol A, et al. (2007)
Comparison of Direct and Indirect Effects of Non-Thermal AtmosphericPressure Plasma on Bacteria. Plasma Processes and Polymers 4: 370–375.
28. Weng C-C, Wu Y-T, Liao J-D, Kao C-Y, Chao C-C, et al. (2009) Inactivation of
bacteria by a mixed argon and oxygen micro-plasma as a function of exposure
time. International Journal of Radiation Biology 85: 362–368.
29. Olofsson BA, Kelly CM, Kim J, Hornsby SM, Azizkhan-Clifford J (2007)
Phosphorylation of Sp1 in response to DNA damage by ataxia telangiectasiamutated kinase. Mol Cancer Res 5: 1319–1330.
1. Vargo JJ (2004) Clinical applications of the argon plasma coagulator.
Gastrointest Endosc 59: 81–88.
2. Sladek REJ, Stoffels E (2005) Deactivation of Escherichia coli by the plasma
needle. Journal of Physics D: Applied Physics 38: 1716–1721.
3. Goree J, Bin l, Drake D, Stoffels E (2006) Killing of S. mutans Bacteria Using a
Plasma Needle at Atmospheric Pressure. IEEE Transactions on Plasma Science
34: 1317–1324.
4. Stoffels E, Kieft AIE, Sladek AREJ, Zandvoort V, Slaaf DW (2008) Cold
gas plasma in biology and medicine. In: d’Agostino R, Favia P, Kawai Y,
Ikegami H, Sato N, et al. (2008) Advanced Plasma Technology. Weinheim:
Wiley-VCH. pp 301–318.
5. Stoffels E (2006) Gas plasmas in biology and medicine. Journal of Physics D:
Applied Physics 39.
6. Laroussi M, Tendero C, Lu X, Alla S, Hynes WL (2006) Inactivation of Bacteria
by the Plasma Pencil. Plasma Processes and Polymers 3: 470–473.
7. Laroussi M, Mendis DA, Rosenberg M (2003) Plasma interaction with microbes.
New Journal of Physics 5: 41.41–41.10.
8. Laroussi M, Leipold F (2004) Evaluation of the roles of reactive species, heat,
and UV radiation in the inactivation of bacterial cells by air plasmas at
atmospheric pressure. International Journal of Mass Spectrometry 233: 81–86.
9. Laroussi M, Alexeff I, Kang WL (2000) Biological decontamination by
nonthermal plasmas. Plasma Science, IEEE Transactions on 28: 184–188.
10. Laroussi M (2005) Low Temperature Plasma-Based Sterilization: Overview and
State-of-the-Art. Plasma Processes and Polymers 2: 391–400.
11. Fridman G, Peddinghaus M, Ayan H, Fridman A, Balasubramanian M, et al.
(2006) Blood Coagulation and Living Tissue Sterilization by Floating-Electrode
Dielectric Barrier Discharge in Air Plasma Chemistry and Plasma Processing 26:
425–442.
12. Kieft IE, Kurdi M, Stoffels E (2006) Reattachment and Apoptosis After PlasmaNeedle Treatment of Cultured Cells. Plasma Science, IEEE Transactions on 34:
1331–1336.
13. Kieft IE, Darios D, Roks AJM, Stoffels E (2005) Plasma treatment of
mammalian vascular cells: a quantitative description. Plasma Science, IEEE
Transactions on 33: 771–775.
14. Kalghatgi S, Fridman G, Cooper M, Nagaraj G, Peddinghaus M, et al. (2007)
Mechanism of Blood Coagulation by Nonthermal Atmospheric Pressure
Dielectric Barrier Discharge Plasma. Plasma Science, IEEE Transactions on
35: 1559–1566.
PLoS ONE | www.plosone.org
10
January 2011 | Volume 6 | Issue 1 | e16270
Effects of Non-Thermal Plasma on Mammalian Cells
42. Dobrynin D, Fridman G, Friedman G, Fridman A (2009) Physical and biological
mechanisms of direct plasma interaction with living tissue. New Journal of
Physics. pp 115020.
43. Gebicki S, Gebicki JM (1993) Formation of peroxides in amino acids and
proteins exposed to oxygen free radicals. Biochem J 289(Pt 3): 743–749.
44. Abraham RT (2004) PI 3-kinase related kinases: ’Big’ players in stress-induced
signaling pathways. DNA Repair 3: 883–887.
45. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NMB, et al. (2004)
Identification and Characterization of a Novel and Specific Inhibitor of the
Ataxia-Telangiectasia Mutated Kinase ATM. Cancer Res 64: 9152–9159.
46. Ward IM, Minn K, Chen J (2004) UV-induced Ataxia-telangiectasia-mutated
and Rad3-related (ATR) Activation Requires Replication Stress. Journal of
Biological Chemistry 279: 9677–9680.
47. Fiers W, Beyaert R, Declercq W, Vandenabeele P (1999) More than one way to
die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18: 7719–7730.
48. Majno G, Joris I (1995) Apoptosis, Oncosis, and Necrosis - an Overview of CellDeath. American Journal of Pathology 146: 3–15.
49. Shiloh Y (2006) The ATM-mediated DNA-damage response: taking shape.
Trends Biochem Sci 31: 402–410.
50. Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin
domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:
905–916.
51. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA
doublestranded breaks induce histone H2AX phosphorylation on serine 139.
Journal of biological chemistry 273: 5858–5868.
52. Ward IM, Chen J (2001) Histone H2AX Is Phosphorylated in an ATRdependent Manner in Response to Replicational Stress. The Journal of
Biological Chemistry 276: 47759–47762.
30. Lehnert BE, Iyer R (2002) Exposure to low-level chemicals and ionizing
radiation: reactive oxygen species and cellular pathways. Hum Exp Toxicol 21:
65–69.
31. Rogakou E, Boon C, Redon C, Bonner W (1999) Megabase chromatin domains
involved in DNA double-strand breaks in vivo. J Cell Biol 146: 905–916.
32. Ayan H, Fridman G, Staack D, Gutsol AF, Vasilets VN, et al. (2009) Heating
Effect of Dielectric Barrier Discharges for Direct Medical Treatment. Plasma
Science, IEEE Transactions on 37: 113–120.
33. Fridman A (2008) Plasma Chemistry: Cambridge University Press.
34. Kogelschatz U, Becker KH, Schoenbach KH, Barker RJ (2004) NonEquilibrium Air Plasmas at Atmospheric Pressure: Taylor & Francis.
35. Fridman A (2008) Plasma Biology and Plasma Medicine. New York: Cambridge
University Press.
36. Mirzoeva OK, Petrini JH (2001) DNA damage-dependent nuclear dynamics of
the Mre11 complex. Mol Cell Biol 21: 281–288.
37. Fitch ME, Cross IV, Ford JM (2003) p53 responsive nucleotide excision repair
gene products p48 and XPC, but not p53, localize to sites of UV-irradiationinduced DNA damage, in vivo. Carcinogenesis 24: 843–850.
38. Bomgarden RD, Lupardus PJ, Soni DV, Yee MC, Ford JM, et al. (2006)
Opposing effects of the UV lesion repair protein XPA and UV bypass
polymerase eta on ATR checkpoint signaling. EMBO J 25: 2605–2614.
39. Rogakou E, Pilch D, Orr A, Ivanova V, Bonner W (1998) DNA double-stranded
breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273:
5858–5868.
40. Fridman A (2008) Plasma Biology and Plasma Medicine. Plasma Chemistry.
New York, NY: Cambridge University Press. pp 848–857.
41. Fridman A, Chirokov A, Gutsol A (2005) Non-thermal atmospheric pressure
discharges. Journal of Physics D: Applied Physics 38: R1–R24.
PLoS ONE | www.plosone.org
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