Dose-response of initial G2-chromatid breaks induced ...

int. j. radiat. biol 2001, vol. 77, no. 2, 165± 174

Dose–response of initial G2-chromatid breaks induced in normal human Ž broblasts by heavy ions T. KAWATA†*, M. DURANTE‡, Y. FURUSAWA§, K. GEORGE†¶, N. TAKAI§, H. WU†# and F. A. CUCINOTTA† (Received 17 May 2000; accepted 7 September 2000) Abstract. Purpose: To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of diÚ erent LET. Material and methods: Exponentially growing human Ž broblast cells AG1522 were irradiated with c-rays, energetic carbon (13 keV/ mm, 80 keV/mm), silicon (55 keV/mm) and iron (140 keV/mm, 185 keV/mm, 440 keV/mm) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. Results: The dose–response curves for total chromatid breaks were linear regardless of radiation type. The relative biological eÚ ectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55–80 keV/mm and decreasing at higher LET. The dose–response curves for isochromatid-type breaks were linear for high-LET radiations, but linear–quadratic for c-rays and 13 keV/mm carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13 keV/mm (about 7) and 80 keV/mm carbon (about 71), and decreased gradually until 440 keV/mm iron ions (about 66). Conclusions : High-LET radiations are more eÚ ective at inducing isochromatid breaks, while low-LET radiations are more eÚ ective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.

1. Introduction Biological eÚ ects induced by accelerated heavy ions diÚ er from those induced by low linear energy transfer (LET) radiations. For most biological endpoints, high-LET heavy ions have a higher relative biological eÚ ectiveness (RBE) than low-LET radiations such as X-rays or c-rays. High-LET radiations * Author for correspondence. e-mail: [email protected] † NASA Lyndon B. Johnson Space Center, Radiation Biophysics Laboratory, Mail Code SN, Houston, Texas, 77058, USA. (T. K. is on leave of the absence from Department of Radiology, School of medicine, Keio University, Tokyo, Japan.) ‡ Department of Physics, University ‘Federico II’, Naples, Italy. §International Space Radiation Laboratory, National Institute of Radiological Sciences, Chiba, Japan. ¶ Wyle Laboratories, 1290 Hercules Drive, Houston, Texas 77058, USA. #Kelsey-Seybold Clinic, Johnson Space Center, Houston, Texas, 77058, USA.

are more lethal to mammalian cells than sparsely ionizing radiation such as c- or X-rays (Barendsen 1968, Cox and Masson 1979, Suzuki et al. 1989, Raju et al. 1991, Napolitano et al. 1992, Furusawa et al. 2000). Investigations of the HPRT-locus (Cox et al. 1977, Thacker et al. 1979, Hei et al. 1988, Kranert et al. 1990, Tsuboi et al. 1992) showed higher levels of mutations induced by high-LET radiation when compared with low-LET radiations. Neoplastic cell transformation is also more eæ ciently induced by heavy ions (Yang et al. 1985, Suzuki et al. 1989). For many endpoints, the trend shows RBE values increasing as a function of LET, with a peak at around 100–200 keV/mm. With the introduction of the premature chromosome condensation (PCC) technique ( Johnson and Rao 1970, Hittelman and Rao 1974, Cornforth and Bedford 1983) it has become possible to study early radiation-induced chromosome damage in interphase cells while the repair process is still progressing. Previous studies have shown that dose–response curves generated from chromosome breaks in G0/G1 cells are linear with radiation dose (Waldren and Johnson 1974, Cornforth and Bedford 1983, Pantelias and Maillie 1984, Goodwin et al. 1996, Durante et al. 1998a). Comparison studies have been conducted for low- and high-LET radiations and the results have shown that high-LET radiation produces a higher frequency of initial chromosome breaks than similar doses of c-rays (Bedford and Goodhead 1989, Watanabe et al. 1992, Cornforth and Bedford 1993, Goodwin et al. 1994, Loucas and Geard 1994). The RBE values for the induction of initial PCC breaks vary from 1.25 to 2.4. The PCC technique has also been used to study chromosome aberrations induced in the G2 phase of the cell cycle (Hittelman and Rao 1974, Hieber and Lu¨ck-Huhle 1983). When Hieber and Lu¨cke-Huhle (1983) exposed Chinese hamster V79 cells to 120 keV/mm a-particles and examined the chromosome damage in G2-PCC, they observed chromatid gaps and breaks similar to those induced by X-rays (Hittelman and Rao 1974). However, they also

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online © 2001 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/09553000010007686

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observed more severe lesions in the decondensed chromatin of chromatids from cells exposed to aparticles. It has been suggested that the multiple chromatid breaks induced in a single chromosome by a-particles prevent the condensation of chromatin in G2-PCC. Very little additional information concerning chromosome aberrations in G2-PCC is available in the literature. In the present study, the authors employ a chemically induced PCC technique with calyculin-A to estimate the initial chromosome damage in human Ž broblasts condensed in G2 phase after exposure to diÚ erent types of high-LET radiations. Calyculin-A, an inhibitor of protein serine/threonine phosphatases, can induce PCC in diÚ erent phases of the cell cycle (Gotoh et al. 1995, Durante et al. 1998b, Alsbeih and Raaphorst 1999), and G2-condensed chromosomes are especially easy to obtain (Gotoh et al. 1999). Previously (Kawata et al. 2000), it was shown that high-LET radiations produce more isochromatid breaks than c-rays and it was suggested that the increased production of isochromatid breaks may be a signature of exposure to high-LET radiations. 2. Materials and methods 2.1. Cell culture Human Ž broblast AG1522 cells (provided by the NIA Cell Repository, Camden, NJ) were grown in a-MEM medium supplemented with 10% fetal calf serum at 37 ß C in 5% CO 2 atmosphere with 95% humidiŽ cation. 2.2. Irradiation Exponentially growing AG1522 cells were irradiated with c-rays or heavy ions. Irradiation with c-rays was performed using a 137 Cs source with a dose rate of 10 Gy/min at Baylor College of Medicine, Texas Medical Center, Houston, Texas. Irradiation with heavy ions was performed at HIMAC, Chiba, Japan or at Brookhaven National Laboratory, NY. One experiment was undertaken for each ion beam. The details of the beam energy, LET values and doses used for each radiation are shown in table 1. 2.3. Premature chromosome condensation and chromosome preparation The PCC technique was used as described elsewhere (Gotoh et al. 1999, Kawata et al. 2000). Calyculin-A from Wako Chemicals ( Japan) was dissolved in 100% ethanol as 1 mm stock solution; 50 nm of calyculin-A was added to the cell cultures 5 min before irradiation to score the initial chromatid

breaks. The highest yield of chromatid breaks and the lowest yield of exchanges is observed when calyculin-A is added 5 min before exposure. After irradiation, cells were incubated for a further 25 min at 37 ß C. Chromosome spreads were then harvested by swelling cells in 75 mm KCl for 20 min at 37 ß C and Ž xing with methanol:glacial acetic acid (3:1 vol/vol). A Ž nal wash and Ž xation in the same Ž xative was completed before dropping cells onto a glass slide and air-drying. 2.4. Observation and scoring after Giemsa staining Chromosomes were stained with Giemsa in phosphate buÚ ered saline ( pH 6.8), mounted, and more than 40 G2-phase cells were scored for each data point, using standard scoring criteria (Savage 1975). Chromatid discontinuity, misalignment of the segment distal to the lesion, or a non-stained region longer than the chromatid width was classiŽ ed as a break. Isochromatid breaks and chromatid-type breaks were scored separately. One chromatidtype break was scored as one break, and one isochromatid break was scored as two breaks. The total chromatid breaks were calculated by summing the yields of chromatid-type breaks and isochromatid breaks. The yields of isochromatid breaks were measured from the excess number of chromosomes ( > 46) observed. 2.5. Cross-sectional area of G2 cells The average cross-sectional area of a G2 cell was determined in order to calculate the average number of particles traversing a G2 nucleus. AG 1522 cells were synchronized at the G1/S phase border by double-thymidine block technique as described elsewhere ( Jackman and O’Connor 1998). The addition of a high concentration of thymidine to a cell population is a reliable and widely used method for synchronizing the cell cycle (Bootsma et al. 1964, Stein and Borun 1972, Heintz et al. 1983). Exponentially growing AG 1522 cells were incubated in a-MEM containing 2 mm thymidine for 12 h at 37 ß C. Then cells were washed twice with PBS before being cultured in a-MEM for 16 h. Then cells were again incubated in a-MEM containing 2 mm thymidine for a further 14 h. During the 14-h incubation with thymidine most of the cells are arrested at the G1/S border. Cells were washed twice with PBS and cultured in a-MEM medium containing 0.1 mg/ml colcemid. Colcemid was added to block cells at metaphase. After incubation for 7 h, G2/M phase cell populations were Ž xed with 100% methanol.

Initial G2 breaks induced by heavy ions Table 1.

Radiation

Gamma-rays and charged particles studied in the present experiment.

Source Baylor Medical College, TX

c-rays

167

Energy (MeV/ nucleon 0.662

Shielding (mm water equivalent) 0

LET (keV/mm) 0.6

Dose rate (Gy/min)

Dose (Gy)

10

1.2, 2.2, 3.0, 4.0

Carbon particles

HIMAC, Chiba, Japan

290

0

13

1

0.5, 1.0, 1.5

Silicon particles

HIMAC, Chiba, Japan

490

0

55

1

0.1, 0.25, 0.5, 1.0

Carbon particles

HIMAC, Chiba, Japan

290

147

80

1

0.5, 1.0, 1.5

Iron particles

Brookhaven National Laboratory, Upton, NY

1000

0

140

1

1.0

Iron particles

Brookhaven National Laboratory, Upton, NY

600

0

185

1

1.0, 1.5

Iron particles

HIMAC, Chiba, Japan

200

0

440

1

0.1, 0.5, 1.0, 1.5

Cells were then stained with Giemsa and the G2 nuclear cross-sectional area was measured. 3. Results 3.1. Examples of G2-PCC breakage Chemically induced PCC technique with calyculin-A was used to determine the initial yield of chromatid-type breaks and isochromatid breaks in G2 human Ž broblast cells exposed to c-rays or each of six ion beams. Examples of chromatid damage observed after exposure to heavy ions are shown in Ž gures 1a and 1b. Figure 1a is an example of chromo-

Figure 1.

somes exposed to 1 Gy of 55 keV/mm silicon ions, where a number of chromatid-type breaks but no isochromatid breaks are observed. Figure 1b is an example of chromosomes exposed to 1.5 Gy of 80 keV/mm carbon ions; here eight excess G2fragments are observed. Some of the isochromatid breaks are shown by arrowheads. 3.2. G2-nuclear cross-sectional area The average cross-sectional area of the G2 nucleus was determined using double-thymidine block and colcemid block techniques. The mean cross-sectional

G2-PCC examples after exposure to 1 Gy of 55 keV/mm silicon particles (a) and 1.5 Gy of 80 keV/mm carbon particles (b). Arrows show chromatid-type breaks and arrowheads show isochromatid breaks.

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area in 400 cells was found to be 242 Ô 4 mm2 (mean Ô standard error). 3.3. Initial chromatid-type breaks after low- or high-LET irradiation Figure 2a shows the number of chromatid-type breaks per cell as a function of dose. Linear dose– responses were observed regardless of radiation type. The data were Ž tted using the linear regression analysis to determine the slopes of best Ž tting lines. RBE values for the induction of chromatid-type breaks were calculated using the linear component (a) of each dose–response curve, and the RBE curve as a function of LET is shown in Ž gure 2b. The RBE curve shows a rapid increase until 55 keV/mm silicon ions (RBE 5 2.44 Ô 0.11) and then decreases until 440 keV/mm iron ions. The RBE for 140 keV/ mm, 185 keV/mm and 440 keV/mm iron ions is 1.12 Ô 0.08, 1.21 Ô 0.05 and 0.68 Ô 0.06, respectively. 3.4. Initial isochromatid breaks (G2-fragments) after lowor high-LET irradiation Figure 3a shows the number of isochromatid breaks (G2-fragments) per cell as a function of dose. Isochromatid breaks induced by high-LET radiations increased linearly with dose. On the other hand, isochromatid breaks were found to increase in a

(a)

Figure 2.

linear–quadratic manner after exposure to c-rays (linear component: a 5 0.055 Ô 0.042, quadratic component: b 5 0.15 Ô 0.02) and 13 keV/mm carbon ions (a 5 0.37 Ô 0.16, b 5 0.27 Ô 0.15). At low doses, isochromatid breaks may be caused by a single track and the probability of inducing isochromatid breaks is proportional to dose. Therefore, the linear components of each dose–response curve were compared to assess the low-dose eÚ ects. Results for the induction of isochromatid breaks are shown in Ž gure 3b, where the linear component (a) is shown on the left y-axis and the RBE on the right y-axis. The curve shows a rapid increase until 80 keV/mm carbon ions (a 5 3.94 Ô 0.13) and then decreased gradually until 440 keV/mm iron ions (a 5 3.66 Ô 0.23). The RBE was 71.6 at 80 keV/mm carbon ions and 66.5 at 440 keV/mm iron ions. In order to assess the highdose eÚ ects, the average dose of each type of radiation that produces one isochromatid break per cell was measured from Ž gure 3a and the RBE values were determined. RBE values obtained from doses producing an average of one isochromatid break per cell ranged from 1.8 to 9.3 (table 2). 3.5. Initial total chromatid breaks after low- or high-LET irradiation Figure 4a shows the number of total chromatid breaks per cell as a function of dose. The RBE values

(b)

(a) Dose–response curves for chromatid-type breaks after exposure to radiations of diÚ erent LET values. Bars are standard errors calculated from the total number of cells scored. (b) RBE curve as a function of LET.

Initial G2 breaks induced by heavy ions

(a)

169

(b)

Figure 3. (a) Dose–response curves for isochromatid breaks after exposure to radiations of diÚ erent LET values. Bars are standard errors calculated from the total number of cells scored. (b) Curve for linear components and RBE as a function of LET. Table 2. Fitted parameters of dose-response curves for isochromatid break induction and dose producing one isochromatid break.

Radiation c-rays

LET (keV/mm) 0.6

a (GyÕ

1

)

Dose producing one isochromatid break (Gy)

0.055 Ô 0.042 (b 5 0.15 Ô 0.02)

2.4 (RBE 5 1)

Carbon

13

0.37 Ô 0.16 (b 5 0.27 Ô 0.15)

1.3 (RBE 5 1.8)

Silicon

55

2.54 Ô 0.20

0.39 (RBE 5 6.0)

Carbon

80

3.94 Ô 0.13

0.25 (RBE 5 9.3)

Iron

140

3.81 Ô 0.07

0.26 (RBE 5 9.0)

Iron

185

3.82 Ô 0.04

0.26 (RBE 5 9.0)

440

3.66 Ô

0.27 (RBE 5 8.5)

Iron

0.23

a, Linear component of the Ž tting curve. b, Quadratic component of the Ž tting curve.

calculated from linear components of dose–response curves show a LET dependence having a gradual increase until 13 keV/mm carbon ions (RBE 5 1.32 Ô 0.08), and then a sharp rise to a peak at 55 keV/mm silicon ions (RBE 5 2.68 Ô 0.17) and 80 keV/mm carbon ions (RBE 5 2.70 Ô 0.12) (Ž gure 4b). Although there is a decrease in the eÚ ectiveness of break induction at higher LET levels, heavy ions used here were more eÚ ective at inducing total breaks than c-rays. The RBE was 2.00 Ô 0.14

for 140 keV/mm iron, 2.11 Ô 0.09 for 185 keV/mm iron and 1.50 Ô 0.11 for 440 keV/mm iron ions. In Ž gure 5, the percentage of initial chromatid-type breaks and isochromatid breaks is shown. Each yield was determined from the linear components of dose–response curves (Ž gure 2a and Ž gure 3a). The percentage of isochromatid breaks increased with increasing LET. For 440 keV/mm iron ions, more than 50% of initial breaks were isochromatid breaks. 3.6. Distribution of initial isochromatid breaks following low-dose irradiation Figure 6 shows frequency distribution of isochromatid breaks in cells exposed to 0.1 Gy silicon ions (55 keV/mm) or iron ions (440 keV/mm). The number of cells scored was 45 for silicon ions and 192 for iron ions. Mean particle number traversing a nucleus (N) can be determined from particle  uence (F ) using the equation (Loucas and Geard 1994) N 5 6.25 Ö A (mm2 ) Ö D (Gy)/LET (keV/mm) (1) where A is a cross-sectional area (mm2 ) and D is dose (Gy). G2 nuclear cross-sectional area (A) was 242 Ô 4 mm2 (mean Ô SE). The average track traversals per nucleus from 0.1 Gy were calculated to be 2.75 Ô 0.05 for 55 keV/mm silicon ions and 0.34 Ô 0.01 for 440 keV/mm iron ions, respectively. Experimental data in Ž gure 6 show that in silicon-irradiated

T. Kawata et al.

170

(a)

Figure 4.

(b)

(a) Dose–response curves for total chromatid breaks after exposure to radiations of diÚ erent LET values. Bars are standard errors calculated from the total number of cells scored. (b) RBE curve as a function of LET.

isochromatid breaks, 27.1% had from one to four isochromatid breaks, and 0.5% had seven isochromatid breaks. The probability of particle hits from each ion was calculated from the Poisson distribution and shown in Ž gure 6. 3.7. Average number of isochromatid breaks from a single track traversal Figure 7 shows the yields of isochromatid breaks/ nucleus from a single particle traversal as a function of LET, which were determined from the linear components of dose–response curves of isochromatid break induction and equation (1). A clear increase in isochromatid break induction with increasing LET was shown. At 440 keV/mm iron ions, 1.05 Ô 0.04 isochromatid breaks were induced by a single particle. 4. Discussion Figure 5. Percentage of isochromatid breaks and chromatidtype breaks in cells as a function of LET. Yields of chromatid-type breaks and isochromatid breaks were determined from the linear components of the dose– response curves (Ž gure 2a and Ž gure 4a).

samples, 86.7% of cells had no isochromatid breaks and 13.3% had one or two isochromatid breaks. In iron-irradiated samples, 72.4% of cells had no

After exposing normal human Ž broblasts to c-rays and diÚ erent types of high-LET radiations, a chemically induced PCC technique was used to score initial chromatid breaks. In order to assess the biological eÚ ects of high-LET radiation accurately, isochromatid breaks and chromatid-type breaks were scored separately and results show that initial chromatid breaks induced by high-LET radiation are diÚ erent from those induced by low-LET radiation. It has

Initial G2 breaks induced by heavy ions

Figure 6.

171

Frequency distribution of isochromatid breaks in cells exposed to 0.1 Gy silicon ions (55 keV/mm) or iron ions (440 keV/mm). The probability of hits calculated from Poisson distribution is shown in the Ž gure.

Figure 7. The average number of isochromatid breaks/nuclear traversal as a function of LET. Bars are standard errors of the mean. The line is Ž tted by eye.

been found that chromatid-type breaks predominate in low-LET irradiated cells, whereas for high-LET radiation isochromatid-type breaks predominate (Ž gure 5). An increased production of isochromatid

breaks by high-LET radiation has also been reported by Durante and colleagues (1994) and Griæ n and colleagues (1994): both groups found that isochromatid deletions in metaphase rodent cells are more frequently produced by a-particles than X-rays. With the use of the chemically induced PCC technique, it has become possible to assess the initial chromosomal damages in G2 cells following irradiation. As was the case for other studies on G0/G1 chromosomes (Waldren and Johnson 1974, Cornforth and Bedford 1983, Pantelias and Maillie 1984, Goodwin et al. 1996, Durante et al. 1998a), chromatidtype breaks and total chromatid breaks showed a linear dose–response regardless of radiation type (Ž gure 2a and Ž gure 4a). This linear dose–response indicates that initial chromatid breaks in G2 cells occur in direct proportion to absorbed dose. The RBE curve for the total break induction shown in Ž gure 4b has a peak at 55–80 keV/mm (2.7) and then decreases until 440 keV/mm (1.5). A LET-dependent trend toward higher frequencies of initial breaks was in good agreement with Goodwin and colleagues (1994) and Loucas and Geard (1994). Goodwin and colleagues (1994), using various kinds of energetic particles, reported that initial breaks in G1 CHO cells were LET-dependent, reaching a peak at 100– 200 keV/mm and then decreasing continuously until

172

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2700 keV/mm, and that the peak RBE was 1.5 when compared with X-rays. Loucas and Geard (1994) measured fragments in non-cycling AG1522 cells after exposure to a-particles and reported an RBE of 2 in comparison with X-rays. Compared with their results, the peak RBE for total chromatid breaks in this study is relatively high (2.7). Since isochromatid breaks were more commonly observed in highLET irradiated samples, this would appear to raise the RBE value for total breaks in G2 cells. However, chromatid-type breaks were less eÚ ectively produced by very high-LET radiation. The RBE curve showed a peak at 55 keV/mm silicon ions (RBE 5 2.44 Ô 0.11) and then a rapid decrease (Ž gure 2b). At 440 keV/mm iron ions, the RBE was about 0.7. These results suggest that the scoring of a chromatid-type break alone leads to underestimation of high-LET radiation damage induced in G2 cells. The dose–responses of isochromatid break induction after high-LET exposures were linear with dose. However, c-rays and 13 keV/mm carbon ions were found to induce a linear–quadratic dose– response (Ž gure 3a). For c-rays, the linear component (0.055 Ô 0.042) is very small but the quadratic component (0.15 Ô 0.02) is relatively high, suggesting that most isochromatid breaks resulted from two separate breaks on sister chromatids induced by independent electron tracks. When compared to the peak RBE for total chromatid breaks (2.7), the peak RBE for the induction of isochromatid breaks calculated from the linear component is very high (about 71). Even when doses producing an average of one isochromatid break per cell are compared, RBE values for heavy ions ranged from 1.8 to 9.3 (table 2). This high RBE value may be attributed to the structure of G2 chromosomes and the ionization clusters produced by energetic heavy ions. In the G2 phase of the cell cycle, sister chromatids are tightly attached to one another (Murray and Hunt 1993) and the two chromatid breaks that lead to an isochromatid break would be in close proximity. The probability of a single track of low-LET radiation producing two breaks on both sister chromatids is small because ionizations would be spaced further apart than the distance between sister chromatids, and in this case chromatid-type breaks would predominate. However, the probability of an isochromatid break occurring from a single track of heavy ions would be proportional to the LET of the charged particles because the distance between the ionization clusters decreases with increasing LET. When the average distance of ionizing clusters becomes comparable to the distance between sister chromatids, the number of isochromatid breaks per particle traversal would probably reach a plateau, although this is not conclusive from

Ž gure 7. Further experiments with radiations of higher-LET would be necessary to evaluate this hypothesis. Further insights into the eæ ciency of heavy ions in producing initial isochromatid breaks can be obtained from the distribution of isochromatid breaks after low-dose exposures (Ž gure 6). The probability of track numbers of 0.1 Gy silicon ions (55 keV/mm) and 0.1 Gy iron ions (440 keV/mm) traversing a G2 nucleus was calculated assuming Poisson distribution and shown in Ž gure 6. The average track traversal (N) per nucleus is also shown in Ž gure 6. Experimental results are in good agreement with the calculated distribution of hit cells. Cells with no isochromatid breaks, cells with from one to four isochromatid breaks, and cells with seven isochromatid breaks following iron irradiation were 72.4%, 27.1%, and 0.5%, respectively. This result suggests that one iron particle (440 keV/mm) traversing a nucleus can produce a maximum of three or four isochromatid breaks. This data also suggests that one silicon particle is less eÚ ective at producing isochromatid breaks compared with one iron particle. One silicon particle out of four or more particles crossing a nucleus has a possibility of producing one isochromatid break. 5. Conclusion The spatial distribution of energy deposition plays an important role in the induction of isochromatid breaks in G2 chromosomes. Owing to the close proximity of sister chromatids in the G2 stage of the cell cycle, the induction of an isochromatid break would require an energy deposition in a very small area, i.e. within the width of sister chromatids. Because of the nature of the dense ionization clusters produced by high-LET radiation, the probability of producing isochromatid breaks would be much higher than low-LET radiations. The authors conclude that increased isochromatid break production is a signature of initial G2 chromosome aberrations after high-LET exposure. Acknowledgements This work was supported by NASA Space Radiation Health Program. T. K. is supported by an NRC grant (fellowship no. 9818170). References Alsbeih, G. and Raaphorst, G. P., 1999, DiÚ erential induction of premature chromosome condensation by calyculin A in human Ž broblast and tumor cell lines. Anticancer Research, 19, 903–908.

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