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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
0033-7587/10 $15.00
g 2010 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR1782.1
Wayne State University, Detroit, Michigan; b Mt. Clemens General Hospital, Mt. Clemens, Michigan; and c William Beaumont Hospital,
Royal Oak, Michigan
INTRODUCTION
290
291
292
SHRIDHAR ET AL.
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.
293
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
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
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
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
60
295
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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.
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.
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