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Characteristic 8 keV X Rays Possess Radiobiological Properties of Higher-LET

Radiation
Author(s) :Ravi Shridhar, William Estabrook, Mark Yudelev, Joseph Rakowski, Jay Burmeister, George
D. Wilson, and Michael C. Joiner
Source: Radiation Research, 173(3):290-297. 2010.
Published By: Radiation Research Society
DOI: http://dx.doi.org/10.1667/RR1782.1
URL: http://www.bioone.org/doi/full/10.1667/RR1782.1

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RADIATION RESEARCH

173, 290297 (2010)

0033-7587/10 $15.00
g 2010 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR1782.1

Characteristic 8 keV X Rays Possess Radiobiological Properties of


Higher-LET Radiation
Ravi Shridhar,a William Estabrook,a Mark Yudelev,b Joseph Rakowski,a Jay Burmeister,a George D. Wilsonc and
Michael C. Joinera,1
a

Wayne State University, Detroit, Michigan; b Mt. Clemens General Hospital, Mt. Clemens, Michigan; and c William Beaumont Hospital,
Royal Oak, Michigan

(,50 keV) X-ray sources are typically contained within


the tips of needles that are inserted into the tissue to be
irradiated. Multiple needles may be used in the same way
that multiple catheters are used in remote afterloading.
Key advantages of these systems over radioisotopes are
their ability to provide selective and precise control of the
radiation exposure (turn-on and turn-off), the comparatively low cost of the apparatus, and the ability to
perform precision radiotherapy in the operating room
with lower collateral radiological exposure to patients
and staff and less hazard to the environment.
Gutman et al. (1) have developed an electronic
brachytherapy technique that uses a two-stage production of X rays. A beam of low-energy (typically 22 keV)
X rays is produced externally by a conventional compact
portable X-ray tube, then collimated through a tube
with conditioning optics onto a secondary target
embedded in a needle tip or other applicator. This
produces a fluorescent X-ray beam that irradiates the
surrounding tissue. A further feature of this X-ray
needle system is the ability to vary the intensity and
shape of the X-ray output from the needle by altering
the energy and intensity of the primary X-ray beam and
the target shape. Also, by changing the secondary target
material, it is possible to vary the energy of monochromatic fluorescent X-ray output from 521 keV.
In this lower-energy X-ray spectrum, the radiobiology
is not as well defined as for the high-energy external X-ray
beams that are more widely used in radiotherapy. At even
lower energies (,1.5 keV), a series of papers by Raju,
Schillaci, Carpenter, Goodhead and colleagues (24)
reported on cell survival after irradiation by ultrasoft X
rays in hamster V79 and mouse 10TK cells. The notable
conclusions from this work were that with decreasing Xray energy, relative biological effectiveness (RBE) could
increase and oxygen enhancement ratio (OER) could
decrease, which are both characteristics of higher-LET
radiations. Hill et al. (5) found an RBE of 1.8 for copper
L characteristic X rays (956 eV) relative to hard X rays in
the cell survival response of V79-4 hamster cells. More
recently, Endo et al. (6) reported the frequency of

Shridhar, R., Estabrook, W., Yudelev, M., Rakowski, J.,


Burmeister, J., Wilson, G. D. and Joiner, M. C. Characteristic
8 keV X Rays Possess Radiobiological Properties of HigherLET Radiation. Radiat. Res. 173, 290297 (2010).
Electronic brachytherapy systems are being developed that
can deliver X rays of varying energy depending on the material
of a secondary target. A copper target produces characteristic
8 keV X rays. Our aim was to determine whether 8 keV X rays
might deliver greater biological effectiveness than megavoltage
photons. Cells of the U251 human glioma cell line were used to
compare the biological effects of 8 keV X rays and 60Co c rays in
terms of relative biological effectiveness (RBE), oxygen
enhancement ratio (OER), and DNA damage. The RBE at
50% and 10% survival was 2.6 and 1.9, respectively. At 50%
survival, the OER for cells treated with 8 keV X rays was 1.6
compared with 3.0 for 60Co c rays. The numbers of H2AX foci
per Gy after treatment with 8 keV X rays and 60Co c rays were
similar; however, the size of the foci generated at 8 keV was
significantly larger, possibly indicating more complex DNA
damage. The mean area of H2AX foci generated by 8 keV X
rays was 0.785 mm2 (95% CI: 0.7560.814) compared with
0.491 mm2 (95% CI: 0.4620.520) for 60Co c rays (P < 0.0001).
Characteristic 8 keV X rays produce two to three times the
biological effectiveness of megavoltage photons, with a radiobiological profile similar to higher-LET radiations. g 2010 by Radiation
Research Society

INTRODUCTION

Electronic brachytherapy is gathering considerable


interest, because it could reduce the dependence of this
important cancer therapy on radionuclide sources, the use
of which has become more difficult, costly and inconvenient to the patient due in part to the increased regulatory
burden arising from concerns over national radiological
security. In electronic brachytherapy, miniature Xray sources replace radionuclides. These low-energy
1 Address for correspondence: Gershenson Radiation Oncology
Center, Wayne State University, 4100 John R, Detroit, MI 482012013; e-mail: joinerm@wayne.edu.

290

RADIOBIOLOGY OF 8 keV X RAYS

291

dicentric formation in mouse m5S cells exposed to


characteristic X rays from copper (8 keV), iron
(6.4 keV), chromium (5.4 keV), molybdenum (2.3 keV),
aluminum (1.5 keV), carbon (0.28 keV) and higher-energy
X-ray sources of 50 kVp, 200 kVp, 137Cs (662 keV) and
60
Co (1.25 MeV). At a dose of 2 Gy 8 keV X rays, RBE
relative to 60Co was approximately 2. However, fitting the
dicentric frequency to a linear-quadratic dependence on
dose, the linear and quadratic terms both varied rapidly
and in opposite directions in the energy range from 10
down to 1 keV, indicating a rapidly changing relationship
between RBE and dose in this energy range.
Therefore, while it is known that the RBE of X rays in
causing cell killing and chromosomal aberrations
increases with decreasing X-ray energy, the published
data have varied considerably, and therefore RBE
cannot yet be predicted reliably at the X-ray energies
used in electronic brachytherapy. In this paper, we
report the radiobiological characteristics of 8 keV X rays
with regard to RBE, OER and DNA damage compared
with c rays from a clinical 60Co irradiator (mean energy
1.25 MeV) in cells of a human glioblastoma cell line.
Glioblastoma could be a potential clinical target for lowenergy intraoperative brachytherapy, which might enable higher biologically effective doses to be delivered to
this intractable disease.
MATERIALS AND METHODS
Irradiations
Cells were irradiated at room temperature. For treatment with
megavoltage photons, cells were irradiated with a clinical 60Co
Theratron teletherapy unit (MDS Nordion, Ottawa, Canada) at a
dose rate of 20 cGy min21, chosen to match the dose rate available for
kilovoltage photon irradiation. For treatment with kilovoltage
photons, a 2.7 kW Seifert X-ray tube operating at 58 kVp and
25 mA was used to generate a primary beam of 22 keV characteristic
X rays from a silver anode (1) that then bombarded a copper target,
generating nearly monochromatic copper K characteristic X rays with
an energy of 8.04 keV (Fig. 1A), comprising Ka1 at 8.048 keV and
Ka2 at 8.028 keV.
Dosimetry
For 60Co c-ray exposure, dosimetry was carried out with a 0.6-cm3
Farmer-Baldwin chamber with calibration traceable to the National
Institute of Standards and Technology (NIST). The relative dose
within all 60Co irradiation fields varied by no more than 2% maximum
to minimum.
For exposure with 8 keV X rays, dosimetry at the sample position
was performed with an extrapolation chamber (Far West Technology
Inc., model EIC-1). This is a parallel-plate, guarded three-chamber
design with a 1-cm-diameter sensitive volume and plate spacing that is
continuously variable from 0.3 mm to 4.5 mm. The entrance window
is polyethylene at 6.9 mg cm22. The ion chamber currents were
measured with a Keithley model 35617EBS electrometer.
The dosimetry calculation model used was a combination of Burlin
theory and Klevenhagens model for measurements with a gradient
chamber (79). Thus absorbed dose to water was determined by the
equation

FIG. 1. Setup for exposure to low-energy X rays (panel A). A


primary beam of 22 keV X rays from a silver anode bombards a
secondary copper target producing 8 keV characteristic X rays.
Dosimetry and field flatness were measured by an extrapolation
ionization chamber and GafchromicTM film, parallel (panel B) and
perpendicular (panel C) to the primary beam.
_ 
PTP
: 1 :
_ w ~ DQ : W
, 1
D
 a z 1 { d : m
e a Ar d : S
DI
en =raw
w
:
:
where Dw is the absorbed dose rate in water, D Q=DI is the
interpolated slope of charge rate per unit electrode separation,

W =e a is the mean energy expended in air per ion pair produced, A is
the chamber active area (determined from cross-calibration in the
60
Co beam), r is the air density, Swa is the average stopping power ratio
of air to water, men =raw is the mass-energy absorption ratio of air to

292

SHRIDHAR ET AL.

water, PTP is the pressure and temperature correction, and d is a


parameter related to cavity size, expressed by the following equation:
d~

1 { e{bL
,
bL

supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM Lglutamine, and non-essential amino acids (MediaTech, Cellgro). For
routine maintenance, cells were grown as a monolayer at 37uC under
5% O2 and 5% CO2 (balance N2) and subcultured once or twice
weekly to maintain exponential growth.

where b is a parameter that satisfies the relationship e{b tmax ~0:04, L


being the mean chord length of the cavity, which is four times the
cavity volume divided by its surface area, and tmax the maximum depth
of electron penetration to which only 1% of the electrons can travel in
the cavity, taken as 95% of the continuous slowing down
approximation (CSDA) range of electrons at their maximum energy
transferred from the characteristic X rays. The values for
a
S w , men =raw , and CSDA were taken from NIST data. The factor d
accounts for the effect of the average reduction in the wall
spectrum and the average buildup of the gas spectrum throughout
the volume of the cavity on the stopping-power ratio.
The absorbed dose to water at the ion chamber window was
determined by extrapolating the chamber volume and the wall
thickness to zero. The wall thickness was varied by adding layers of
A-150 tissue-equivalent plastic for a total of seven distinct thicknesses, from 6.9 mg cm22 (chamber window alone) up to 16.56 mg cm22.
The extrapolation chamber volume was varied using the electrode
spacing. A least-squares regression of charge collected per minute
relative to wall thickness was made for each electrode spacing. The y
intercept of each regression was taken as the charge rate for zero wall
thickness. The slope of the regression of charge rate at zero: wall
thickness as a function of electrode spacing was then taken as D Q=DI.
The denominator in Eq. (1), equal to the Burlin ratio Da =Dw , was
calculated based on the average of four chamber electrode separation
distances of 1, 2, 3 and 4 mm. Absorbed dose rate in water for each
wall thickness was determined using Eq. (1).
An exponential function of absorbed dose rate based on mass
thickness is expressed as
_ (rt) ~ D
_ w : e{(men =r) : rt ,
D

where rt is the wall mass thickness, men =r is the mass energy


absorption coefficient of the wall material for the characteristic
:
photon energy, and Dw is the extrapolated absorbed dose rate in
water without the extrapolation chamber wall influence.
The dose variation across the irradiation field was determined using
GafchromicTM EBT film. A calibration curve was created by exposing
the film to a range of 8 keV X-ray doses at the face of the extrapolation
chamber. Scan profiles parallel and perpendicular to the primary beam,
at a nominal dose of approximately 1 Gy, at the position at which cells
were irradiated, are shown in Fig. 1B and C. Due to the inherent noise
in GafchromicTM film, trend lines were fitted to these data to clarify the
profiles. The 1-cm-diameter sensitive volume of the extrapolation ion
chamber was centered in the profiles; therefore, film calibration data
were measured in a circular region 1 cm in diameter at the position of
the sensitive volume. The film was read with a Microtek ScanMaker
i800 transmission flatbed scanner and analyzed using ImageJ version
1.383 from the National Institutes of Health.
Finally, the dose of 8 keV X rays to the actual cell cultures was
measured by irradiating appropriately sized GafchromicTM film disks
in contact with the bottom of the inside of the culture dish (for
Permanox dishes) or well (for 96-well plates), covered with culture
medium, at the exact position of the cells but without actual cells
present. The film irradiation times used were similar to the actual cell
culture irradiation times. The dose rate of 8 keV X rays to the cell
cultures was 20 cGy min21.
Cell Lines
U251 cells (human glioma) were a gift from Dr. Tom Mikkelson,
Henry Ford Hospital, Detroit, MI. U251 cells were grown in DMEM

Clonogenic Cell Survival Assay


Immediately after exposure to megavoltage or kilovoltage radiation, cells were trypsinized, counted and plated into fresh growth
medium at appropriate numbers to give rise to about 100 colonies per
dish. Six dishes were used per dose, and experiments were repeated
three times. Cells were incubated under standard growth conditions
for 10 days and the resultant colonies were stained with 2% crystal
violet in 95% ethanol. Those colonies consisting of greater than 50
cells were scored as representing surviving cells using ColCount
[Oxford Optronix Ltd., Oxford, UK (10)]. Surviving fractions were
calculated from these data by reference to the mean plating efficiency
of at least six sham-irradiated control plates.
Hypoxia
For experiments comparing irradiation under air and anoxia, cells
were plated on 60-mm Permanox dishes (EMB biosciences) and
irradiated at 50% confluence. Dishes were placed inside a sealed
flexible plastic chamber with two open ports. For irradiation with
8 keV X rays, the plastic chamber also enclosed all of the irradiation
apparatus (primary X-ray beam, copper target and aluminum tray;
see Fig. 1A). One port allowed gas inflow and the other port allowed
atmospheric gas to escape. For hypoxic conditions, cells were exposed
to 100% nitrogen gas at an excess pressure of 2 PSI with a flow rate of
20 liters min21 for 1 h prior to irradiation. Nitrogen gas flow was
maintained during hypoxic radiation treatments. After irradiation,
cells were immediately harvested for clonogenic survival assays. For
irradiations with 8 keV X rays, cells were restricted to a circular area
of 20 mm within the Permanox dish, achieved by initially plating
within a small volume of liquid spread restricted to this area and then
adding additional culture medium after cell attachment had occurred.
This was done to reduce dosimetric variation to less than 9% in these
low-energy irradiations.
Fluorescence Microscopy
After irradiation at doses of 0.5 and 1 Gy for each energy, cells
were incubated for 1 h at 37uC and then fixed with 2%
paraformaldehyde/PBS for 15 min on ice, washed twice with PBS,
and permeabilized with 0.5% Triton-X-100/PBS for 2 min at room
temperature. The cells were then blocked with 3% BSA in Tris
buffered saline (TBS) for 1 h at room temperature. After washing, the
cells were incubated for 1 h at room temperature with anti-phosphohistone H2AX mouse monoclonal antibody (Millipore) at a 1:500
dilution in TBS/1% BSA. The cells were then washed again and
incubated with goat anti mouse-Alexa Fluor 488 diluted 1:5000 in 1%
BSA/TBS for 1 h at room temperature (Invitrogen). Finally, cells were
rinsed and dropped onto slides in ProLong Antifade kit (Invitrogen),
covered with cover slips, and stored at 4uC in a slide box until
analysis.
Analysis of Foci
Foci were analyzed using a Nikon 80i Eclipse microscope at 4003
magnification. Images were captured using a Coolsnap CF 36 Bit,
20 MHz Digital Color Camera with IEEE-1394 interface. Images of
individual cells were analyzed using a custom macro developed in
Image Pro Plus Version 6.2 (MediaCybernetics). The macro used the
following image parameters: a 5 3 5 Laplace edge filter, green
channel extraction to produce a grayscale image, and manual
adjustment of segmentation using the histogram-based model. The

293

RADIOBIOLOGY OF 8 keV X RAYS

TABLE 1
Parameters (a, b) in the Fit of the
Linear-Quadratic Model to Cell Survival as a
Function of Dose of 60Co c Rays or 8 keV
X Rays (Fig. 2)
Radiation

Parameter

Value

SEM

Co c rays
Co c rays
8 keV X rays
8 keV X rays

a
b
a
b

0.109
0.0411
0.529
0.0540

0.011
0.0023
0.023
0.0073

60
60

Lower CL Upper CL
0.085
0.0346
0.465
0.0337

0.133
0.0447
0.592
0.0752

Notes. Surviving fraction 5 exp(2a/D 2 b/D2). Uncertainty is


given as standard error of the mean (SEM) and as lower and upper
95% confidence limits (CL).

FIG. 2. RBE of 8 keV X rays. Comparison of cell survival curves


generated from clonogenic assays of U251 cells irradiated with 60Co c
rays (#) or 8 keV X rays ( ). Data points are means SEM.

resulting numerical data, including number and area, were subsequently exported to Excel (Microsoft) and analyzed further using
JMP Statistical Analysis Software version 6.03 (SAS). Ten to 15
nuclei were scored per dose and energy per individual experiment.
Each experiment was repeated three times.

RESULTS

To determine the RBE, we measured clonogenic cell


survival after irradiation with increasing doses of 60Co
(1.25 MeV) c rays and 8 keV X rays. Cell survival curves
were established from the combined data from up to six
irradiations per dose for each energy in experiments
repeated three times (Fig. 2). The linear-quadratic (LQ)
model of cell survival as a function of dose (11) was
fitted to the data using non-linear least-squares regression (SAS JMP version 7). Table 1 shows the values of a
and b in the LQ model. The surviving fraction after a
dose of 2 Gy (SF2) of 60Co c rays was 0.68, indicative of
a radioresistant cell line, and was 0.28 for 8 keV X rays,
indicating substantial radiosensitization. The survival
curve for 60Co c rays exhibits a larger shoulder (a/b 5
2.65) compared with 8 keV X rays (a/b 5 9.80), so that
RBE will increase with increasing surviving fraction or
decreasing dose. The LQ parameters in Table 1 allow
the direct calculation of RBE at any effect level or dose
of either radiation (12). At surviving fractions of 0.05,
0.1, 0.25 and 0.5, the RBE is 1.82, 1.92, 2.15 and 2.55,
respectively, and the RBE is 2.96 at a c-ray dose of 2 Gy.

We then measured clonogenic survival after irradiation with increasing doses of 60Co c rays and 8 keV X
rays under hypoxic and normally oxygenated conditions. Cells grown on Permanox dishes were exposed to
either 100% nitrogen or normal atmosphere for 1 h in a
hypoxic chamber at room temperature and irradiated
with increasing doses of either megavoltage or kilovoltage X rays. Cell survival curves from combined data
from six irradiations per dose for each energy were
established (Fig. 3). The LQ model of cell survival as a
function of dose was fitted to the data; Table 2 lists the
values of a and b under nitrogen or normal atmosphere,
allowing the calculation of OER at any level of effect or
dose. The OER at 50% survival, was 2.97 and 1.61 for
60
Co c rays and 8 keV X rays, respectively. For 60Co c
rays, the OER was 3.26 at 80% survival and dropped to
2.87 at 30% survival. For 8 keV X rays, the OER
increased as cell survival decreased from 1.43 at 80%
survival to 1.79 at 30% survival.
We compared DNA damage at 1 h after exposure to
60
Co c rays and 8 keV X rays by analyzing phosphoH2AX immunostaining after irradiation. There was no
significant difference in the number of phospho-H2AX
foci detected between 8 keV X rays and 60Co c rays (twotailed Students t test), and the number of foci detected
increased proportionally to the dose delivered (Fig. 4A).
We measured an average of 37.8 foci per gray per cell
produced by 8 keV X rays and 41.6 foci per gray per cell
produced by 60Co c rays (P . 0.25) (Fig. 4B). However,
the foci were significantly larger for 8 keV X rays
compared with 60Co c rays. The mean area of H2AX foci
generated with 8 keV X rays was 0.785 mm2 compared
with 0.491 mm2 for 60Co c rays (Table 3). Figure 5 shows
representative slides of radiation-generated H2AX foci,
illustrating the larger foci produced by the 8 keV X rays.
DISCUSSION

As linear energy transfer (LET) increases, radiation


produces more cell killing per unit dose (increased RBE),
cell survival curves become steeper and straighter with
less shoulder (higher a/b ratio), the DNA damage is more

294

SHRIDHAR ET AL.

TABLE 2
Parameters (a, b) in the Fit of the Linear-Quadratic
Model to Cell Survival as a Function of Dose
Delivered under Normal Atmosphere or Nitrogen
for 60Co c Rays (Fig. 3A) or 8 keV X Rays (Fig. 3B)
Radiation

SEM

Lower
CL

Upper
CL

Parameter

Value

Normal atmosphere
60
Co c rays
60
Co c rays
8 keV X rays
8 keV X rays

a
b
a
b

0.165
0.0447
0.489
0.0868

0.052
0.022 0.315
0.0165 20.0014 0.0919
0.050
0.351 0.631
0.0266
0.0117 0.162

Nitrogen
60
Co c rays
60
Co c rays
8 keV X rays
8 keV X rays

a
b
a
b

0.0412
0.00699
0.369
20.0018

0.0154 20.0015 0.0853


0.00189 0.00161 0.0124
0.016
0.324 0.415
0.0043 20.0141 0.0104

Notes. Surviving fraction 5 exp(2a/D 2 b/D2). Uncertainty is


given as SEM and as lower and upper 95% confidence limits (CL).

FIG. 3. OER of 8 keV X rays. Comparison of cell survival curves


generated from clonogenic assays of U251 cells irradiated under
hypoxic (N2, ) or normally oxygenated (O2, #) conditions for (panel
A) 60Co c rays or (panel B) 8 keV X rays. Data points are means
SEM.

difficult to repair, and the OER decreases (12). The RBE


of X rays should increase with decreasing photon energy
(13). However, even at similar energies, actual measurements of RBE vary widely according to the biological
system studied and the configuration and type of photon
source. RBEs as high as 3 have been reported previously
for low-energy X rays (14). In the present report, we have
confirmed that characteristic 8 keV X rays have a high
RBE, with a value of 2.96 at the SF2 of 60Co c rays. We
have also made the additional observations that 8 keV X
rays exhibit low OER values around 1.6, compared to
around 3 for megavoltage photons, and that 8 keV X rays
also generate more complex DNA damage as seen in the
larger H2AX foci. These observations parallel the greater
biological effectiveness of high-LET radiations compared
to megavoltage photons.
A fundamental difference between low-energy X rays
(below 18 keV) and high-energy X rays (above 50 keV) is
in the physical mechanism of absorption by tissue. At an
X-ray energy of 17 keV, for example, photoelectric
absorption is responsible for 75% of interactions in
water and only 25% of interactions are due to the
Compton effect, which dominates in megavoltage
therapy (15). At 10 keV, this proportion changes to
95% and 5%, respectively. Thus, in the case of 8 keV X
irradiation, the photoelectric process dominates over
other absorption processes.
RBE is defined as the ratio of doses given to achieve
the same biological effect. It depends on both the
energies and the doses of the radiations involved in the
comparison. Using high-voltage (.150 kV) X rays or
60
Co c rays as the reference, RBE increases with
decreasing energy and with decreasing dose or dose rate
of the radiation under scrutiny (12, 16). This situation is
further complicated because radiations may be monochromatic, which is sometimes the case for c-ray
emissions from radionuclides, or may comprise a

RADIOBIOLOGY OF 8 keV X RAYS

FIG. 4. Panel A: U251 cells were irradiated with 0.5 and 1 Gy of


Co c rays or 8 keV X rays and incubated for 1 h at 37uC. Cells were
then fixed and immunostained with anti-phospho-serine139 H2AX
antibody and foci were counted in 10 cells per dose for each energy.
Panel B: The number of H2AX foci normalized to dose. Bars depict
mean values 95% CL.

60

spectrum of energies around an average, which is the


usual situation for bremsstrahlung X rays. Thus it is not
possible to specify a single, definitive value of RBE for a
given energy of radiation. In V79 hamster cells, relative
to 60Co c rays, RBE values of 1.51.6 were measured at
an isoeffect cell survival level of 10% for soft X rays with
effective energies in the range 819 keV (17). The
corresponding 60Co c-ray dose is about 9 Gy in these
cells, and the RBE would be larger at a lower c-ray dose
of 2 Gy used in standard fractionated radiotherapy. At
doses less than 20 cGy, the RBE for soft X rays exceeds
3 for induction of dicentric chromosomes in human
lymphocytes (14). In radioresistant human cells (e.g.
glioblastoma) with typically lower a/b ratios compared
with V79 cells, the RBE should also be higher. These
and other studies therefore suggest that RBE values at
clinically relevant doses of soft X rays might be between
2 and 3, which is confirmed in our experiments using
human glioblastoma cells.

295

In this study, we have demonstrated that 8 keV X rays


have an OER of 1.41.8 depending on the surviving
fraction, with a trend toward lower OER at higher cell
survival. Using the same experimental protocols and cell
line, the OER for 60Co c rays was 2.93.3 with a slight
trend toward higher OER at higher survival. This
magnitude of reduction in OER for the 8 keV X rays
is similar to that expected with radiations of substantial
LET (.50 keV/mm), as described by Barendsen (18). A
reduction in OER at higher cell survival as demonstrated
by Palcic et al. (19) is also expected, especially with
higher-LET radiations, because at lower doses the
response is more dominated by production of irreparable damage (the a component in the cell survival
response), which is more likely to arise from direct than
indirect action. Taken together, these two observations
lend additional support to the characterization of 8 keV
X rays as high-LET. A similar reduction of the OER
has also been reported for 10 keV electron beams (20).
Radiation-induced DNA damage results in the
formation of nuclear foci that are comprised of proteins
that sense double-stranded breaks and initiate DNA
repair (2126). One of those proteins is H2AX
phosphorylated at serine 139 (27). Many studies have
used phospho-H2AX as a marker for double-stranded
DNA breaks (2833). It has been reported that the
number of H2AX foci generated in human cells
irradiated with low-LET ionizing radiation is in the
range of 3639 foci per Gy per cell (34). We obtained
similar results with cells irradiated with 60Co c rays
(Fig. 4). It has generally been accepted that the number
of H2AX foci generated is one per track for high-LET
charged-particle radiation and that more foci are
generated per gray of radiation for low-LET than
high-LET radiation (29). The number of foci we
detected with characteristic 8 keV X rays was not
significantly different compared to 60Co c rays (Fig. 4).
However, the H2AX foci generated with 8 keV X rays
were approximately 1.6 times larger than foci generated
with 60Co c rays (Fig. 5, Table 3). This is consistent with
data from Costes et al. (29), who showed that H2AX
foci generated with nitrogen ions were 1.5 times larger
than foci generated from 320 kVp X rays and supports
the conclusion that 8 keV X rays possess, to a significant
extent, the damage-producing characteristics of highLET radiations.
Conclusion
We have demonstrated that 8 keV fluorescent X rays
possess radiobiological properties similar to high-LET
radiation, which include higher RBE and lower OER
compared with megavoltage photons. This is also the
first report on the comparative effects of such lowenergy X rays that includes measurement of DNA
damage using immunostaining of H2AX foci. The

296

SHRIDHAR ET AL.

TABLE 3
Size of Nuclear Phospho-H2AX Foci in U251 Cells Measured at 1 h after Irradiation
Mean area (mm2) and 95% CI; number of foci scored
Radiation

0.5 Gy

1 Gy

Total

8 keV X rays

0.777 (0.7340.818)
203
0.426 (0.3770.474)
145
,0.0001

0.790 (0.7520.829)
349
0.514 (0.4790.550)
418
,0.0001

0.785 (0.7560.814)
552
0.491 (0.4620.520)
563
,0.0001

60

Co c rays

Note. Foci produced by 8 keV X rays are significantly larger than foci produced by 60Co c rays.

number of H2AX foci produced per unit dose is similar


for 8 keV and 1.25 MV photons, but 8 keV X rays
generate larger foci, which likely reflects more complex
DNA damage that is more difficult to repair and leads
to the higher RBE. The use of low-energy X rays in a
brachytherapy system potentially offers other advantages that include improved dosimetry, less shielding and
higher cost effectiveness compared with the use of

isotopes. The utility of low-energy X-ray brachytherapy


should now be evaluated clinically.

ACKNOWLEDGMENTS
This work was generously supported by research funds from the
Radiation Oncology Department and the Medical Physics Graduate
Program, of Wayne State University. Advanced X-ray Technology,

FIG. 5. H2AX immunofluorescence staining in U251 cells after irradiation with either 8 keV X rays or 60Co c rays. Representative fields of
view of cells irradiated with 0.5 Gy (panel A) or 1 Gy (panel B) of 8 keV X rays 1 h postirradiation and of cells irradiated with 0.5 Gy (panel C) or
1 Gy (panel D) of 60Co c rays 1 h postirradiation.

RADIOBIOLOGY OF 8 keV X RAYS

Inc., kindly donated the low-energy X-irradiation apparatus used in


this study.
18.
Received: March 10, 2009; accepted: November 11, 2009; published
online: January 4, 2010
19.

REFERENCES
1. G. Gutman, E. Sozontov, E. Strumban, F. F. Yin, S. W. Lee and
J. H. Kim, A novel needle-based miniature x-ray generating
system. Phys. Med. Biol. 49, 46774688 (2004).
2. M. R. Raju, S. G. Carpenter, J. J. Chmielewski, M. E. Schillaci,
M. E. Wilder, J. P. Freyer, N. F. Johnson, P. L. Schor, R. J.
Sebring and D. T. Goodhead, Radiobiology of ultrasoft X rays.
I. Cultured hamster cells (V79). Radiat. Res. 110, 396412 (1987).
3. M. E. Schillaci, S. Carpenter, M. R. Raju, R. J. Sebring, M. E.
Wilder and D. T. Goodhead, Radiobiology of ultrasoft X rays.
II. Cultured C3H mouse cells (10T1/2). Radiat. Res. 118, 8392
(1989).
4. S. Carpenter, M. N. Cornforth, W. F. Harvey, M. R. Raju, M. E.
Schillaci, M. E. Wilder and D. T. Goodhead, Radiobiology of
ultrasoft X rays. IV. Flat and round-shaped hamster cells (CHO10B, HS-23). Radiat. Res. 119, 523533 (1989).
5. M. A. Hill, M. D. Vecchia, K. M. Townsend and D. T.
Goodhead, Production and dosimetry of copper L ultrasoft xrays for biological and biochemical investigations. Phys. Med.
Biol. 43, 351363 (1998).
6. S. Endo, M. Hoshi, J. Takada, T. Takatsuji, Y. Ejima, S.
Saigusa, A. Tachibana and M. S. Sasaki, Development, beam
characterization and chromosomal effectiveness of X-rays of
RBC characteristic X-ray generator. J. Radiat. Res. (Tokyo) 47,
103112 (2006).
7. T. E. Burlin, A general theory of cavity ionisation. Br. J. Radiol.
39, 727734 (1966).
8. A. Janssens, G. Eggermont, R. Jacobs and G. Thielens, Spectrum
perturbation and energy deposition models for stopping power
ratio calculations in general cavity theory. Phys. Med. Biol. 19,
619630 (1974).
9. S. C. Klevenhagen, Determination of absorbed dose in highenergy electron and photon radiation by means of an
uncalibrated ionization chamber. Phys. Med. Biol. 36, 239253
(1991).
10. P. R. Barber, B. Vojnovic, J. Kelly, C. R. Mayes, P. Boulton, M.
Woodcock and M. C. Joiner, Automated counting of
mammalian cell colonies. Phys. Med. Biol. 46, 6376 (2001).
11. M. C. Joiner, Quantifying cell kill and cell survival. In Basic
Clinical Radiobiology (M. C. Joiner and and A. J. van der Kogel,
Eds.), pp. 4155. Hodder Arnold, London, 2009.
12. M. C. Joiner, Linear energy transfer and relative biological
effectiveness. In Basic Clinical Radiobiology (M. C. Joiner and
and A. J. van der Kogel, Eds.), pp. 6877. Hodder Arnold,
London, 2009.
13. D. J. Brenner, C. S. Leu, J. F. Beatty and R. E. Shefer, Clinical
relative biological effectiveness of low-energy x-rays emitted by
miniature x-ray devices. Phys. Med. Biol. 44, 323333 (1999).
14. R. P. Virsik, D. Harder and I. Hansmann, The RBE of 30 kV Xrays for the induction of dicentric chromosomes in human
lymphocytes. Radiat. Environ. Biophys. 14, 109121 (1977).
15. F. M. Khan, The Physics of Radiation Therapy. Lippincott
Williams & Wilkins, Philadelphia, 2009.
16. M. C. Joiner and B. Marples, Annex I: Response in vivo to high
LET radiation. In Relative Biological Effectiveness in Ion Beam
Therapy (G. A. Whitmore, Ed.), pp. 7592. International Atomic
Energy Agency, Vienna, 2008.
17. M. Hoshi, S. Antoku, N. Nakamura, W. J. Russell, R. C. Miller,
S. Sawada, M. Mizuno and S. Nishio, Soft X-ray dosimetry and

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

297

RBE for survival of Chinese hamster V79 cells. Int. J. Radiat.


Biol. 54, 577591 (1988).
G. W. Barendsen, Responses of cultured cells, tumours and
normal tissues to radiations of different linear energy transfer.
Curr. Topics Radiat. Res. Q. 4, 293356 (1968).
B. Palcic, J. W. Brosing and L. D. Skarsgard, Survival
measurements at low doses: oxygen enhancement ratio. Br. J.
Cancer 46, 980984 (1982).
W. T. Tobleman and A. Cole, Repair of sublethal damage and
oxygen enhancement ratio for low-voltage electron beam
irradiation. Radiat. Res. 60, 355360 (1974).
T. Haaf, E. I. Golub, G. Reddy, C. M. Radding and D. C. Ward,
Nuclear foci of mammalian Rad51 recombination protein in
somatic cells after DNA damage and its localization in
synaptonemal complexes. Proc. Natl. Acad. Sci. USA 92, 2298
2302 (1995).
Y. Liu, M. Li, E. Y. Lee and N. Maizels, Localization and
dynamic relocalization of mammalian Rad52 during the cell cycle
and in response to DNA damage. Curr. Biol. 9, 975978 (1999).
R. S. Maser, K. J. Monsen, B. E. Nelms and J. H. Petrini,
hMre11 and hRad50 nuclear foci are induced during the normal
cellular response to DNA double-strand breaks. Mol. Cell Biol.
17, 60876096 (1997).
E. Raderschall, E. I. Golub and T. Haaf, Nuclear foci of
mammalian recombination proteins are located at singlestranded DNA regions formed after DNA damage. Proc. Natl.
Acad. Sci. USA 96, 19211926 (1999).
C. L. Reimer, A. M. Borras, S. K. Kurdistani, J. R. Garreau, M.
Chung, S. A. Aaronson and S. W. Lee, Altered regulation of
cyclin G in human breast cancer and its specific localization at
replication foci in response to DNA damage in p53z/z cells. J.
Biol. Chem. 274, 1102211029 (1999).
R. Scully, J. Chen, R. L. Ochs, K. Keegan, M. Hoekstra, J.
Feunteun and D. M. Livingston, Dynamic changes of BRCA1
subnuclear location and phosphorylation state are initiated by
DNA damage. Cell 90, 425435 (1997).
E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova and W. M.
Bonner, DNA double-stranded breaks induce histone H2AX
phosphorylation on serine 139. J. Biol. Chem. 273, 58585868
(1998).
A. S. Balajee and C. R. Geard, Replication protein A and
gamma-H2AX foci assembly is triggered by cellular response to
DNA double-strand breaks. Exp. Cell Res. 300, 320334 (2004).
S. V. Costes, A. Boissiere, S. Ravani, R. Romano, B. Parvin and
M. H. Barcellos-Hoff, Imaging features that discriminate
between foci induced by high- and low-LET radiation in
human fibroblasts. Radiat. Res. 165, 505515 (2006).
K. H. Karlsson and B. Stenerlow, Focus formation of DNA
repair proteins in normal and repair-deficient cells irradiated with
high-LET ions. Radiat. Res. 161, 517527 (2004).
E. L. Leatherbarrow, J. V. Harper, F. A. Cucinotta and P.
ONeill, Induction and quantification of gamma-H2AX foci
following low and high LET-irradiation. Int. J. Radiat. Biol. 82,
111118 (2006).
S. H. MacPhail, J. P. Banath, T. Y. Yu, E. H. Chu, H. Lambur
and P. L. Olive, Expression of phosphorylated histone H2AX in
cultured cell lines following exposure to X-rays. Int. J. Radiat.
Biol. 79, 351358 (2003).
T. T. Paull, E. P. Rogakou, V. Yamazaki, C. U. Kirchgessner, M.
Gellert and W. M. Bonner, A critical role for histone H2AX in
recruitment of repair factors to nuclear foci after DNA damage.
Curr. Biol. 10, 886895 (2000).
K. Rothkamm and M. Lobrich, Evidence for a lack of DNA
double-strand break repair in human cells exposed to very low xray doses. Proc. Natl. Acad. Sci. USA 100, 50575062
(2003).

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