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Static electric fields interfere in the viability of cells exposed to ionising radiation
Joo D. T. Arruda-Neto
ab
; Errol C. Friedberg
c
; Maria C. Bittencourt-Oliveira
d
; Erika Cavalcante-Silva
ad
; Ana
C. G. Schenberg
e
; Tulio E. Rodrigues
a
; Fermin Garcia
f
; Monica Louvison
e
; Claudete R. Paula
e
; Joel Mesa
g
; Michelle M. Moron
a
; Durvanei A. Maria
h
; Godofredo C. Genofre
i
a
Physics Institute - University of So Paulo,
b
FESP - So Paulo Engineering School, So Paulo
c
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
d
ESALQ -
University of So Paulo,
e
Institute for Biomedical Sciences - University of So Paulo,
f
Medical Physics Group
- Santa Cruz State University, Ilhus, Brazil
g
So Paulo State University/UNESP, Botucatu, Brazil
h
Biomedical and Biophysical Laboratories, Butantan Institute, So Paulo
i
CENEPES, Grajau General
Hospital, So Paulo, Brazil
Online Publication Date: 01 April 2009
To cite this Article Arruda-Neto, Joo D. T., Friedberg, Errol C., Bittencourt-Oliveira, Maria C., Cavalcante-Silva, Erika, Schenberg, Ana
C. G., Rodrigues, Tulio E., Garcia, Fermin, Louvison, Monica, Paula, Claudete R., Mesa, Joel, Moron, Michelle M., Maria, Durvanei A.
and Genofre, Godofredo C.(2009)'Static electric fields interfere in the viability of cells exposed to ionising radiation',International
Journal of Radiation Biology,85:4,314 321
To link to this Article: DOI: 10.1080/09553000902781121
URL: http://dx.doi.org/10.1080/09553000902781121
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Static electric elds interfere in the viability of cells exposed to ionising
radiation
JOA

O D. T. ARRUDA-NETO
1,2
, ERROL C. FRIEDBERG
3
,
MARIA C. BITTENCOURT-OLIVEIRA
4
, ERIKA CAVALCANTE-SILVA
1,4
,
ANA C. G. SCHENBERG
5
, TULIO E. RODRIGUES
1
, FERMIN GARCIA
6
,
MONICA LOUVISON
5
, CLAUDETE R. PAULA
5
, JOEL MESA
7
, MICHELLE M. MORON
1
,
DURVANEI A. MARIA
8
, & GODOFREDO C. GENOFRE
9
1
Physics Institute University of Sa o Paulo,
2
FESP Sa o Paulo Engineering School, Sa o Paulo,
3
Department of Pathology,
University of Texas Southwestern Medical Center, Dallas, Texas, USA,
4
ESALQ University of Sa o Paulo,
5
Institute for
Biomedical Sciences University of Sa o Paulo,
6
Medical Physics Group Santa Cruz State University, Ilheus, Brazil,
7
Sa o
Paulo State University/UNESP, Botucatu, Brazil,
8
Biomedical and Biophysical Laboratories, Butantan Institute, Sa o Paulo,
and
9
CENEPES, Grajau General Hospital, Sa o Paulo, Brazil
(Received 14 November 2007; Revised 12 November 2008; Accepted 21 December 2008)
Abstract
Purpose: The interference of electric elds (EF) with biological processes is an issue of considerable interest. No studies
have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined
effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells.
Materials and methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the
interval 0.55 kGy, using a
60
Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V cm
71
static electric eld and viable cells were enumerated. Cultures of Candida albicans were incubated at 368C for 20 h, gamma-
irradiated with doses from 14 kGy, and submitted to an electric eld of 180 V cm
71
. Samples were examined under a
uorescence microscope and the number of unviable (red) and viable (apple green uorescence) cells was determined. For
crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric eld of 1250 V/cm,
incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a uorescence microscope to
quantify nuclei with g-H2AX foci.
Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed
suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of
surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric
eld on the irradiated MRC5 cells the number of nuclei with g-H2AX foci increased 40%, approximately.
Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair
shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation EF suggests that EF likely
inactivate cellular recovering processes. The result for the number of nuclei with g-H2AX foci in MRC5 cells indicates that
an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.
Keywords: Cellular radiobiology, radiation, radiosensitivity, bacteria
Introduction
At the cellular level, phenomena such as transport
processes are induced by local gradients of electric
elds, temperature, or chemical potential (Becker-
man 2005). Endogenous elds are also important in
development (Levin et al. 2002) and wound healing
(Song et al. 2002). In particular, electric eld (EF)
effects in biological systems are of long-standing
interest. For example, small exogenous elds,
static or pulsed (up to 1 GHz) are of interest with
respect to sensory systems, medical applications, and
Correspondence: J. D. T. Arruda-Neto, Universidade de Sao Paulo, Instituto de F sica, Rua do Matao Travessa R. 187, 05508-090, Sao Paulo, SP, Brazil.
Fax: 55(11) 3091 6640. E-mail: arruda@if.usp.br
Int. J. Radiat. Biol., Vol. 85, No. 4, April 2009, pp. 314321
ISSN 0955-3002 print/ISSN 1362-3095 online 2009 Informa Healthcare USA, Inc.
DOI: 10.1080/09553000902781121
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possible human health hazards (Gowrishankar and
Weaver 2003). Indeed, high-intensity external
pulsed elds have been used to stimulate excitable
cells (Aidley 1998, Hille 2001), for electroporation
and for heating tissues in vivo (Prausnitz et al. 1993,
Jaroszeski et al. 2000). Furthermore, the orientation
of cell division is inuenced by small static electric
elds (Zhao et al. 1999).
Over two decades ago Goodman et al. (1983)
demonstrated that very weakly pulsing exogenous
electromagnetic elds (pulse amplitude around
0.15 V/m) induce the transcription of genes in
cultured salivary gland cells of the fruity. Likewise,
basic cellular phenomena such as growth, differ-
entiation, dedifferentiation, and repair have been
reported to be modied by weak electromagnetic
elds (Crombie et al. 1990, Panagopoulos et al.
2002, Dini and Abbro 2004). We note too that the
potential for genotoxicity of electric and magnetic
elds has been discussed by McCann et al. (1998)
in the context of a signicant body of genotoxicity
data. Finally, we draw attention to the fact that
exogenous EF interference is apparently high on the
agenda of biophysical and medical physics research
groups, as recently evidenced by the seminal studies
of Kirson et al. (2004, 2007), demonstrating that
low-intensity alternating elds can kill dividing cells
and slow the growth of brain tumors in cancer
patients.
To our knowledge there are no reported studies on
the combined effect of static EF and ionising
radiation. Here we report the results of studies using
both prokaryote and eukaryote organisms, with an
emphasis on possible synergistic roles played by these
two physical stresses at the cellular level. The
examination of both prokaryotic and eukaryotic
models was driven by several considerations:
(1) Many DNA repair mechanisms in bacteria are
conserved in eukaryotes. Hence, a comparative
study of their responses to combined ionising
radiation and static EF exposure is both
relevant and opportune.
(2) Two of the three organisms selected for study
are both endowed with high levels of radio-
resistance. Using genetically well dened radio-
sensitive organisms such as E. coli and the yeast
S. cerevisiae would likely introduce complexities
associated with additive cytotoxicity.
Finally, we decided to add more evidences on the
possible role played by an exogenous electric eld on
DNA repair mechanisms, vis-a` -vis the results ob-
tained with these prokaryotic and eukaryotic models,
by carrying out an experiment with human cells
where the amount of nuclei with g-H2AX foci were
quantied.
Materials and methods
Cell culture and density counting
(A) Microcystis panniformis BCCUSP100 strain (Cy-
anobacteria) was grown in BG-11 medium (after
a prescription found in Rippka et al. 1979) for a
14:10 hours (light:dark) photoperiod, at 22 +0.58C
with an intensity of 30 +2 mmol photons m
72
s
71
.
Prior to the beginning of the experiments, the pre-
cultures (exponential growth phase) were divided in
10 ml samples and inoculated in 30 ml of new
medium in triplicate. Each total volume of 40 ml was
housed in a glass tube. The cultures were initiated
with 2.6 610
6
cells.ml
71
and incubated as de-
scribed above. The irradiations were carried out
with a
60
Co gamma source facility (Gammabeam,
model 650 from MSD Nordion, Otawa, Canada).
All cell samples in glass tubes were irradiated with
doses in the interval 0.55 kGy at a rate of 0.94 kGy/
h. Sets of three glass tubes previously irradiated were
exposed for 2 h to a 20 V cm
71
static electric eld
between the plates of a capacitor, immediately after
irradiation. The control tubes were exposed either
only to irradiation or only to the static electric eld.
Total cells were enumerated by microscopic
counts of culture samples stained with Lugols 4%
solution in a Fuchs Rosenthal haemacytometer
(Optik Labor, Friedrichsdorf, Germany).
The average number of counted cells ranged from
6001000 in the rst and second days after irradia-
tion. It should be noticed that a total of 400 counted
cells is required to obtain an error of *10% at a
condence level of 95% (Guillard 1973). In this
sense, errors obtained in this work were considerably
better than 10% for each single sample. All
measurements were performed in triplicate. The
results were averaged, and it was found that their
dispersion did not exceed 10% (the same was
veried in the case of C. albicans).
(B) Candida albicans (strain ICB-12-A) was
inoculated in a tube containing Sabouraud agar,
incubated at 368C for 20 h, gamma irradiated with
doses from 14 kGy, and then submitted to a static
electric eld with net intensity (inside the medium)
equal to 180 V cm
71
for 1 h and 30 min. A total of
1 ml of the culture was diluted in 200 ml of PBS
(Phosphate Buffered Saline), to which 200 ml of
ethidium bromide (Sigma, St Louis, MO, EUA) and
200 ml of uorescein diacetate (Sigma) were added.
A drop of the suspension was set between slide and
cover slip and examined under a uorescence
microscope (Leika, DMLB, Wetzlar, Germany),
coupled to a digital camera (Hitachi, KCP-D581,
color, Tokyo, Japan). The number of unviable (red)
and viable (apple green uorescence) cells was
counted by means of the fungal-cell viability method
in a solution of uorescein diacetate (Calich et al.
Electric interference in viability of irradiated cells 315
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1978). One hundred uorescent cells were counted
on each prepared slide, three counts were performed
for every sample, and the results are presented as
averages of these measurements.
Peculiarities of the elected counting techniques
We used microscopy techniques to count the
number of total cells, which include those that might
divide and those that will die after the treatment.
In the case of M. panniformis, the counting was
carried out by direct optical recognition of total cells
in the sample solutions. In order to discriminate
those that might divide from those that will die, cell
growth was monitored during 10 days as a criterion,
since in the 10th day the cell titer decreased by three
orders of magnitude (fraction 10
73
).
With C. albicans, intact cells were identied by
their green color, since those with red color present
membrane rupture. However, some of the cells
turning green could have already suffered clonogenic
death.
Therefore, in both cases our results represent
lower limits of the observed effect, that is, the effect
should be more intense. However, lower limits are
appropriate for the purposes of the present work.
Human cells MRC5 strain of lung cells
Plated cultures of these cells were irradiated with
2 Gy and immediately exposed to a static electric
eld of 1250 V/cm. Besides all the conventional
procedures, the cells were incubated overnight with
the anti-body anti-phospho-histone H2AX (Billerica,
MA, USA) dilution 1: 200. The cell plates were
covered with 1 mg/ml of DAPI (4
0
, 6
0
-diamidino-2-
fenilindol-Invitrogen), examined under a uores-
cence microscope (Zeiss model Axiovert 200
1000 X. Go ttingen, Niedersachsen, Germany), and
nuclei with g-H2AX foci were quantied using the
program IMAGEJ 1.38. It should be noted that given
both the much higher complexity and the huge
genome size of the mammalian cells, a more intense
electric eld was used.
Results
Cellular survival in Candida albicans and Microcystis
panniformis
The survival of C. albicans cells following exposure to
ionising radiation with and without an exogenous EF
is shown in Figure 1, in which the logarithm of the
viable cells fraction (S) is plotted as a function of
gamma ray dose (D). The survival curve following
irradiation exhibits the well-known initial DNA
repair shoulder, characterised by a slow decrease of
viable cells, followed by a faster decrease at doses
greater than *2 kGy. However, when irradiated
cells were exposed to static EF the shoulder in the
survival curve disappeared (Figure 1). The number
of viable cells in non-irradiated control samples was
Figure 1. Survival curves of C. albicans as a function of g-rays doses in the range 04 kGy. Irradiated samples were also submitted to a static
electric eld of 180 V cm
71
(net intensity inside the medium) for 1 h and 30 min. Open circles irradiation only. Full circles irradiation
plus application of the electric eld (EF). The ordinate axis provides the logarithm of the viable cells fraction (S). This fraction is given by
SN/N
0
, where N represents the number of cells surviving after irradiation (or after irradiation plus electric eld), and N
0
is the number of
cells before irradiation. Results for control samples (zero dose) are represented by an open square (without EF application) and by a full
square (with EF application). The error bars indicate the standard error of the mean (SEM) for three independent experiments.
316 J. D. T. Arruda-Neto et al.
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unchanged following the application of a static EF
(Figure 1).
Similar to the results observed with C. albicans, the
survival of M. panniformis following exposure to
irradiation alone also exhibits an initial shoulder and
a rapid decrease above *2 kGy (Figure 2). How-
ever, in contrast to C. albicans, application of a static
EF to the irradiated cells reduced but did not
completely abolish the DNA repair shoulder com-
ponent of the survival curves (Figure 2).
Growth kinetics of M. panniformis
In addition to the studies of survival in C. albicans
and M. panniformis as a function of dose, we
examined the effects of EF interference on the
growth of M. panniformis as a function of incuba-
tion times up to several days, at a xed dose of
3 kGy. This was facilitated by the availability of a
unique method to measure cell growth in this
organism (see Materials and methods). As shown
in Figure 3A, the application of an EF (20 V/cm)
did not affect cell growth in control experiments.
The average of the relative differences between the
Figure 2. Survival curves of M. panniformis as a function of g-rays
doses in the range 05 kGy. Irradiated samples were also
submitted to a static electric eld of 20 V cm
71
(net intensity
inside the medium) for 2 h. Full squares irradiation only. Full
circles irradiation plus application of the electric eld (EF).
Results for control samples (zero dose) are in Figure 4A. The error
bars are of the size of the data points, and they indicate the SEM
for three independent experiments.
Figure 3. Growth curves of M. panniformis. (A) Growth curves for the control and control plus the application of an electric eld, as function
of the incubation time. (B) Growth curves following g-rays irradiation with a single dose of 3 kGy, with and without the subsequent
application of an electric eld. (C) Ratio between the two data sets shown in (B). The ordinate axis of Figure 2A (and in Figure 2B) provides
the fraction of the cells. The 0th day corresponds to the beginning of the experiment. The lines connecting the data points are only to guide
the eyes. The error bars are of the size of the data points, and they indicate the SEM for three independent experiments.
Electric interference in viability of irradiated cells 317
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two sets of data points shown is negligible. However,
when the same EF was applied for 2 h immediately
following radiation exposure, there was a signicant
decrease in cell growth (Figure 3B). This result is
better appreciated in Figure 3C, where the ratio of the
two data sets in Figure 3B is plotted.
Cell death rate of M. panniformis
The cell death rate, the number of killed cells per unit
of time, can be dened as r
de
(t) DN
de
(t) /Dt , where
DN
de
(t) N(t) N(t Dt) is the number cells killed
in the time interval Dt, and N(t) is the number of
cells of M. panniformis measured at time t (in days)
Figure 3. Considering Dt 1 day, we have r
de
(t)
DN
de
(t) Figure 4.
Our denition for r
de
refers to the mean cell death
rate in the time interval from t to t Dt, yielding an
observation time of t Dt/2. The cell death rate is a
useful quantity to identify since it provides the rate at
which cells are killed daily.
Quantication of nuclei with g-H2AX foci in MRC5
strain of lung cells
Fluorescence results for the MRC5 cells are shown
in Figure 5. We observe that the non-subjective
quantication of the g-H2AX foci, carried out by
means of the routine IMAGEJ 1.38, provided
statistical counting error equal or smaller than 10%.
We note in Figure 5 that the number of g-H2AX
foci counted in cells growing under normal condi-
tions (378C, 5% CO
2
) was equal to the one when
these cells were submitted to a static electric eld of
1250 V/cm by 1 h, within the statistical error.
On the other hand, in cells irradiated with 2 Gy
and submitted to a static electric eld of 1250 V/cm
by 1 h, the number of g-H2AX foci was nearly 40%
higher than in cells only irradiated with 2 Gy.
Discussion and conclusion
Choice of dose and electric eld exposure time
Figure 3B reveals that for 3 kGy of ionising radiation
exposure, about 80% of cells of the bacterium M.
panniformis counted at t 24 h have disappeared by
the following day (t 48 h). We conclude that 3 kGy
is an appropriate dose to consider because the 1/e
( 0.37) fraction lies between the two most im-
portant observation days (the rst and the second
days). It is also relevant to point out that the dose
corresponding to 37% of survivors is equal to (1/
a) 6100, where a is the inactivation factor, since this
represents the dose required to deliver one inactivat-
ing event per cell (Alpen 1990). Regarding the
optimal duration of exposure to a static EF, we
determined that 2 h was sufcient to hinder cell
recovery processes, since longer exposure time did
not increase cell death.
Puzzling results and questions
The data presented in Figures 1 and 2 reveals the
following relevant and surprising results: (a) Deple-
tion of the so-called repair shoulder in the survival
curves of cells exposed to both irradiation and an EF
demonstrates that the EF substantially increased the
radiosensitivity of the organisms examined. For C.
albicans, in particular, this effect is similar to that
resulting from exposure to dense ionising radiations
such as neutrons and alpha particles, which generate
clusters of double strand breaks in DNA (Alpen
1990). In contrast, for M. panniformis the shoulder is
depleted but not suppressed; and (b) For the
prokaryote M. panniformis the effect of EF on cell
growth is profound. As it can be seen in Figure 3C,
Figure 4. Cell death rates of M. panniformis for the same two
experimental situations shown in Figure 3B see text for details.
The error bars are of the size of the data points, and they indicate
the SEM for three independent experiments.
Figure 5. Fluorescence results for MRC5 cells. Control: cells
growing under normal conditions (378C, 5% CO
2
). Control EF:
cells growing under normal conditions 1250 V/cm of static
electric eld by 1 h. 2 Gy (1 h): cells irradiated with 2 Gy and then
cultivated by 1 h under normal conditions. 2 Gy (1 h) EF: cells
irradiated with 2 Gy 1250 V/cm of static electric eld by 1 h.
318 J. D. T. Arruda-Neto et al.
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the number of surviving cells on the second day after
irradiation was more than 12 times greater than when
an EF was applied.
These observations pose the following relevant
questions:
(1) Why does a static EF have no cytotoxic effect
on cells, but when associated with exposure to
radiation cytotoxicity increases substantially?
(2) What is the mechanism by which DNA repair
(reected in the shoulder of the survival curves
for both C. albicans and M. panniformis) is
depleted when cells are exposed to a non-
cytotoxic agent, like a static electric eld?
Answers and a model proposition
With respect to question 1, rst phrase, it is well-
known that static EF of the intensities used in this
study are not cytotoxic. As noted in the literature for
example, electroporation is only achieved with high-
intensity external pulsed elds (from several hundreds
to thousands of V/cm (see e.g., Prausnitz et al. 1993,
Jaroszeski et al. 2000). The answer to question 2 (and
question 1, second phrase) on the other hand, led us
to the proposition of a model, as depicted below.
Double strand breaks (DSBs) generate quadru-
pole-like static electric elds, as revealed by experi-
ments of perturbed angular correlations of gamma-
rays (PAC), an alternative and elegant experimental
technique to study the molecular dynamics of DNA
(Kalfas et al. 1984, 1994). These static electric
(quadrupole) elds persist until completion of repair.
Because of e.g. the presence of salt ions in the
cytoplasm, one would argue that the net charge at
each DSB site is gradually screened during the
signaling-repairing time interval. However, although
undergoing reorientation and attraction by the
electric eld toward the DSB site, ion movement is
not propelled as in a repair protein. Therefore, while
the former moves diffusively, the latter moves
processively (Okada and Hirokawa 1999).
Our model states that the electric dipole of the
repair proteins senses the endogenous static electric
eld at the damage and uses it as a navigation cue.
This is consistent with the fact that repair proteins
can locate damaged sites within seconds after double
strand break (DSB) formation (Jakob et al. 2005),
although those sites are constituted by only a few
base pairs in the entire genome. Also, our premise
provides answer to the long-standing question of
Bartek and Lukas: How do proteins throughout the
cell nucleus respond in a coordinated way, and how
are a few DSBs within three billion base pairs
recognised? (Bartek and Lukas 2003).
When irradiated cells are exposed to an exogenous
EF stronger than the endogenous EF, repair proteins
would tend gradually to align their electric dipoles
with the direction of this exogenous EF, as pictorially
represented in Figure 6. Consequently, the majority
of these proteins would be unable to reach damaged
sites in DNA. Such a possibility is corroborated by
our results for the H2AX histones (Figure 5). In fact,
these results show that by the action of a static
electric eld on the irradiated cells the number of
nuclei with g-H2AX foci increased 40%, approxi-
mately. This indicated that the static electric eld
impeded the action of 40% of the H2AX histones
Figure 6. (a) Pictorial representation of DSB recognition by repair proteins (our hypothesis). These proteins usually have huge electric dipole
momenta p
!
(represented in [b] with more details), which are oriented toward the damaged site by the static electric eld E
!
ds
produced by
the electric imbalance at the strand breaks. (c) An external static electric eld E
!
ext
stronger than E
!
dss
would reorient the repair proteins
displacement all along its direction.
Electric interference in viability of irradiated cells 319
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pool initially recruited for the DNA damage sites. In
fact, they were still phosphorilating, indicating that
they were not used in the repair nal process. Also,
since 2 Gy is a dose near the repairing shoulder of the
MRC5 cells, this result is an additional evidence that
the electric eld action is more effective at doses
where cells are much more repair efcient.
Thus, many of the damages were not repaired
because the H2AX histones are crucial players of the
repairing cascade. Since a single DSB is lethal if
unrepaired (Friedberg 2003), the predictable nal
consequence for a persistent high-level of unrepaired
DSBs would be cell death, as suggested by the
experimental results obtained in this study.
The increasing cytotoxicity in C. albicans and M.
panniformis, when irradiation is combined with ex-
posure toa static EF, is expectedsince the fundamental
mechanisms of DNA repair are similar in both
microorganisms. However, the most striking observa-
tion is shoulder suppression in C. albicans compared to
shoulder depletionM. panniformis. Froma radiological
point of view, suppression of the DNArepair shoulder
component of the survival curve reects the persistence
of DSBs. At the lower-dose regime (shoulder region of
the survival curve) the concentration of damage
inicted to the DNA, for a given dose, is proportional
to the amount of targeting material (base pairs)
exposed to the radiation more precisely, the effective
radiation interaction cross section. In this sense, our
results suggest that damage clustering may be higher in
C. albicans compared to M. panniformis. This is
consistent with the fact that in prokaryotes the genome
is constituted by a single circular DNA molecule,
about a millimeter in length, while in eukaryotes
genomic DNA is densely wrapped as chromatin.
Our ability to monitor cell death rate for M.
panniformis was facilitated by the availability of appro-
priate methodology (see Materials and methods). While
following irradiation alone the average cell death rate
from the 0th to the rst day is roughly r
de
(0.5)
0.35 6N(0), i.e., the initial cell populations N(0)
was killed at a rate of about 35% per day. In the case
of irradiation plus EF we obtained r
de
(0.5)
0.75 6N(0), i.e., 75% of the total N(0) is killed per
day. Besides its massive killing rate, the combinationof
irradiation plus EF is considerably faster, exhibiting a
peak at t 0.5d. Actually, 3/4 of the entire cell
population is killed in the rst 12 h (t 0.5d),
explainingwhytheratedrops soabruptlyinsubsequent
days. In contrast, when only irradiation is applied, the
death rate peaks at about 2436 h (t 11.5 d).
In conclusion, the results of these studies demon-
strate that the application a static EF following
exposure of both radioresistant prokaryotic and
eukaryotic cells to ionising radiation greatly increases
cell death. The effect is similar to that observed when
a highly ionising radiation is used. Our results also
suggest that EF likely act by inactivating cellular
DNA repair processes. In fact, the observation of a
40% surplus of nuclei with g-H2AX foci in MRC5
strain of lung cells, when irradiated and exposed to
an intense EF, points into this direction.
Acknowledgements
We are very grateful to the key collaboration of Prof.
Carlos F. Martins Menck and Dr Luis F. Zirnherger
Batista, particularly by suggesting, discussing and
giving support to the experiment with the histone
H2AX. This work was supported by grants from
FAPESP and CNPq, Brazilian agencies for the
promotion of science.
Declaration of interest: The authors report no
conicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
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