RADIATION RESEARCH
164, 571–576 (2005)
0033-7587/05 $15.00 q 2005 by Radiation Research Society. All rights of reproduction in any form reserved.
Cytogenetic Effects of High-Energy Iron Ions: Dependence on Shielding Thickness and Material M. Durante,a,b,1 K. George,c G. Gialanella,a,b G. Grossi,a,b C. La Tessa,d L. Manti,a,b J. Miller,e M. Pugliese,a,b P. Scampolia,b and F. A. Cucinottaf d
a Department of Physics, University Federico II, Naples, Italy; b INFN, Sezione di Napoli, Naples, Italy; c Wyle Laboratories, Houston, Texas; Chalmers University of Technology, Gothenburg, Sweden; e Lawrence Berkeley Laboratory, Berkeley, California; and f NASA Lyndon B. Johnson Space Center, Houston, Texas
shielding that is within the weight constraints necessary for space flight, long-term interplanetary missions cannot be undertaken. Model calculations are commonly used for designing spacecraft shielding (1–4), but it is generally acknowledged that physical (5) and especially biological (6) measurements are urgently needed to validate the predictions resulting from these codes. The U.S. National Academy of Sciences has determined that research on the effect of shielding thickness and composition is a high priority for radiation protection during deep-space missions (7). To address this issue, a large international collaboration was begun in 1999 to study the influence of shielding on the induction of chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to accelerated heavy ions (8). Preliminary results have already been reported (9, 10). In this paper, we summarize all data gathered so far using the projectile 56Fe, which was produced either at the Heavy-Ion Medical Accelerator (HIMAC) in Chiba (Japan) or at the NASA Space Radiation Laboratory (NSRL) or the Alternating Gradient Synchrotron (AGS) at the Brookhaven National Laboratory (BNL) in Long Island, NY.
Durante, M., George, K., Gialanella, G., Grossi, G., La Tessa, C., Manti, L., Miller, J., Pugliese, M., Scampoli, P. and Cucinotta, F. A. Cytogenetic Effects of High-Energy Iron Ions: Dependence on Shielding Thickness and Material. Radiat. Res. 164, 571–576 (2005). We report results for chromosomal aberrations in human peripheral blood lymphocytes after they were exposed to highenergy iron ions with or without shielding at the HIMAC, AGS and NSRL accelerators. Isolated lymphocytes were exposed to iron ions with energies between 200 and 5000 MeV/ nucleon in the 0.1–1-Gy dose range. Shielding materials consisted of polyethylene, lucite (PMMA), carbon, aluminum and lead, with mass thickness ranging from 2 to 30 g/cm2. After exposure, lymphocytes were stimulated to grow in vitro, and chromosomes were prematurely condensed using a phosphatase inhibitor (calyculin A). Aberrations were scored using FISH painting. The yield of total interchromosomal exchanges (including dicentrics, translocations and complex rearrangements) increased linearly with dose or fluence in the range studied. Shielding decreased the effectiveness per unit dose of iron ions. The highest RBE value was measured with the 1 GeV/nucleon iron-ion beam at NSRL. However, the RBE for the induction of aberrations apparently is not well correlated with the mean LET. When shielding thickness was increased, the frequency of aberrations per particle incident on the shield increased for the 500 MeV/nucleon ions and decreased for the 1 GeV/nucleon ions. Maximum variation at equal mass thickness was obtained with light materials (polyethylene, carbon or PMMA). Variations in the yield of chromosomal aberrations per iron particle incident on the shield follow variations in the dose per incident particle behind the shield but can be modified by the different RBE of the mixed radiation field produced by nuclear fragmentation. The results suggest that shielding design models should be benchmarked using both physics and biological data. q 2005 by Radiation Research Society
MATERIALS AND METHODS Irradiations A summary of the beam characteristics and shielding data for each experiment is provided in Table 1. The 500 MeV/nucleon iron particles were accelerated using the HIMAC. Due to the presence of scatterers (1.6 mm lead plus 0.2 mm tantalum) and the exit window, the actual energy of the extracted beam was 414 MeV/nucleon. The target area was shielded with blocks of polymethylmethacrylate (PMMA, lucite), aluminum or lead. The 200 MeV/nucleon iron particles were also accelerated using the HIMAC, and the actual energy in air was around 115 MeV/nucleon. The experiments at the NSRL used the 1 GeV/nucleon iron-ion beam, and one experiment (performed at the BNL AGS) used a 5 GeV/nucleon beam without shielding. We also performed one study of unshielded 5 GeV/ nucleon iron particles accelerated at NSRL. Shield blocks of low-density polyethylene (LDPE) and carbon (CCAT CC-1, carbon filled-carbon) were used for the NSRL beams. The same experimental setup was used at HIMAC and NSRL. Dosimetry at HIMAC has been described in detail elsewhere (9, 10), and a similar procedure was used at NSRL. The dose at the target was measured using ionization chambers, while the fluence of primary ions incident on the shield was measured using CR-39 plastic
INTRODUCTION
Radiation is one of the major barriers to safe human exploration of the solar system. Without effective radiation 1 Address for correspondence: Dipartimento di Scienze Fisiche, Universita` Federico II Monte S. Angelo, Via Cintia, 80126 Napoli, Italy; e-mail:
[email protected].
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TABLE 1 Summary of the Experiments Performed with 56Fe Ions Energya (MeV/ nucleon)
Rangeb (mm)
Shield materialc
200 500 500 500 500 500 500 1000 1000 1000 1000 1000 1000 1000 5000
8.0 71.6 8.0 24.0 47.3 8.1 14 290 52 158 71 117 231 100 1780
— — PMMAe PMMA PMMA Aluminum Lead — PMMA LDPEf Aluminum Lead Carbong Carbon —
DoseShield TrackShield mass averaged thickness LETd averaged LETd Dose per incident thickness (keV/mm) (keV/mm) particle (mGy cm2) (mm) (g/cm2) — — 56 43 23 30 10 — 198 150 96 26 30 120 —
— — 6.5 5.0 2.7 8.1 11.3 — 23.0 13.8 26.0 29.5 5.0 19.8 —
442 202 394 277 228 442 354 147 163 129 179 170 143 141 135
440 200 294 225 201 336 318 142 52 77 78 90 88 62 92
0.71 0.32 0.56 0.39 0.33 0.55 0.52 0.23 0.14 0.13 0.18 0.22 0.20 0.138 0.23
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.007 0.01
a (Gy21) 0.29 0.68 0.59 0.39 0.61 0.42 0.41 1.38 0.94 0.72 1.10 0.79 0.69 0.62 1.30
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
0.04 0.07 0.18 0.28 0.27 0.04 0.12 0.05 0.12 0.09 0.21 0.39 0.10 0.10 0.11
s (mm2) 20.4 21.7 35.8 27.2 21.2 23.4 21.9 32.5 13.1 8.6 19.3 17.3 14.0 8.7 29.5
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
0.7 2.1 0.9 5.2 5.5 1.3 3.6 3.4 3.0 2.1 4.2 5.5 2.9 0.7 2.5
Notes. Beams at 200 and 500 MeV/nucleon were accelerated at HIMAC (Japan), while 1 and 5 GeV/nucleon were accelerated at NSRL (U.S.). Parameters a and s are defined in Eqs. (3) and (4). Uncertainties on these fitting parameters are calculated from x2 minimization. Uncertainty on the dose/particle is propagated from the standard error of the mean value of fluence measurements by CR-39. a Acceleration energy in vacuum. b Residual range in water of the primary ions calculated at the sample position. c Shield material between the exit window and the sample. Further passive absorbers are located along the beam line. d LETs in bold type were calculated only, using a Monte Carlo code based on cross sections generated by NUCFRAG2 (5). The other values were estimated from measurements of the spectrum distributions. e Polymethylmethacrylate or lucite (r 5 1.16 g/cm3). f Polyethylene (r 5 0.92 g/cm3). g CCAT CC-1, carbon-filled carbon (r 5 1.65 g/cm3). nuclear track detectors. For comparison with heavy-ion data, we used a dose–response curve for the induction of chromosome aberrations by X rays (250 kVp, 19 mA, 1 mm copper shielding) at a dose rate of approximately 1 Gy/min. The X-ray experiments were repeated four separate times, with four different blood donors over time, and the results were pooled. Linear Energy Transfer (LET) Measurements The energy (or LET) spectrum of fragments produced by the nuclear interactions in the shield was measured by silicon detectors as described in ref. (11). The first sampling moment of the LET distribution is known as track-averaged LET (^LET&T), while the ratio between second and first moment is the dose-averaged LET (^LET&D). From the measured LET spectrum f (L), these quantities are then evaluated as ^LET&T 5
^LET&D 5
E E
L · f (L) dL;
(1)
L 2 · f (L) dL ^LET&T
.
(2)
Dosimetry measurements were not available for a few of the experiments, and in those cases we performed a full simulation using a Monte Carlo code based on cross sections calculated using the NUCFRAG2 nuclear fragmentation model (5). Cells Venous blood from healthy volunteers was drawn into a Vacutainer CPT (Becton-Dickinson, Lincoln Park, NJ). In most cases a different volunteer was used for each experiment. All volunteers gave informed
consent for their blood samples to be used in these experiments. The experiments were approved by the BNL Institutional Review Board (IRB), following guidelines outlined in the Federal Policy for the Protection of Human Subjects. No significant sensitivity differences were observed in X-irradiated lymphocytes from the different donors. The vacutainer tube was centrifuged for 30 min at 3000 rpm, and the buffy coat was carefully removed, resuspended in PBS, and spun for 10 min at 1500 rpm. After two further washes in PBS, the white blood cells were resuspended in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 20% fetal bovine serum. A 2-ml volume of the suspension, at a concentration of approximately 106 cells/ml, was loaded by syringe into a specially constructed lucite holder. Both the loading chamber and the holder wall facing the beam were 1 mm thick. The shield material was placed between the beam and the blood holder and was attached directly to the holder wall to allow secondary fragments emitted at large angles with respect to the beam direction to reach the target. Cells were exposed in air at room temperature. The dose rate ranged from 0.1 to 3 Gy/min. Immediately after exposure, lymphocytes were aspirated from the holder using a syringe and placed in a T-25 tissue culture flask containing 10 ml of RPMI-1640 medium supplemented with 1% phytohemagglutinin (Gibco). Premature Chromosome Condensation Lymphocytes were incubated at 378C, and 48 h later chromosomes were prematurely condensed using the phosphatase inhibitor calyculin A following the protocol developed by Durante et al. (12). Briefly, a 5-ml volume of a 0.1 mM calyculin A stock solution (Wako Chemicals, Japan) was added to each 10-ml culture, and the cells were incubated at 378C for an additional hour. Tubes were then centrifuged, and the pellet was resuspended in 75 mM KCl. After a 20-min treatment in hypotonic so-
CHROMOSOME ABERRATIONS AND SHIELDING
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lution, the cells were fixed in methanol:acetic acid (3:1) and spread on humid, warm slides. FISH Slides were aged for 3 days at room temperature before being hybridized in situ with whole-chromosome human DNA probes (Vysis, Downers Grove, IL) following the protocol recommended by the manufacturer. Combinations of two or three chromosomes were analyzed using probes for chromosomes 1, 2, 4 or 5. Different cocktails were sometimes used for different experiments. Slides were scored at an epifluorescence Axioskop microscope (Carl Zeiss, Germany) connected to a video camera. The slide was scanned and chromosomes located automatically using the Metafer4 software (MetaSystem, Althlusseim, Germany), and chromosome images were automatically acquired in three colors and stored using the ISIS image analysis software (MetaSystem). Chromosome analysis was performed off-line on the saved images. The number of cells scored for each data point ranged from 67 to 4727. All chromosome aberration types were scored separately. However, for the sake of clarity, we present here only the data relative to total chromosomal interchanges involving the painted fraction of the genome, i.e. dicentrics, translocations, plus complex-type exchanges (including incomplete-type). Aberration frequencies were normalized to the whole genome using the formula of Lucas et al. (13). Hence all results presented here refer to whole-genome equivalents of total interchromosomal exchanges. Statistical Analysis Standard errors on the aberration frequency values were evaluated assuming Poisson statistics. All dose– and fluence–response curves were fitted using the functions Y 5 Y0 1 aD;
(3)
Y 5 Y0 1 sF;
(4)
Y is the measured frequency of total exchanges (whole-genome equivalents) per cell; Y0 is the (fitted) background yield; a (Gy21) is the (fitted) yield of total exchanges (whole-genome equivalents) per unit dose; D is the measured dose in Gy; s (mm2) the (fitted) yield of aberrations per unit iron-particle fluence; and F the measured fluence of primary iron particles hitting the shield. The dose–response curves were adequately fitted with linear functions, including the X-ray curve, within the lowdose range explored in these experiments. We also have measurements at a higher dose that point to deviation from linearity, and those data points are not included. Weighted fits were performed using the Origin 6.0 software (Microcal Software, Northampton, MA). Y0 was forced to positive values and was compatible with zero within the errors in each fit. Relative biological effectiveness for each beam and shielding was calculated as RBE 5 a/aX, where aX 5 0.126 6 0.018 Gy21 is the average slope form the four separate X-ray experiments. Errors on RBE were propagated from fitting errors on a and aX. Fitted values of a and s are reported in Table 1.
RESULTS
The dose–response curves for the induction of total interchromosomal exchanges in human lymphocytes exposed to iron ions plus secondary fragments are summarized in Fig. 1. Figure 1A shows the results of the experiments without shielding, although these beams should not be considered as ‘‘pure’’ iron-particle beams. Even when no shield blocks are placed on the beam line, some fragments are produced by beam interaction with scatterers, monitors, exit windows, air, etc. Beam characterization measurements show that the NSRL beam is 96–98% iron ions. Another
FIG. 1. Frequency of aberrations (total interchromosomal exchanges, whole-genome equivalents) as a function of the dose absorbed by lymphocytes exposed to accelerated iron ions or X rays. Panel A: Iron-ion beams without shielding. The curve for X rays is also shown for comparison with heavy-ion data. Panel B: HIMAC experiments with 500 MeV/nucleon iron ions shielded with PMMA, aluminum or lead. Panel C: NSRL experiments with 1 GeV/nucleon iron ions shielded with PMMA, aluminum, lead, LDPE or carbon. Shielding thicknesses are shown on each panel. Bars are standard errors of the mean values.
indication of the beam quality is that the measured LET of the 1 GeV/nucleon iron-particle beam is 149 keV/mm, and the track-averaged and dose-averaged LETs are 142 and 147 keV/mm, respectively (Table 1). Figure 1B includes the shielding experiments at the HIMAC (projectile iron particle 500 MeV/nucleon) and Fig. 1C the NSRL experiments (projectile iron particle 1 GeV/nucleon). In all cases, the shielding decreased the effectiveness of the primary beam,
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FIG. 2. RBE for the induction of total exchanges in lymphocytes as a function of dose- (panel A) or track- (panel B) averaged LET. Symbols for the different shielding materials are shown on each panel. Values of LET and of the a coefficient (Eq. 1) used to calculate the RBE are reported in Table 1. The curve is a guide for the eye.
and the effect was more pronounced at 1 GeV/nucleon than at 500 MeV/nucleon. Figure 2 shows the calculated RBE (a/aX) for every set of data, plotted as a function of dose- (Eq. 2) or trackaveraged (Eq. 1) LET. The maximum effectiveness in the induction of aberrations is reached around 140 keV/mm, corresponding to the unshielded 1 GeV/nucleon NSRL beam. A wide scatter in the data points is observed that is caused, at least in part, by the statistical uncertainties on the fitting parameters. However, significant differences are observed among data points with similar LETT or LETD values, suggesting that LET is not a perfect parameter for describing these experimental values. Figure 3A shows the fluence–response curves for the induction of aberrations by iron-particle beams at different acceleration energies. Also plotted in Fig. 3 is the yield of exchanges (whole-genome equivalents) as a function of the fluence of primary 500 MeV/nucleon (Fig. 3B) or 1 GeV/ nucleon (Fig. 3C) iron ions hitting the shielding. The slopes s of the best-fitting lines (Eq. 4) are plotted in Fig. 4 as a function of the shielding mass thickness for the 500 MeV/ nucleon (Fig. 4B) and 1 GeV/nucleon (Fig. 4C) beams. It is clear that shielding increases the effectiveness per unit incident 500 MeV/nucleon iron particle (Fig. 4B) but decreases the effectiveness per unit incident 1 GeV/nucleon
FIG. 3. Frequency of aberrations (total interchromosomal exchanges, whole-genome equivalents) as a function of the fluence of primary accelerated iron ions measured on the shielding in front of the biological sample. Panel A: Iron-ion beams without shielding. Panel B: HIMAC experiments with 500 MeV/nucleon iron ions shielded with PMMA, aluminum or lead. Panel C: NSRL experiments with 1 GeV/nucleon iron ions shielded with PMMA, aluminum lead, LDPE or carbon. Symbols are the same as in Fig. 1. Bars are standard errors of the mean values.
iron particle (Fig. 4C). This altered behavior pattern is related to the different physical properties of the two beams. In Fig. 4A we plotted the ratio between the measured dose (in the sample position) and measured fluence of iron ions (incident on the shield). The plot is similar to the trends observed in Fig. 4B and C. However, for the 1 GeV/nucleon iron-particle beam, thick targets of low-Z material reduce the cross section s to about 25% of the unshielded value, while the dose is reduced no more than a factor of 2 (compare panels A and C). For comparison, we also show in Fig. 4 the measured Bragg curves in water for the HIMAC 500 MeV/nucleon and NSRL 1 GeV/nucleon ironparticle beams.
CHROMOSOME ABERRATIONS AND SHIELDING
FIG. 4. Dose or aberrations per incident iron ion as a function of the shielding mass thickness (in g/cm2). Panel A: Measured dose (in the sample position) per unit fluence of primary iron ions (incident on the shield). Both HIMAC (500 MeV/nucleon) and NSRL (1 GeV/nucleon) data are shown. The cross sections s (Eq. 4, Table 1) are plotted as a function of shielding mass thickness for the HIMAC 500 MeV/nucleon (panel B) and NSRL 1 GeV/nucleon (panel C) experiments. Curves are measured Bragg curves in water for the HIMAC or NSRL iron-ion beams, normalized at the value measured (in each plot) at zero thickness. Symbols are the same as in Fig. 2.
DISCUSSION
We have reported the results of several accelerator experiments aimed at studying the influence of shielding on the biological effectiveness of high-energy iron ions. The yield of total chromosomal exchanges in human peripheral blood lymphocytes was examined as a function of the dose (at the sample position) and the fluence of primary iron ions incident on the shield (Figs. 1 and 3).
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Fragmentation modifies the quality of the incident beam. At a given absorbed dose, samples behind the shield are exposed to a mixed radiation field, including fast projectile fragments with 1 # Z # 25 and neutrons. Considering that the mean free path for fragmentation of iron particles in water is around 10 cm, it is clear that after the beam has traversed the thick shields used in the experiments described here, only a few primary particles will survive, whereas a wide spectrum of secondary fragments will be produced. The average LET can be used to characterize the effectiveness of the mixed radiation field. In microdosimetry, the dose mean lineal energy, which is the microscopic analog of ^LET&D, is used to describe mixed radiation fields (14). The highest RBE measured in the present study corresponds to the unshielded 1 GeV/nucleon iron-particle beam at NSRL, which is consistent with previous studies using several different ions (15). Oddly enough, we note that neither ^LET&D nor ^LET&T adequately describes the quality of the mixed beam produced by fragmentation. In fact, experiments with similar dose- or track-averaged LET gave different RBE values. Other parameters, perhaps related to the track structure of the secondary particles produced, seem to be necessary to specify radiation quality in these cases (16). Recent experiments on the induction of mammary tumors in rats exposed to energetic charged particles also indicate that mean LET is not a good parameter to describe risk of stochastic effects in mixed charged-particle radiation fields (17). It is useful to evaluate the effectiveness per particle incident on the shield to compare biological results with current models. Here nuclear fragmentation plays an important role, as seen in Fig. 4A. Clearly, for 1 GeV/nucleon ironparticle beams, nuclear fragmentation is dominant, and the dose (behind the shield) per particle (hitting the shield) decreases when shield thickness increases. At lower energy (500 MeV/nucleon), the increase in LET caused by the slowdown of the primary iron ion compensates for fragmentation, and the Bragg curve increases with thickness. This effect has been observed previously (5, 11). Here we note that the effectiveness in the induction of chromosomal aberrations per particle incident on the shield (cross section s) basically follows the same trend. Quite simply, for a given fluence incident on the shield, the beam that produces a higher dose will also produce more biological damage. However, relative variation of aberrations/particle and dose/ particle is not the same at the same shield mass thickness (in g/cm2). This is caused by a combination of the physical decrease in dose/particle (Fig. 4A) and of the decreased RBE of the beam with low track-averaged LET (Fig. 2B). The effect is dependent on the shielding material, with lowZ material (LDPE, PMMA, CCAT CC-1) always being more efficient than heavier shields (aluminum, lead) in increasing (for low-energy projectiles) or decreasing (for relativistic projectiles) the biological effect. The results from the present study demonstrate that biological effects are determined by both physical beam
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transport through the targets and biological effectiveness of the mixed charged-particle radiation field, following a complex pattern that cannot be described by LET alone. This suggests that present and future shielding design models should be benchmarked using both physics and biological data such as those presented here.
6.
7.
ACKNOWLEDGMENTS This work was generously supported by the Italian Space Agency (ASI grants I/R/99-00-01), Italian National Institute for Nuclear Physics (INFN group V, grant SHIELD), and NASA (NRA-02-OBPR-02). We are very grateful to the crews of the HIMAC and NSRL accelerators for their skillful and patient cooperation during the many accelerator runs described in these experiments. Dr. Marc Cohen (NASA Ames Research Center) kindly supplied the CCAT CC-1 carbon shields for the experiments at NSRL. The authors are indebted to Dr. Cary Zeitlin for assistance in dosimetry and transport calculations and to Prof. Lembit Sihver for useful discussions. We also thank Ornella Ortenzia, Danilo Esposito, Rosanna Spera and Charo Del Genio for their contributions in the chromosomal aberration analysis. Finally, special thanks to Dr. Walter Schimmerling for his encouragement and support to this research project. Received: July 28, 2004; accepted: December 2, 2004
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