F2.1-0017-02
CHROMOSOMAL ABERRATIONS INDUCED BY HIGH-ENERGY
IRON IONS WITH SHIELDING
G. Grossi1, M. Durante1, G. Gialanella1, M. Pugliese1, P. Scampoli1, Y. Furusawa2
1
Department of Physical Sciences, University of Naples Federico II, Via Cintia, I-80126 Napoli, Italy
National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263-8555, Japan
2
ABSTRACT
Biophysical models, based on Monte Carlo codes, are commonly used to evaluate the effectiveness of shielding in
reducing the biological damage caused by cosmic radiation in space flights. To improve and validate these codes
biophysical experiments are needed. We have measured the induction of chromosomal aberrations in human
peripheral blood lymphocytes exposed in vitro to 500 MeV/n iron ion beams (dose range 0.1-1 Gy) after traversing
shields of different material (lucite, aluminum, or lead) and thickness (0 to 11.3 g/cm2). For comparison, cells were
exposed to 200 MeV/n iron ions and to X-rays. Chromosomes were prematurely condensed by a phosphatase
inhibitor (calyculin A) to avoid cell-cycle selection produced by the exposure to high-LET heavy-ion beams.
Aberrations were scored in chromosomes 1, 2, and 4 following fluorescence in situ hybridization. The fraction of
aberrant lymphocytes has been evaluated as a function of the dose at the sample position, and of the fluence of
primary 56Fe ions hitting the shield. The influence of shield thickness on the action cross-section for the induction
of exchange-type aberrations has been analyzed, and the dose average-LET measured as a function of the shield
thickness. These preliminary results prove that shielding can increase the effectiveness of heavy ions, and the
damage is dependent upon shield thickness and material.
INTRODUCTION
For terrestrial radiation workers, protection against radiation exposure can be provided through shielding of the
radiation source. In extra-terrestrial space, shielding is effective against trapped protons, but its efficiency is poor or
problematic against galactic cosmic radiation (GCR) penetration. Indeed, high-energy particle radiation is very
penetrating: a thin or moderate shielding is generally efficient in reducing the equivalent dose, but as the thickness
increases, shield effectiveness drops (Wilson et al., 1997). This is the result of the nuclear interactions of the GCR
with the nuclei in the shield, which produce a large number of secondary particles, including neutrons. These
particles have generally lower energy, but can have higher quality factors than incident cosmic primary particles
(Cucinotta and Wilson, 1997).
To calculate GCR doses in different shielding configurations for the International Space Station (ISS) and
Mars mission, NASA is using the analytical code HZETRN coupled to NUCFRG2 (Wilson et al., 1997; Wilson et
al., 1991), developed at the Langley Research Center. HZETRN is based on the solution of the Boltzmann transport
equation, and provides the fluence of primary and secondary ions and the dose in each point inside a spacecraft and
in the human body. The dose can be used to calculate the equivalent dose, using the ICRP-60 (ICRP, 1991)
recommendations, or to estimate action cross-sections for specific biological effects, using biophysical models of
charged particle radiation action. Using ICRP-60 or the Katz’ track structure model (Katz et al., 1996), Wilson and
co-workers (Wilson et al., 1995a; Wilson et al., 1995b; Wilson et al., 1997) estimated that low-Z materials are more
efficient than massive shields in attenuating GCR. In fact, thin shields of high-Z materials, such as Pb, can
substantially increase the dose equivalent or the frequency of neoplastic cell transformation caused by GCR
exposure. In addition, their calculations show that the biological effect depends on the biophysical model used for
calculations, and on the biological endpoint.
In fact, very few biophysical experiments are available to improve and validate these models. Some
experiments were performed at Berkeley (Schimmerling, 1992), and scattered data are available for
1
F2.1-0017-02
cataractogenesis (Medvedovsky et al., 1994) and chromosomal aberrations (Yang et al., 1998). Recently, a large
international collaboration (involving Italian, US, and Japanese groups) is measuring different biological endpoints
in human cells exposed to different heavy ions with or without shielding of different materials (Durante, 2001a;
Durante, 2002). In this paper, we will present data relative to induction of chromosomal aberrations in human
peripheral blood lymphocytes exposed to accelerated 56Fe-ions unshielded and shielded with PMMA (lucite, a
common plastic material), Al and Pb. We are focusing on chromosomal aberrations, because those are considered a
reliable biomarker of radiation-induced stochastic effects (Bonassi et al., 2000; Kodama et al. 2001; Durante et al.,
2001b). Not surprisingly, the National Academy of Sciences (National Academy of Sciences, 1997) has identified
as an important research issue to be urgently addressed the measurement of cell killing and chromosomal
aberrations as a function of thickness and composition of the shielding.
MATERIALS AND METHODS
Irradiation
Lymphocytes were isolated from whole human blood by centrifugation in Vacutainer CPT tubes (BecktonDickinson, Lincoln Park, USA). After two washes in PBS medium, cells were resuspended in RPMI-1640 medium
(Gibco-BRL, Grand Island, NY) supplemented with 20% calf serum, and loaded by a syringe into specially
constructed 1ml PMMA holders. Both the loading chamber and the holder wall exposed to the beam were 1 mm
thick. Cells were exposed in air at room temperature. Doses measured at the sample position by ionization
chambers ranged from 0.1 Gy to 1 Gy. During the irradiation, the blood holder wall exposed to the beam was
attached the shield, to so that secondary fragments emitted at large angles in the laboratory are hitting the biological
target. The 56Fe- ions were accelerated at the HIMAC synchrotron of the National Institute for Radiological
Sciences in Chiba (Japan). Physical characteristics of the beams used in the experiments described here are given in
Table 1. The fluence of heavy particles incident on the shield was measured by CR-39 plastic nuclear track
detectors attached to the shield and facing the beam. The irradiated plastics were etched in 7N NaOH for 1-11 h.
Results of fluence measurements by nuclear track detectors were consistent with the data of the monitor ionization
chambers.
Table 1. Physical characteristics of the iron beams used at the HIMAC accelerator. Fluence in the last column
provides the numer of 56Fe ions per unit area incident on the shield to get a dose of 1 Gy at the sample position
Energy in
vacuum
(MeV/n)
Energy on
sample
(MeV/n)
Shield
Thickness
mm (g/cm2)
500
500
500
500
500
500
200
414
115
Al
PMMA
PMMA
PMMA
Pb
-
30 (8.1)
23 (2.8)
43 (5.2)
56 (6.7)
10 (11.3)
-
Residual range Dose-average
in H2O
LET
(mm)
(keV/m)
72
8.0
56
24
8.0
8.0
8.0
200
378
221
268
394
-----442
Fluence on
the shield
(particles·
cm-2·Gy-1·106)
3.1
1.8
3.0
2.6
1.8
1.9
1.5
Chromosomal aberration analysis
To avoid the cell-cycle selection in the harvested cell population produced by the exposure to high-LET radiation,
we elected to use the novel technique of premature chromosome condensation with phosphatase inhibitors
(calyculin A) to visualize the chromosomes in different stages of the cell-cycle (Durante et al., 1999). Immediately
after exposure, cells were transferred in tissue culture flasks in RPMI-1640 medium supplemented with 20% calf
serum and 2% phytohaemmaglutinin (PHA, Gibco-BRL, Grand Island, NY). Flasks were incubated in vertical
position for 48 h at 37 °C. Calyculin A (Wako chemicals, Japan) at a final concentration of 50 nM was then added
for 1 h, and cells containing prematurely condensed chromosomes (PCCs) in G1, S, G2, and M-phase were
harvested as previously described (Durante et al., 1998). To remove residual cytoplasmic membrane and
organelles, slides were incubated with 100 g/ml RNase A (Sigma Aldrich) for 1 h at 37°C. After denaturation in
70% formamide/2x SSC (Sigma Aldrich), spreads were hybridized in situ with whole-chromosome DNA probes
(Vysis, Downers Grove, IL) specific for human chromosomes 1, 2, and 4, according to manufacturer’s instructions.
4,6-diamidino-2-phenylindole (DAPI) in antifade (Vysis) was applied as a counterstain. Slides were visualized at a
2
F2.1-0017-02
Zeiss Axioscope epi-fluorescent microscope. All kinds of chromosomal aberrations (dicentrics, translocations,
complex-type exchanges, rings, acentric fragments) were scored separately in samples ranging from 150 to 3000
cells per dose. In the figures we plot the fraction of cells containing aberrations involving chromosomes 1, 2, and 4.
RESULTS AND DISCUSSION
The fraction of aberrant lymphocytes (i.e., the fractions of scored cells displaying any type of visible structural
aberration involving painted chromosomes 1, 2, and 4) is plotted in Figure 1 vs. the radiation dose at the sample
position. It can be noted that iron beams are more efficient per unit dose than X-rays in the induction of
chromosomal aberrations, and the 500 MeV/n beam is more efficient then the 200 MeV/n beam in the same doserange (0.1-1 Gy). No significant differences are observed for the 500 MeV/n iron ions beams unshielded or
shielded with 30 mm Al or 56 mm PMMA (lucite) at the same dose.
0.35
200 MeV/n
500 MeV/n
0.3
500 MeV/n + PMMA
Fraction of aberrant cells
500 MeV/n + Al
X-rays
0.25
0.2
0.15
0.1
0.05
0
0
20
40
60
80
100
Dose (cGy)
Fig. 1. Fraction of aberrant lymphocytes exposed to accelerated iron ions, plotted vs. the dose at the sample
position. The shield thickness is 0 mm for () and (■), or 56 mm PMMA for (▼) and 30 mm Al for (▲). X-ray data
are shown for comparison. Bars are standard errors of the mean values. Physical characteristics of the beams are
provided in Table 1.
The same data for the fraction of aberrant lymphocytes are plotted in Figure 2 as a function of the 56Fe ions
fluence incident on the shield. The 200 MeV/n and 500 MeV/n unshielded beams display similar efficiency per
incident particle. The PMMA and Al shields increase the effectiveness per incident ion of the 500 MeV/n 56Fe ions
beam, and the cytogenetic damage behind the 56 mm PMMA seems to be slightly higher than behind the 30 mm Al
shield.
3
F2.1-0017-02
0.35
0.3
Fraction of aberrant cells
0.25
0.2
0.15
0.1
200 MeV/n
500 MeV/n
0.05
500 MeV/n + PMMA
500 MeV/n + Al
0
0
0.5
1
1.5
2
2.5
2
3
3.5
6
Fluence (particles/cm) x 10
Fig. 2. Fraction of aberrant lymphocytes exposed to accelerated iron ions, plotted vs. the fluence of iron ions
incident on the shield. The shield thickness is 0 mm for () and (■), or 56 mm PMMA for (▼) and 30 mm Al for
(▲). X-ray data are shown for comparison. Bars are standard errors of the mean values. Physical characteristics of
the beams are provided in Table 1.
The effect of different shield material and thickness on the effectiveness of the 500 MeV/n iron beam is
shown in Figure 3. Fluence-response curves (such as those in Figure 2) appeared to be linear in the measured doserange, and were fitted by the function: Y = F, where Y is the frequency of exchanges in the painted genome
(chromosomes 1, 2, and 4) per lymphocyte, F is the fluence (in particles/m2) of iron ions hitting the shield, and 
is the action cross-section for the induction of exchange-type aberrations. The action cross-section represents the
product of the cross-sectional area of the sensitive target and the probability of aberration induction per ion hit on
the shielding. Clearly, the cross-section increases by increasing the shield thickness in this range, and the increase
appears to be caused by the increased dose-rate induced by the shield.
Data in Figure 1 basically show that the fragmentation produced by the shield does not significantly change
the quality factor of the beam at the sample position. In fact, cytogenetic damage is similar at the same radiation
dose absorbed by the sample. However, when plotted as a function of the number of ions hitting the shield (Figures
2 and 3), the curves are separated and the shield increases the effectiveness per unit ion. A lower number of 56Fe
ions is required to produce a certain dose at the sample when it is shielded either with Al or PMMA.
The dose average-LET measured for different PMMA thickness is shown in Figure 4. Despite the loss of
heavy primary ions from the beam by increasing depth, the dose-averaged LET increases in the range studied. As a
consequence of this increase, the dose delivered by a single ion hitting the attenuator is increased by increasing the
shield thickness. The increased dose-rate observed when the shield is posed between the primary beam and the
4
F2.1-0017-02
target appears to be responsible for the increase in the action cross-section reported in Figure 3. These data can be
used to benchmark the codes used to calculate shielding in space vessels.
18
PMMA
Al
Pb
2
Action cross-section (µm )
16
14
12
10
8
6
0
2
4
6
8
10
12
2
Shield thickness (g/cm)
Fig. 3. Action cross-section for the induction of chromosomal aberrations involving prematurely condensed human
chromosomes 1, 2, and 4 in blood lymphocytes exposed to 500 MeV/n 56Fe ions attenuated by different thickness
of PMMA, 30 mm Al and 10 mm Pb. Bars are standard errors of the mean values. The line is an interpolation of the
data points.
5
F2.1-0017-02
10000
Primary plus secondary ions
LET (keV/m)
Primary ions only
1000
100
0
20
40
60
80
100
PMMA depth (mm)
Fig. 4. Dose-average LET for primary ions only and primary and secondary ions produced by nuclear
fragmentation of 500 MeV/n beam in the target plotted vs. depth in PMMA.
ACKNOWLEDGEMENTS
We are grateful to Dr. T. Kanai (NIRS) for his assistance with the physics measurements at the HIMAC
accelerator. This work was supported by the Italian Space Agency.
REFERENCES
Bonassi, S., L. Hagmar, U. Stromberg, A.H. Montagud, H. Tinnerberg, A. Forni, P. Heikkila, S. Wanders, P.
Wilhardt, I.L. Hansteen, L.E. Knudsen and H. Norppa, Chromosomal aberrations in lymphocytes predict
human cancer independently of exposure to carcinogens, Cancer. Res., 60, 1619-1625, 2000.
Cucinotta F.A. and J.W. Wilson, Assessment of current shielding issues, in Shielding Strategies for Human Space
Exploration, edited by J.W. Wilson, J. Miller, A. Konradi, and F.A. Cucinotta, pp. 447-467, NASA-CP 3360,
Washington, DC, 1997.
Durante, M., Y. Furusawa, and E. Gotoh, A simple method for simultaneous interphase-metaphase chromosome
analysis in biological dosimetry, Int. J. Radiat. Biol., 74, 457-462, 1998.
Durante, M., Y. Furusawa, H. Majima, T. Kawata, and E. Gotoh, Association beteween G2-phase block and repair
of radiation-induced chromosome fragments in human lymphocytes, Radiat. Res., 151, 670-676, 1999.
Durante, M., Italian Space Radiobiology Program: influence of the shielding on the biological effects of heavy
ions, in Exploring Future Research Strategies in Space Radiation Sciences, edited by H. Majima and K.
Fujitaka, pp. 79-85, Iryokagakusha, Tokyo, 2000.
6
F2.1-0017-02
Durante, M., Influence of the shielding on the space radiation biological effectiveness, Phys. Med., 17, S269-S271,
2001a.
Durante, M., S. Bonassi, K. George, and F.A. Cucinotta, Risk estimation based on chromosomal aberrations
induced by radiation, Radiat. Res., 156, 662-667, 2001b.
ICRP, Recommendations of the International Commission on Radiological Protection, Annals of the ICRP 21,
Publication 60, Pergamon Press, Oxford, 1991.
Katz, R., F.A. Cucinotta, and C.X. Zhang, The calculation of radial dose from heavy ions: predictions of biological
action cross sections, Nucl. Instr. Meth., B107, 287-291, 1996.
Kodama, Y., D. Pawel, N. Nakamura, D. Preston, T. Honda, M. Itoh, M. Nakano, K. Ohtaki, S. Funamoto, and
A.A. Awa, Stable chromosome aberrations in atomic bomb survivors: results from 25 years of investigation,
Radiat. Res., 156, 337-346, 2001.
Medvedovsky, C., B.V. Worgul, Y. Huang, D.J. Brenner, F. Tao, J. Miller, C. Zeitlin, and E.J. Ainsworth, The
influence of dose, dose-rate and particle fragmentation on cataract induction by energetic iron ions, Adv. Space
Res., 14, 475-482, 1994.
National Academy of Sciences, Radiation Hazards to Crews on Interplanetary Missions, Report of the Task Group
on the Biological Effects of Space Radiation, National Academy Press, Washington, DC, 1997.
Schimmerling, W., Ground-based measurements of galactic cosmic ray fragmentation in shielding, Adv. Space
Res., 12, 445-459, 1992.
Wilson, J.W., L.W. Townsend, W. Schimmerling, G.S. Khandelwal, F. Khan, J.E. Nealy, F.A. Cucinotta, L.C.
Simonsen, J.L. Shinn, and J.W. Norbury, Transport methods and interactions for space radiations. NASA RP1257, Springfield, VA, 1991.
Wilson, J.W., M. Kim, W. Schimmerling, F.F. Badavi, S.A. Thibeault, F.A. Cucinotta, J.L. Shinn, and R. Kiefer,
Issues in space radiation protection: galactic cosmic rays, Health Phys., 68, 50-58, 1995a.
Wilson, J.W., S.A. Thibeault, F.A. Cucinotta, J.L. Shinn, M. Kim, R. Kiefer, and F.F. Badavi, Issues in protection
from galactic cosmic rays, Radiat. Environ. Biophys., 34, 217-222, 1995b.
Wilson, J.W., A. Konradi, J. Miller, and F.A. Cucinotta, Shielding strategies for human space exploration, NASA
CP-3360, Springfield, VA, 1997.
Yang, T.C., K. George, H. Wu, D. Miller, and J. Miller, Cytogenetic effects of energetic ions with shielding, Adv.
Space Res., 22, 1683-1690, 1998.
E-mail address of the corresponding author Gianfranco Grossi
7
gianfranco.grossi@na.infn.it