- Email: [email protected]
Chromosomal aberrations induced by 12C6+ heavy ion irradiation in spermatogonia and spermatocytes of mice
Mutation Research 398 Ž1998. 27–31
Chromosomal aberrations induced by 12 C 6q heavy ion irradiation in spermatogonia and spermatocytes of mice H. Zhang a , R.L. Zheng b,) , R.Y. Wang c , Z.Q. Wei a , W.J. Li a , Q.X. Gao b, W.Q. Chen a , Z.H. Wang b, G.W. Han a , J.P. Liang a a
c
Department of Application of HeaÕy Ions, Institute of Modern Physics, The Chinese Academy of Sciences, P.O. Box 31, Lanzhou 730000, People’s Republic of China b Department of Biology, Lanzhou UniÕersity, Lanzhou 730000, People’s Republic of China Department of Gynecology and Obstetrics, The First Hospital, Lanzhou Medical College, Lanzhou 730000, People’s Republic of China Received 23 July 1997; revised 9 October 1997; accepted 16 October 1997
Abstract The testes of Kun-Ming strain mice were radiated with different doses of 12 C 6q ion or 60 Co g-ray. Chromosomal aberrations induced in spermatogonia and spermatocytes were analyzed by the air-drying method. The relative biological effectiveness ŽRBE. of 12 C 6q ion was calculated with respect to 60 Co g-ray for the induction of chromosomal aberrations. The 12 C 6q ion and 60 Co g-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models. The results showed that there was an increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values were 1.67 for aberrations of spermatogonia and 1.66 for aberrations of spermatocytes for a dose of 2.0 Gy. Moreover, a different distribution of the various types of aberrations has been found for 12 C 6q ion and 60 Co g-ray irradiations. The dose–response relationships for 12 C 6q ion and 60 Co g-ray exhibited negative curvature in both spermatogonia and spermatocytes groups: the frequencies of aberrations increased sharply at low doses and exhibited less sharp increases for higher doses, which may be related to an interaction between the chromosomal damage and a block in cell cycle. Our results may provide useful information for the assessment of genetic risks of humans exposed to heavy ions. q 1998 Elsevier Science B.V. Keywords:
12
C 6q heavy ion; Chromosomal aberration; Spermatogonium; Spermatocyte; Mouse
1. Introduction In higher organisms, the DNA is organized in chromosomes, and the induced aberrations of these chromosomes can lead to cellular lethality and mutation. Ionizing radiation has been confirmed to be one ) Corresponding author. Tel.: q86 Ž931. 891-2563; fax: q86 Ž931. 891-1100; E-mail: [email protected]
of important exogenous inducers of chromosomal aberrations w1–3x. Comparing with X-ray or g-ray, the track of a heavy ion is complex, energy is not only deposited by the primary interaction, but also by secondary electrons that may travel considerable distances from the core. This heavy ion with high linear energy transfer ŽLET. and high relative biological effectiveness ŽRBE. is also significantly more deleterious on the cellular or molecular level than
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 6 - 4
28
H. Zhang et al.r Mutation Research 398 (1998) 27–31
low LET ionizing irradiation, such as X-ray or g-ray. For example, heavy ion irradiation induces irreparable breaks in DNA more readily than low-LET irradiation w4x. It has been repeatedly demonstrated that heavy ion irradiations induce chromosomal aberrations of somatic cells w5–8x; but, chromosomal aberrations induced by irradiation in germ cells, which differ from those in somatic cells, not only indicate the cellular damage of radiated individuals, but are also partly transmitted to offspring and result in genetic effects, i.e. abnormalities, sterility and malignant diseases. Moreover, the testis is one of the most radiosensitive organs of the body and is the critical organ in the radiotherapy of Hodgkin’s disease and seminoma w9,10x. With the advent of new radiotherapy modalities, such as fast neutrons and heavy ions, there is a considerable improvement in the survival rate of cancer patients, and these radiotherapy patients of child-bearing age are concerned about risk of future children. Thus, it is important to ascertain whether the high LET radiation exposure increases the risk of chromosomal aberrations in gametes, so as to pay more attention to the reproductive potential and the possible genetic alteration in the germ cells of these patients. The spermatogonia and spermatocytes are ones of the most radiosensitive cells of the body. Moreover, radiation-induced chromosomal aberrations of spermatogonia and spermatocytes were demonstrated to be transmitted into spermatozoa w11x, and these chromosomally abnormal sperm fertilize eggs, as well as normal sperm w12x. Hence the aim of the present study is to investigate the frequency and characteristic of chromosomal aberrations induced by 12 C 6q ion in spermatogonia and spermatocytes of mice.
2. Materials and methods 2.1. Animals Male mice Ž10–12 weeks. of the Kun-Ming strain provided by Lanzhou Medical College ŽLanzhou, China. were used under identical breeding conditions. They were randomly divided with 5 animals in each group.
2.2. Irradiation procedure The mouse was positioned in a chamber which was fixed to the irradiation equipment at the Heavy Ion Research Facility in Lanzhou ŽHIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China. w13x. The abdomen Ž12 mm diameter around the scrotum. of the mouse was radiated with 12 C 6q ion beam at energy 50 MeVrU and LET 45 keVrmm in the water generated from HIRFL, with a dose rate of 0.2 Gyrmin. The remainder of the body was shielded with lead plate. The acquisition of data was automatically accomplished using a microcomputer during irradiation. Doses of the beams were determined with air ionization chamber. Animals irradiated by 60 Co g-ray were similarly given abdomen irradiation by a FTC-50H model 60 Co teletherapy machine ŽShanghai Nuclear Equipment Factory, China. at a source to surface distance ŽSSD. of 75 cm, with a dose rate 0.4 Gyrmin. The doses used for each kind of irradiation Ž 12 C 6q ion or 60 Co g-ray. were 0, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 Gy.
2.3. Preparation and analysis of chromosome Preparation and analysis of chromosome were done according to the air-drying method of Evans et al. w14x. The mice were injected Ži.p.. with colchicine Ž3 mgrkg. at 8 h after irradiation and 5 h later they were killed by cervical dislocation. The testes were transferred to 2.5 ml of a 2.2% citrate solution in fresh Petri dishes and the tunica removed. The contents of the tubule were gently teased out with curved forceps. The cell suspension produced was centrifuged at 1000 = g for 5 min. The supernatant was discarded, and the pellet was resuspended in 2 ml of hypotonic solution Ž0.4% KCl. at 378C. After 15 min, the suspension was centrifuged for 5 min at 1000 = g, and the supernatant then was removed. The cells were fixed 3 times with Carnoy’s solution Ž1:3 of glacial acetic acid and methanol.. The precleaned slides were kept cold in distilled water before being used. Three to four droplets of the final suspension were on a slide and blown upon to dry, and two slides per animal were made. Slides were
H. Zhang et al.r Mutation Research 398 (1998) 27–31 Table 1 Chromosome aberrations induced by Group
12
C 6q ion or
60
Co g-ray in spermatogonia of mice
Distribution of aberrations Žno. Ž%.. Gap
Control 0 Gy 0 12 6q C ŽGy. 0.05 0 0.1 2 Ž0.8 " 1.78. 0.2 5 Ž2.0 " 0.89. 0.5 6 Ž2.4 " 1.10. 1.0 9 Ž3.6 " 3.29. 2.0 10 Ž4.0 " 2.19. 60 Co ŽGy. 0.05 1 Ž0.4 " 0.89. 0.1 2 Ž0.8 " 1.10. 0.2 4 Ž1.6 " 1.67. 0.5 4 Ž1.6 " 0.89. 1.0 8 Ž3.2 " 3.03. 2.0 10 Ž4.0 " 2.45.
Chromatid break 1 Ž 0.4 " 0.89.
29
Total no. of aberrations Ž%.
No. of cells with aberrations Ž%.
Chromosome break 1 Ž 0.4 " 0.89.
0
2 Ž 0.8 " 1.10. 3 Ž 1.2 " 1.79. 7 Ž 2.8 " 2.28. 16 Ž 6.4 " 2.97. 30 Ž12.0 " 3.16. 57 Ž22.8 " 5.02.
3 Ž 1.2 " 1.79. 5 Ž 2.0 " 2.00. 9 Ž 3.6 " 1.67. 23 Ž 9.2 " 2.45. 41 Ž16.4 " 4.15. 70 Ž28.0 " 6.07.
2 Ž 0.8 " 1.09. 3 Ž 1.2 " 1.10. 7 Ž 2.8 " 3.29. 14 Ž 5.6 " 3.03. 25 Ž10.0 " 2.97. 40 Ž16.0 " 6.92.
0 0 0 5 Ž 2.0 " 2.00. 14 Ž 5.6 " 4.34. 32 Ž12.8 " 6.23.
5 Ž 2.0 " 2.83. 10 Ž 4.0 " 1.41. b 21 Ž 8.4 " 3.63. b 45 Ž18.0 " 4.34. c 80 Ž32.0 " 7.01. c 137 Ž54.8 " 11.05. c 3 Ž 1.2 " 5 Ž 2.0 " 11 Ž 4.4 " 23 Ž 9.2 " 47 Ž18.8 " 82 Ž32.8 "
1.10. 1.41. a 2.61. a 4.69. b 5.76. c 9.12. c
1 Ž 0.4 " 0.89. 5 Ž 2.0 " 2.83. 10 Ž 4.0 " 1.41. 21 Ž 8.4 " 3.63. 45 Ž18.0 " 4.34. 61 Ž24.4 " 3.85. 102 Ž40.8 " 4.38. 3 Ž 1.2 " 1.10. 5 Ž 2.0 " 1.41. 11 Ž 4.4 " 2.61. 23 Ž 9.2 " 4.69. 47 Ž18.8 " 5.76. 66 Ž26.4 " 6.07.
Results based on 250 analyzed cells from 5 samples. Data represent mean " SD. The significance of differences between radiated groups and controls was determined by Student’s t-test. a p - 0.05, b p - 0.01, c p - 0.001 vs. control.
stained in 10% Giemsa for 8 min. Fifty cells at diakinesis-metaphase spermatogonia or spermatocytes for each animal were respectively scored miTable 2 Chromosome aberrations induced by Group
12
C 6q ion or
60
Co g-ray in spermatocytes of mice
Distribution of aberrations Žno. Ž%.. Gap
Control 0 Gy 1 Ž0.4 " 0.89. 12 6q C ŽGy. 0.05 1 Ž0.4 " 0.89. 0.1 3 Ž1.2 " 1.79. 0.2 3 Ž1.2 " 1.09. 0.5 6 Ž2.4 " 0.89. 1.0 11 Ž4.4 " 2.61. 2.0 8 Ž3.2 " 3.03. 60 Co ŽGy. 0.05 2 Ž0.8 " 1.10. 0.1 2 Ž0.8 " 1.79. 0.2 4 Ž1.6 " 0.89. 0.5 7 Ž2.8 " 2.28. 1.0 6 Ž2.4 " 2.61. 2.0 9 Ž3.6 " 2.19.
Chromatid break 1 Ž 0.4 " 0.89.
croscopically at 1000-fold magnification. Abnormal chromosomes were classified into gap, chromatid break and chromosome break Žincluding fragments..
Total no. of aberrations Ž%.
No. of cells with aberrations Ž%.
Chromosome break 0
4 Ž 1.6 " 1.67. 5 Ž 2.0 " 2.83. 12 Ž 4.8 " 3.63. 22 Ž 8.8 " 6.00. 29 Ž11.6 " 3.03. 49 Ž19.6 " 4.34.
3 Ž 1.2 " 1.79. 7 Ž 2.8 " 2.68. 14 Ž 5.6 " 2.61. 31 Ž12.4 " 4.90. 50 Ž20.0 " 3.85. 91 Ž36.4 " 9.86.
3 Ž 1.2 " 1.79. 6 Ž 2.4 " 2.61. 6 Ž 2.4 " 1.67. 15 Ž 6.0 " 2.45. 27 Ž10.8 " 4.34. 45 Ž18.0 " 3.35.
0 1 Ž 0.4 " 0.89. 4 Ž 1.6 " 1.67. 9 Ž 3.6 " 1.41. 19 Ž 7.6 " 4.82. 35 Ž14.0 " 6.23.
2 Ž 0.8 " 1.10. 8 Ž 3.2 " 2.28. a 15 Ž 6.0 " 2.61. b 29 Ž11.6 " 5.02. b 59 Ž23.6 " 6.84. c 90 Ž36.0 " 7.35. c 148 Ž59.2 " 11.44. c 5 Ž 2.0 " 9 Ž 3.6 " 14 Ž 5.6 " 30 Ž12.0 " 52 Ž20.8 " 89 Ž35.6 "
2.00. 2.19. a 3.29. a 3.63. b 4.15. c 8.65. c
2 Ž 0.8 " 1.10. 8 Ž 3.2 " 2.28. 15 Ž 6.0 " 2.61. 29 Ž11.6 " 5.02. 59 Ž23.6 " 6.84. 67 Ž26.8 " 5.40. 107 Ž42.8 " 9.10. 5 Ž 2.0 " 2.00. 9 Ž 3.6 " 2.19. 14 Ž 5.6 " 3.29. 30 Ž12.0 " 3.63. 51 Ž20.4 " 4.56. 70 Ž28.0 " 3.35.
Results based on 250 analyzed cells from 5 samples. Data represent mean " SD. The significance of differences between radiated groups and controls was determined by Student’s t-test. a p - 0.05, b p - 0.01, c p - 0.001 vs. control.
30
H. Zhang et al.r Mutation Research 398 (1998) 27–31
3. Results and discussion The frequencies and distributions of chromosomal aberrations observed in spermatogonia and spermatocytes induced by 12 C 6q ion and 60 Co g-ray are shown in Tables 1 and 2, respectively. There was an increase in frequency of chromosomal aberrations in all the treated groups compared to controls. There were 54.8 and 59.2% chromosomal aberrations for spermatogonia and spermatocytes exposed to 2 Gy of 12 C 6q ion, and 32.8 and 35.6% in the spermatogonia and spermatocytes which received 2 Gy of 60 Co g-ray. The RBE of 12 C 6q ion was calculated with respect to 60 Co g-ray for induction of chromosomal aberrations. The RBE values were 1.67 for aberrations of spermatogonia and 1.66 for aberrations of spermatocytes for a dose of 2.0 Gy. The majority of aberrations observed were chromatid break and chromosome break. A different distribution of the various types of aberrations has been found for 12 C 6q ion and 60 Co g-ray irradiations: in 60 Co g-ray irradiation, the distribution was dominated by the chromatid break; in 12 C 6q ion irradiation, the distribution of aberrations was clearly dominated by chromosome break and fragments, even complete disintegrations of parts of chromosomes, which is thought to be a manifestation of the high and local energy density in a particle track w7x. Furthermore, at low doses of 0.05–1 Gy for 60 Co g-ray or 0.05–0.5 Gy for 12 C 6q ion, the frequencies of cells with chromosomal aberrations were equal to the total frequencies of chromosomal aberrations. At higher doses of ) 1 Gy for 60 Co g-ray or ) 0.5 Gy for 12 C 6q ion, increasing numbers of cells with multiple aberrations were observed in both spermatogonia and spermatocytes Žthe frequencies of cells with aberrations became lower than those of total aberrations., which demonstrated that higher doses of ionizing irradiation increase not only frequency of cells with aberrations, but also the amount of induced chromosomal aberrations per cell, and this also suggested that with increasing doses, the degree of damage of radiated cells increases. From Tables 1 and 2 it can be seen that the frequencies of chromosomal aberrations induced by both 12 C 6q ion or 60 Co g-ray were higher in spermatocytes than in spermatogonia. This variation might be due to differences in the sensitivity to mutagenic
agents of male germ cells in different stages of the cell development. Male germ cells in meiosis and postmeiosis phases are generally more sensitive to mutagenic agents than those in other phases. The 12 C 6q ion and 60 Co g-ray dose–response relationships for chromosomal aberrations of spermatogonia and spermatocytes were plotted by linear quadratic models in Fig. 1. The equations obtained for spermatogonia and spermatocytes were, respectively, Y s 0.547 q 36.670 D y 4.792 D 2 and Y s 1.781 q 43.590 D y 7.523 D 2 in 12 C 6q ion exposure and Y s 0.200 q 20.107 D y 1.888 D 2 and Y s 1.052 q 22.774 D y 2.762 D 2 in 60 Co g-ray exposure Ž Y is chromosomal aberrations per cell and D is the irradiation dose in Gy.. As shown in Fig. 1, the dose–response relationships exhibited negative curvature, and the slopes of these curves decrease
Fig. 1. Dose–response relationships of chromosomal aberrations induced by 12 C 6q ion Žv . and 60 Co g-ray ŽB. in spermatogonia ŽA. and spermatocytes ŽB. of mice. Data represent mean"SD, ns 5.
H. Zhang et al.r Mutation Research 398 (1998) 27–31
with increasing doses. The frequencies of aberrations increased sharply at low doses and exhibited a less sharp increases for higher doses, especially for 12 C 6q ion irradiation. It might be possible that the curvature of dose–response relationships is due to an interaction between the chromosomal damage and a block in cell cycle w15,16x, which arrests the majority of heavily damaged cells in the G2 phase. Cells with severe chromosome damage cannot proceed to mitosis or meiosis. This interference between cell cycle effects and expression of chromosomal damage should be minimal at low doses w7x. In conclusion, 12 C 6q ion irradiation significantly increased the frequencies of chromosomal aberrations in spermatogonia and spermatocytes of mice, even at low dose of 0.05 Gy. Our results may provide useful information for assessment of genetic risks of human exposed to heavy ions. Radiosensitivity of germ cells is higher in human than in mouse. Hence there is a need for further investigation of the relationship of chromosomal aberrations induced by heavy ions between human and mouse germ cells, and finding out reasonable comparable rules among species.
w3x
w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
Acknowledgements This project was supported by China Postdoctoral Science Foundation and Institute Head’s Science Foundation of The Chinese Academy of Sciences. We express our thanks to the accelerator crew at the HIRFL, National Laboratory of Heavy Ion Accelerator in Lanzhou.
w12x
w13x
w14x
References w15x w1x N. Kamada, The effects of radiation on chromosomes of bone marrow cells. 1. Type and frequency of rat chromosome aberrations induced by fast neutrons X-ray and gamma rays, Acta Haematol. Jap. 32 Ž1969. 212–235. w2x Y. Matsuda, T. Yamada, I. Tobar, A. Ohkawa, Preliminary study on chromosomal aberrations in eggs of mice fertilized
w16x
31
in vitro after X-irradiation, Mutation Res. 121 Ž1983. 125– 130. Y. Kodama, J.-i. Kushiro, Y. Hirai, Y. Kusunoki, N. Nakamura, M. Akiyama, A.A. Awa, Frequent involvement of visible chromosomal deletion in X-Ray-induced mutations at the HLA-A locus in human T-lymphocytes, Mutation Res. 309 Ž1994. 63–72. M.A. Ritter, J.E. Cleaver, C.A. Tobias, High-LET radiations induce a large proportion of non-rejoining DNA breaks, Nature 266 Ž1977. 653–655. W. Muller, H.G. Miltenburger, G. Kraft, Chromosome aberrations in Chinese hamster cells cultures induced by heavy ion irradiation, GSI-Scientific Report 1982, GSI, Darmstadt, 1983, 197 pp. W. Muller, H.G. Miltenburger, G. Kraft, Chromosome aberrations in V79 Chinese hamster cells produced by heave charged particles, GSI-Scientific Report 1983, GSI Darmstadt, 1984, 202 pp. C.R. Geard, Charged particle cytogentics: effects of LET, fluence, and particle separation on chromosome aberrations, Radiat. Res. 104 Ž1985. S112–S121. G. Kraft, Radiological effects of very heavy ions: inactivation, induction of chromosome aberrations and strand breaks, Nucl. Sci. Appl. 3 Ž1987. 1–28. R.D. Amelar, L. Dubin, R.S. Hotchkiss, Restoration of fertility following unilateral orchiectomy and radiation therapy for testicular tumors, J. Urol. 106 Ž1971. 714–718. B. Speiser, P. Rubin, G. Cassarett, Aspermia following lower truncal irradiation in Hodgkin’s disease, Cancer 32 Ž1973. 692–698. R.H. Matin, K. Hildebrand, J. Yamamoto, A. Rademaker, M. Barnes, G. Douglas, K. Arthur, T. Ringrose, I.S. Brown, An increased frequency of human sperm chromosomal abnormalities after radiotherapy, Mutation Res. 174 Ž1986. 225– 291. C.J. Epstein, B. Travis, Preimplantation lethality of monosomy for mouse chromosome 19, Nature 280 Ž1979. 144– 145. Z.Q. Wei, Y.Y. Liu, G.L. Wang, X.B. Chen, An irradiation equipment for investigation on biological effects of heavy ions, Nucl. Tech. 14 Ž1991. 341–346. E.P. Evans, G. Breckon, C.E. Ford, An air-drying method for meiotic preparations for mammalian testes, Cytogenetics 3 Ž1964. 289–294. C. Lucke-Huhle, E.A. Blakely, P.Y. Chang, C.A. Tobias, Drastic G2 arrest in mammalian cells after irradiation with heavy-ion beams, Radiat. Res. 79 Ž1979. 97–112. M.R. Raju, T.S. Johnson, N. Tokita, S. Carpenter, J.H. Jett, Differences in cell-cycle progression delays after exposure to 238 Pu particles compared to X-rays, Radiat. Res. 84 Ž1980. 16–24.
Chromosomal aberrations induced by 12 C 6q heavy ion irradiation in spermatogonia and spermatocytes of mice H. Zhang a , R.L. Zheng b,) , R.Y. Wang c , Z.Q. Wei a , W.J. Li a , Q.X. Gao b, W.Q. Chen a , Z.H. Wang b, G.W. Han a , J.P. Liang a a
c
Department of Application of HeaÕy Ions, Institute of Modern Physics, The Chinese Academy of Sciences, P.O. Box 31, Lanzhou 730000, People’s Republic of China b Department of Biology, Lanzhou UniÕersity, Lanzhou 730000, People’s Republic of China Department of Gynecology and Obstetrics, The First Hospital, Lanzhou Medical College, Lanzhou 730000, People’s Republic of China Received 23 July 1997; revised 9 October 1997; accepted 16 October 1997
Abstract The testes of Kun-Ming strain mice were radiated with different doses of 12 C 6q ion or 60 Co g-ray. Chromosomal aberrations induced in spermatogonia and spermatocytes were analyzed by the air-drying method. The relative biological effectiveness ŽRBE. of 12 C 6q ion was calculated with respect to 60 Co g-ray for the induction of chromosomal aberrations. The 12 C 6q ion and 60 Co g-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models. The results showed that there was an increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values were 1.67 for aberrations of spermatogonia and 1.66 for aberrations of spermatocytes for a dose of 2.0 Gy. Moreover, a different distribution of the various types of aberrations has been found for 12 C 6q ion and 60 Co g-ray irradiations. The dose–response relationships for 12 C 6q ion and 60 Co g-ray exhibited negative curvature in both spermatogonia and spermatocytes groups: the frequencies of aberrations increased sharply at low doses and exhibited less sharp increases for higher doses, which may be related to an interaction between the chromosomal damage and a block in cell cycle. Our results may provide useful information for the assessment of genetic risks of humans exposed to heavy ions. q 1998 Elsevier Science B.V. Keywords:
12
C 6q heavy ion; Chromosomal aberration; Spermatogonium; Spermatocyte; Mouse
1. Introduction In higher organisms, the DNA is organized in chromosomes, and the induced aberrations of these chromosomes can lead to cellular lethality and mutation. Ionizing radiation has been confirmed to be one ) Corresponding author. Tel.: q86 Ž931. 891-2563; fax: q86 Ž931. 891-1100; E-mail: [email protected]
of important exogenous inducers of chromosomal aberrations w1–3x. Comparing with X-ray or g-ray, the track of a heavy ion is complex, energy is not only deposited by the primary interaction, but also by secondary electrons that may travel considerable distances from the core. This heavy ion with high linear energy transfer ŽLET. and high relative biological effectiveness ŽRBE. is also significantly more deleterious on the cellular or molecular level than
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 6 - 4
28
H. Zhang et al.r Mutation Research 398 (1998) 27–31
low LET ionizing irradiation, such as X-ray or g-ray. For example, heavy ion irradiation induces irreparable breaks in DNA more readily than low-LET irradiation w4x. It has been repeatedly demonstrated that heavy ion irradiations induce chromosomal aberrations of somatic cells w5–8x; but, chromosomal aberrations induced by irradiation in germ cells, which differ from those in somatic cells, not only indicate the cellular damage of radiated individuals, but are also partly transmitted to offspring and result in genetic effects, i.e. abnormalities, sterility and malignant diseases. Moreover, the testis is one of the most radiosensitive organs of the body and is the critical organ in the radiotherapy of Hodgkin’s disease and seminoma w9,10x. With the advent of new radiotherapy modalities, such as fast neutrons and heavy ions, there is a considerable improvement in the survival rate of cancer patients, and these radiotherapy patients of child-bearing age are concerned about risk of future children. Thus, it is important to ascertain whether the high LET radiation exposure increases the risk of chromosomal aberrations in gametes, so as to pay more attention to the reproductive potential and the possible genetic alteration in the germ cells of these patients. The spermatogonia and spermatocytes are ones of the most radiosensitive cells of the body. Moreover, radiation-induced chromosomal aberrations of spermatogonia and spermatocytes were demonstrated to be transmitted into spermatozoa w11x, and these chromosomally abnormal sperm fertilize eggs, as well as normal sperm w12x. Hence the aim of the present study is to investigate the frequency and characteristic of chromosomal aberrations induced by 12 C 6q ion in spermatogonia and spermatocytes of mice.
2. Materials and methods 2.1. Animals Male mice Ž10–12 weeks. of the Kun-Ming strain provided by Lanzhou Medical College ŽLanzhou, China. were used under identical breeding conditions. They were randomly divided with 5 animals in each group.
2.2. Irradiation procedure The mouse was positioned in a chamber which was fixed to the irradiation equipment at the Heavy Ion Research Facility in Lanzhou ŽHIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China. w13x. The abdomen Ž12 mm diameter around the scrotum. of the mouse was radiated with 12 C 6q ion beam at energy 50 MeVrU and LET 45 keVrmm in the water generated from HIRFL, with a dose rate of 0.2 Gyrmin. The remainder of the body was shielded with lead plate. The acquisition of data was automatically accomplished using a microcomputer during irradiation. Doses of the beams were determined with air ionization chamber. Animals irradiated by 60 Co g-ray were similarly given abdomen irradiation by a FTC-50H model 60 Co teletherapy machine ŽShanghai Nuclear Equipment Factory, China. at a source to surface distance ŽSSD. of 75 cm, with a dose rate 0.4 Gyrmin. The doses used for each kind of irradiation Ž 12 C 6q ion or 60 Co g-ray. were 0, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 Gy.
2.3. Preparation and analysis of chromosome Preparation and analysis of chromosome were done according to the air-drying method of Evans et al. w14x. The mice were injected Ži.p.. with colchicine Ž3 mgrkg. at 8 h after irradiation and 5 h later they were killed by cervical dislocation. The testes were transferred to 2.5 ml of a 2.2% citrate solution in fresh Petri dishes and the tunica removed. The contents of the tubule were gently teased out with curved forceps. The cell suspension produced was centrifuged at 1000 = g for 5 min. The supernatant was discarded, and the pellet was resuspended in 2 ml of hypotonic solution Ž0.4% KCl. at 378C. After 15 min, the suspension was centrifuged for 5 min at 1000 = g, and the supernatant then was removed. The cells were fixed 3 times with Carnoy’s solution Ž1:3 of glacial acetic acid and methanol.. The precleaned slides were kept cold in distilled water before being used. Three to four droplets of the final suspension were on a slide and blown upon to dry, and two slides per animal were made. Slides were
H. Zhang et al.r Mutation Research 398 (1998) 27–31 Table 1 Chromosome aberrations induced by Group
12
C 6q ion or
60
Co g-ray in spermatogonia of mice
Distribution of aberrations Žno. Ž%.. Gap
Control 0 Gy 0 12 6q C ŽGy. 0.05 0 0.1 2 Ž0.8 " 1.78. 0.2 5 Ž2.0 " 0.89. 0.5 6 Ž2.4 " 1.10. 1.0 9 Ž3.6 " 3.29. 2.0 10 Ž4.0 " 2.19. 60 Co ŽGy. 0.05 1 Ž0.4 " 0.89. 0.1 2 Ž0.8 " 1.10. 0.2 4 Ž1.6 " 1.67. 0.5 4 Ž1.6 " 0.89. 1.0 8 Ž3.2 " 3.03. 2.0 10 Ž4.0 " 2.45.
Chromatid break 1 Ž 0.4 " 0.89.
29
Total no. of aberrations Ž%.
No. of cells with aberrations Ž%.
Chromosome break 1 Ž 0.4 " 0.89.
0
2 Ž 0.8 " 1.10. 3 Ž 1.2 " 1.79. 7 Ž 2.8 " 2.28. 16 Ž 6.4 " 2.97. 30 Ž12.0 " 3.16. 57 Ž22.8 " 5.02.
3 Ž 1.2 " 1.79. 5 Ž 2.0 " 2.00. 9 Ž 3.6 " 1.67. 23 Ž 9.2 " 2.45. 41 Ž16.4 " 4.15. 70 Ž28.0 " 6.07.
2 Ž 0.8 " 1.09. 3 Ž 1.2 " 1.10. 7 Ž 2.8 " 3.29. 14 Ž 5.6 " 3.03. 25 Ž10.0 " 2.97. 40 Ž16.0 " 6.92.
0 0 0 5 Ž 2.0 " 2.00. 14 Ž 5.6 " 4.34. 32 Ž12.8 " 6.23.
5 Ž 2.0 " 2.83. 10 Ž 4.0 " 1.41. b 21 Ž 8.4 " 3.63. b 45 Ž18.0 " 4.34. c 80 Ž32.0 " 7.01. c 137 Ž54.8 " 11.05. c 3 Ž 1.2 " 5 Ž 2.0 " 11 Ž 4.4 " 23 Ž 9.2 " 47 Ž18.8 " 82 Ž32.8 "
1.10. 1.41. a 2.61. a 4.69. b 5.76. c 9.12. c
1 Ž 0.4 " 0.89. 5 Ž 2.0 " 2.83. 10 Ž 4.0 " 1.41. 21 Ž 8.4 " 3.63. 45 Ž18.0 " 4.34. 61 Ž24.4 " 3.85. 102 Ž40.8 " 4.38. 3 Ž 1.2 " 1.10. 5 Ž 2.0 " 1.41. 11 Ž 4.4 " 2.61. 23 Ž 9.2 " 4.69. 47 Ž18.8 " 5.76. 66 Ž26.4 " 6.07.
Results based on 250 analyzed cells from 5 samples. Data represent mean " SD. The significance of differences between radiated groups and controls was determined by Student’s t-test. a p - 0.05, b p - 0.01, c p - 0.001 vs. control.
stained in 10% Giemsa for 8 min. Fifty cells at diakinesis-metaphase spermatogonia or spermatocytes for each animal were respectively scored miTable 2 Chromosome aberrations induced by Group
12
C 6q ion or
60
Co g-ray in spermatocytes of mice
Distribution of aberrations Žno. Ž%.. Gap
Control 0 Gy 1 Ž0.4 " 0.89. 12 6q C ŽGy. 0.05 1 Ž0.4 " 0.89. 0.1 3 Ž1.2 " 1.79. 0.2 3 Ž1.2 " 1.09. 0.5 6 Ž2.4 " 0.89. 1.0 11 Ž4.4 " 2.61. 2.0 8 Ž3.2 " 3.03. 60 Co ŽGy. 0.05 2 Ž0.8 " 1.10. 0.1 2 Ž0.8 " 1.79. 0.2 4 Ž1.6 " 0.89. 0.5 7 Ž2.8 " 2.28. 1.0 6 Ž2.4 " 2.61. 2.0 9 Ž3.6 " 2.19.
Chromatid break 1 Ž 0.4 " 0.89.
croscopically at 1000-fold magnification. Abnormal chromosomes were classified into gap, chromatid break and chromosome break Žincluding fragments..
Total no. of aberrations Ž%.
No. of cells with aberrations Ž%.
Chromosome break 0
4 Ž 1.6 " 1.67. 5 Ž 2.0 " 2.83. 12 Ž 4.8 " 3.63. 22 Ž 8.8 " 6.00. 29 Ž11.6 " 3.03. 49 Ž19.6 " 4.34.
3 Ž 1.2 " 1.79. 7 Ž 2.8 " 2.68. 14 Ž 5.6 " 2.61. 31 Ž12.4 " 4.90. 50 Ž20.0 " 3.85. 91 Ž36.4 " 9.86.
3 Ž 1.2 " 1.79. 6 Ž 2.4 " 2.61. 6 Ž 2.4 " 1.67. 15 Ž 6.0 " 2.45. 27 Ž10.8 " 4.34. 45 Ž18.0 " 3.35.
0 1 Ž 0.4 " 0.89. 4 Ž 1.6 " 1.67. 9 Ž 3.6 " 1.41. 19 Ž 7.6 " 4.82. 35 Ž14.0 " 6.23.
2 Ž 0.8 " 1.10. 8 Ž 3.2 " 2.28. a 15 Ž 6.0 " 2.61. b 29 Ž11.6 " 5.02. b 59 Ž23.6 " 6.84. c 90 Ž36.0 " 7.35. c 148 Ž59.2 " 11.44. c 5 Ž 2.0 " 9 Ž 3.6 " 14 Ž 5.6 " 30 Ž12.0 " 52 Ž20.8 " 89 Ž35.6 "
2.00. 2.19. a 3.29. a 3.63. b 4.15. c 8.65. c
2 Ž 0.8 " 1.10. 8 Ž 3.2 " 2.28. 15 Ž 6.0 " 2.61. 29 Ž11.6 " 5.02. 59 Ž23.6 " 6.84. 67 Ž26.8 " 5.40. 107 Ž42.8 " 9.10. 5 Ž 2.0 " 2.00. 9 Ž 3.6 " 2.19. 14 Ž 5.6 " 3.29. 30 Ž12.0 " 3.63. 51 Ž20.4 " 4.56. 70 Ž28.0 " 3.35.
Results based on 250 analyzed cells from 5 samples. Data represent mean " SD. The significance of differences between radiated groups and controls was determined by Student’s t-test. a p - 0.05, b p - 0.01, c p - 0.001 vs. control.
30
H. Zhang et al.r Mutation Research 398 (1998) 27–31
3. Results and discussion The frequencies and distributions of chromosomal aberrations observed in spermatogonia and spermatocytes induced by 12 C 6q ion and 60 Co g-ray are shown in Tables 1 and 2, respectively. There was an increase in frequency of chromosomal aberrations in all the treated groups compared to controls. There were 54.8 and 59.2% chromosomal aberrations for spermatogonia and spermatocytes exposed to 2 Gy of 12 C 6q ion, and 32.8 and 35.6% in the spermatogonia and spermatocytes which received 2 Gy of 60 Co g-ray. The RBE of 12 C 6q ion was calculated with respect to 60 Co g-ray for induction of chromosomal aberrations. The RBE values were 1.67 for aberrations of spermatogonia and 1.66 for aberrations of spermatocytes for a dose of 2.0 Gy. The majority of aberrations observed were chromatid break and chromosome break. A different distribution of the various types of aberrations has been found for 12 C 6q ion and 60 Co g-ray irradiations: in 60 Co g-ray irradiation, the distribution was dominated by the chromatid break; in 12 C 6q ion irradiation, the distribution of aberrations was clearly dominated by chromosome break and fragments, even complete disintegrations of parts of chromosomes, which is thought to be a manifestation of the high and local energy density in a particle track w7x. Furthermore, at low doses of 0.05–1 Gy for 60 Co g-ray or 0.05–0.5 Gy for 12 C 6q ion, the frequencies of cells with chromosomal aberrations were equal to the total frequencies of chromosomal aberrations. At higher doses of ) 1 Gy for 60 Co g-ray or ) 0.5 Gy for 12 C 6q ion, increasing numbers of cells with multiple aberrations were observed in both spermatogonia and spermatocytes Žthe frequencies of cells with aberrations became lower than those of total aberrations., which demonstrated that higher doses of ionizing irradiation increase not only frequency of cells with aberrations, but also the amount of induced chromosomal aberrations per cell, and this also suggested that with increasing doses, the degree of damage of radiated cells increases. From Tables 1 and 2 it can be seen that the frequencies of chromosomal aberrations induced by both 12 C 6q ion or 60 Co g-ray were higher in spermatocytes than in spermatogonia. This variation might be due to differences in the sensitivity to mutagenic
agents of male germ cells in different stages of the cell development. Male germ cells in meiosis and postmeiosis phases are generally more sensitive to mutagenic agents than those in other phases. The 12 C 6q ion and 60 Co g-ray dose–response relationships for chromosomal aberrations of spermatogonia and spermatocytes were plotted by linear quadratic models in Fig. 1. The equations obtained for spermatogonia and spermatocytes were, respectively, Y s 0.547 q 36.670 D y 4.792 D 2 and Y s 1.781 q 43.590 D y 7.523 D 2 in 12 C 6q ion exposure and Y s 0.200 q 20.107 D y 1.888 D 2 and Y s 1.052 q 22.774 D y 2.762 D 2 in 60 Co g-ray exposure Ž Y is chromosomal aberrations per cell and D is the irradiation dose in Gy.. As shown in Fig. 1, the dose–response relationships exhibited negative curvature, and the slopes of these curves decrease
Fig. 1. Dose–response relationships of chromosomal aberrations induced by 12 C 6q ion Žv . and 60 Co g-ray ŽB. in spermatogonia ŽA. and spermatocytes ŽB. of mice. Data represent mean"SD, ns 5.
H. Zhang et al.r Mutation Research 398 (1998) 27–31
with increasing doses. The frequencies of aberrations increased sharply at low doses and exhibited a less sharp increases for higher doses, especially for 12 C 6q ion irradiation. It might be possible that the curvature of dose–response relationships is due to an interaction between the chromosomal damage and a block in cell cycle w15,16x, which arrests the majority of heavily damaged cells in the G2 phase. Cells with severe chromosome damage cannot proceed to mitosis or meiosis. This interference between cell cycle effects and expression of chromosomal damage should be minimal at low doses w7x. In conclusion, 12 C 6q ion irradiation significantly increased the frequencies of chromosomal aberrations in spermatogonia and spermatocytes of mice, even at low dose of 0.05 Gy. Our results may provide useful information for assessment of genetic risks of human exposed to heavy ions. Radiosensitivity of germ cells is higher in human than in mouse. Hence there is a need for further investigation of the relationship of chromosomal aberrations induced by heavy ions between human and mouse germ cells, and finding out reasonable comparable rules among species.
w3x
w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
Acknowledgements This project was supported by China Postdoctoral Science Foundation and Institute Head’s Science Foundation of The Chinese Academy of Sciences. We express our thanks to the accelerator crew at the HIRFL, National Laboratory of Heavy Ion Accelerator in Lanzhou.
w12x
w13x
w14x
References w15x w1x N. Kamada, The effects of radiation on chromosomes of bone marrow cells. 1. Type and frequency of rat chromosome aberrations induced by fast neutrons X-ray and gamma rays, Acta Haematol. Jap. 32 Ž1969. 212–235. w2x Y. Matsuda, T. Yamada, I. Tobar, A. Ohkawa, Preliminary study on chromosomal aberrations in eggs of mice fertilized
w16x
31
in vitro after X-irradiation, Mutation Res. 121 Ž1983. 125– 130. Y. Kodama, J.-i. Kushiro, Y. Hirai, Y. Kusunoki, N. Nakamura, M. Akiyama, A.A. Awa, Frequent involvement of visible chromosomal deletion in X-Ray-induced mutations at the HLA-A locus in human T-lymphocytes, Mutation Res. 309 Ž1994. 63–72. M.A. Ritter, J.E. Cleaver, C.A. Tobias, High-LET radiations induce a large proportion of non-rejoining DNA breaks, Nature 266 Ž1977. 653–655. W. Muller, H.G. Miltenburger, G. Kraft, Chromosome aberrations in Chinese hamster cells cultures induced by heavy ion irradiation, GSI-Scientific Report 1982, GSI, Darmstadt, 1983, 197 pp. W. Muller, H.G. Miltenburger, G. Kraft, Chromosome aberrations in V79 Chinese hamster cells produced by heave charged particles, GSI-Scientific Report 1983, GSI Darmstadt, 1984, 202 pp. C.R. Geard, Charged particle cytogentics: effects of LET, fluence, and particle separation on chromosome aberrations, Radiat. Res. 104 Ž1985. S112–S121. G. Kraft, Radiological effects of very heavy ions: inactivation, induction of chromosome aberrations and strand breaks, Nucl. Sci. Appl. 3 Ž1987. 1–28. R.D. Amelar, L. Dubin, R.S. Hotchkiss, Restoration of fertility following unilateral orchiectomy and radiation therapy for testicular tumors, J. Urol. 106 Ž1971. 714–718. B. Speiser, P. Rubin, G. Cassarett, Aspermia following lower truncal irradiation in Hodgkin’s disease, Cancer 32 Ž1973. 692–698. R.H. Matin, K. Hildebrand, J. Yamamoto, A. Rademaker, M. Barnes, G. Douglas, K. Arthur, T. Ringrose, I.S. Brown, An increased frequency of human sperm chromosomal abnormalities after radiotherapy, Mutation Res. 174 Ž1986. 225– 291. C.J. Epstein, B. Travis, Preimplantation lethality of monosomy for mouse chromosome 19, Nature 280 Ž1979. 144– 145. Z.Q. Wei, Y.Y. Liu, G.L. Wang, X.B. Chen, An irradiation equipment for investigation on biological effects of heavy ions, Nucl. Tech. 14 Ž1991. 341–346. E.P. Evans, G. Breckon, C.E. Ford, An air-drying method for meiotic preparations for mammalian testes, Cytogenetics 3 Ž1964. 289–294. C. Lucke-Huhle, E.A. Blakely, P.Y. Chang, C.A. Tobias, Drastic G2 arrest in mammalian cells after irradiation with heavy-ion beams, Radiat. Res. 79 Ž1979. 97–112. M.R. Raju, T.S. Johnson, N. Tokita, S. Carpenter, J.H. Jett, Differences in cell-cycle progression delays after exposure to 238 Pu particles compared to X-rays, Radiat. Res. 84 Ž1980. 16–24.