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Chromosomal aberrations induced by 12C6+ ions and 60Co γ-rays in mouse immature oocytes
Mutation Research 595 (2006) 37–41
Chromosomal aberrations induced by 12C6+ ions and 60 Co ␥-rays in mouse immature oocytes Hong Zhang ∗ , Xin Duan, Zhigang Yuan, Wenjian Li, Guangming Zhou, Qingming Zhou, Liu Bing, Fengling Min, Xiaoda Li, Yi Xie Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China Received 26 July 2005; received in revised form 23 September 2005; accepted 10 October 2005 Available online 7 December 2005
Abstract The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of 12 C6+ ion or 60 Co ␥-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of 12 6+ C ion was calculated with respect to 60 Co ␥-ray for the induction of chromosomal aberrations. The 12 C6+ ion and 60 Co ␥-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for 12 C6+ ions relative to 60 Co ␥-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for 12 C6+ ion and 60 Co ␥-ray irradiations. The dose–response relationships for 12 C6+ ion and 60 Co ␥-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation. © 2005 Elsevier B.V. All rights reserved. Keywords:
12 C6+
heavy ion; Chromosomal aberration; Oocytes; Mouse
1. Introduction The relatively high sensitivity of oocytes to irradiation is manifested by ovarian failure and infertility following pelvic radiotherapy [1,2]. There is abundant evidence from the animals that exposure of female germ cells to ionizing radiation causes dose-related increases in the incidences of numerical and structural chromosome anomalies [3–9]. Most of these studies were performed with X-ray or gamma (␥)-ray irradiation. Comparing with X-ray or ␥-ray, the track of a heavy ion is complex, ∗
Corresponding author. Tel.: +86 931 4969344; fax: +86 931 8272100. E-mail address: [email protected] (H. Zhang). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.10.010
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 (LET) and high relative biological effectiveness (RBE) is also significantly more deleterious on the cellular or molecular level than low LET ionizing irradiation, such as X-ray or ␥-ray. For example, heavy ion irradiation induces irreparable breaks in DNA more readily than low-LET irradiation [10]. It has been repeatedly demonstrated that heavy ion irradiations induce chromosomal aberrations of somatic cells [11–14]. However, 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
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H. Zhang et al. / Mutation Research 595 (2006) 37–41
offspring and result in genetic effects, such as abnormalities, sterility and malignant diseases [15]. Moreover, the ovary is the radiosensitive organs of the body and is the critical organ affected in the radiotherapy of Hodgkin’s disease, cervical and rectal cancers [2]. 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 women 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 immature oocyte is the highly radiosensitive germ cell in female mammals [16–18]. Moreover, radiation-induced chromosomal aberrations of immature oocytes may be transmitted into mature oocytes, and these chromosomally abnormal eggs may also be fertilized. Hence, the aim of the present study is to investigate the frequency and characteristic of chromosomal aberrations induced by 12 C6+ ion and 60 Co ␥-rays in immature oocytes of mice. 2. Materials and methods 2.1. Animals Female outbreed Kun-Ming mice (3 weeks) provided by Lanzhou Medical College (Lanzhou, China) were used under identical breeding conditions. They were randomly divided with 7 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 (HIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China). The lower abdomen of the mouse was radiated with 12 C6+ ion beam at energy 80 MeV/u and LET 31.3 keV/m in the water generated from HIRFL, with a dose rate of 0.5 Gy/min. The remainder of the body was shielded with lead plate. The acquisition of data (preset numbers converted by doses of irradiation) was automatically accomplished using a microcomputer during irradiation. Doses of the beams were determined with air ionization chamber. Animals irradiated by 60 Co ␥-ray were similarly given lower abdomen irradiation by a FTC-50H model 60 Co teletherapy machine (Shanghai Nuclear Equipment Factory, China) at a source to surface distance (SSD) of 75 cm, with a dose rate 0.4 Gy/min. The doses used for each kind of irradiation (12 C6+ ion or 60 Co ␥-ray) were 0, 0.5, 1.0, 2.0, 4.0 or 6.0 Gy.
2.3. Preparation and analysis of chromosome Preparation and analysis of chromosome were done according to the air-drying method of Tarkowski [19]. The mice were injected (i.p.) with 5 IU of pregnant mare’s serum gonadotrophin (PMSG) followed 48 h later by 5 IU of human chorionic gonadotrophin (HCG) at 7 weeks after irradiation, which interval was used to observe the effects of exposure of mouse immature oocytes with irradiations on chromosomal aberrations after sexual maturity. At 16 h after HCG, the mice were injected intraperitoneally with colcemid (0.002 mg/kg) and 2 h later they were killed by cervical dislocation. The oocytes were collected from the oviducts and treated with hypotonic solution (1% sodium citrate) for 5–10 min at room temperature. The cells were fixed with 1:3 of methanol: glacial acetic acid. Three to four droplets of the final suspension were on a slide and dried by air. Slides were stained with lactic–acetic–orcein. Intact and well-spread metaphase II oocytes were analyzed. Abnormal chromosomes were classified into gap, chromatid break and chromosome break (including fragments) [6,16].
3. Results and discussion The frequencies and distributions of chromosomal aberrations observed in mouse immature oocytes induced by 12 C6+ ion and 60 Co ␥-ray are shown in Table 1. The total number of aberrations in the 0.5 60 Co ␥ group did not differ statistically (p > 0.05) from the controls (see Table 1). There was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. Despite there were reports that immature oocytes were essentially refractory to radiation-induced to specific-locus mutations in mouse [20,21] and guinea-pig [22], preovulatory phase of mouse oogenesis was more radiosensitive than earlier stages [3,23] and the rate of chromosomal anomalies in mouse oocytes induced by irradiation was larger in old than young females [6], our data indicated both 12 C6+ ion and 60 Co ␥-ray induced significant increases in chromosomal aberrations in mouse immature oocytes. This data are consistent with earlier results from some researchers [4,5] and provided support for the conclusion that follicles in primordial stages are highly radiosensitive [16–18]. Moreover, chromosomal aberrations induced by 12 C6+ were higher than that induced by 60 Co ␥-ray (Table 1), for example there were 55.3% chromosomal aberrations exposed to 6 Gy of 12 C6+ ion and only 41.8% in the oocytes which received 6 Gy of 60 Co ␥-ray. The RBE of 12 C6+ ion was calculated with respect to 60 Co ␥ray for induction of chromosomal aberrations. The RBE values were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses
H. Zhang et al. / Mutation Research 595 (2006) 37–41
39
Table 1 Mean numbers of oocytes and chromosome aberrations induced by 12 C6+ ion or 60 Co ␥-ray in immature oocytes of mice Group
Number of aberrations per cell (%)
Control 0 Gy 12 C6+
Gap
Chromatid break
1 (0.7 ± 0.80)
0
Total number of aberrations per cell (%) Chromosome breaks (including fragments) 0
Number of oocytes ovulated/collected per mouse
Total number of cells analyzed
1 (0.7 ± 0.80)
20.7 ± 4.15
145
(Gy) 1 (0.8 ± 0.91) 4 (3.4 ± 1.09) 4 (4.0 ± 2.17) 6 (8.0 ± 3.25) 4 (10.5 ± 4.20)
4 (3.0 ± 1.81) 3 (2.5 ± 1.44) 5 (5.0 ± 2.96) 6 (8.0 ± 3.25) 6 (15.8 ± 3.25)
2 (1.5 ± 1.10) 5 (4.2 ± 3.23) 12 (11.9 ± 4.14) 14 (18.4 ± 4.68) 11 (29.0 ± 7.10)
7 (5.3 ± 2.05)b 12 (10.1 ± 2.15)c 21 (20.8 ± 4.12)c 26 (34.2 ± 5.01)c 21 (55.3 ± 6.83)c
19.0 ± 4.40 17.0 ± 4.16a 14.4 ± 3.51a 10.9 ± 2.67b 5.4 ± 2.30c
133 119 101 76 38
(Gy) 0.5 1 (0.7 ± 1.03) 1.0 3 (2.2 ± 1.34) 2.0 7 (5.8 ± 2.84) 4.0 7 (7.7 ± 2.39) 6.0 8 (14.6 ± 4.01)
2 (1.4 ± 1.64) 3 (2.2 ± 1.34) 6 (5.0 ± 2.62) 9 (9. 9 ± 4.69) 8 (14.6 ± 4.01)
0 0 3 (2.5 ± 1.09) 6 (6.6 ± 3.81) 6 (10.9 ± 4.25)
3 (2.1 ± 1.21) 6 (4.4 ± 3.21)a 16 (13.2 ± 5.07)c 22 (24.2 ± 6.17)c 23 (41.8 ± 8.14)c
20.3 ± 4.82 19.4 ± 3.87 17.3 ± 3.15 13.0 ± 4.24b 7.9 ± 2.85c
142 136 121 91 55
0.5 1.0 2.0 4.0 6.0 60 Co
Data represent mean ± S.D. 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.
of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, and showed decrease with increase of radiation doses. It may be possible that at high doses, there is an interaction between the chromosomal damage and a block in cell cycle, especially in irradiation of 12 C6+ ion which induces more unrepairable breaks in DNA than 60 Co ␥-ray. The majority of heavily damaged cells was arrested in the G2 phase and cannot proceed to mitosis or meiosis. This interference between cell cycle effects and expression of chromosomal damage should be minimal at low doses [13]. The majority of aberrations observed were chromatid break and chromosome break (including fragments). A different distribution of the various types of aberrations has been found for 12 C6+ ion and 60 Co ␥-ray irradiations (Table 1): in 60 Co ␥-ray irradiation, the distribution was dominated by the chromatid break; whereas in 12 C6+ 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 [13]. The 12 C6+ ion and 60 Co ␥-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models in Fig. 1. The equations were Y = 1.711 + 8.253 D + 0.095 D2 in 12 C6+ ion exposure and Y = 0.567 + 4.658 D + 0.364 D2 in 60 Co ␥-ray exposure (Y is the chromosomal aberrations per cell and D
is the irradiation dose in Gy). And the curvature (D2 ) of dose–response relationship was less for 12 C6+ ion irradiation than for 60 Co ␥-ray irradiation. This may be interpreted that DNA lesions induced by low LET irradiation are repaired partly by cell and lesions induced by high LET irradiation, however, are unrepairiable [14]. As shown in Fig. 1, the dose–response relationships exhibited positive correlations (r2 = 0.995 for 12 C6+ ion exposure and r2 = 0.990 for 60 Co ␥-ray exposure), and
Fig. 1. Dose–response relationships of chromosomal aberrations induced by 12 C6+ ion () and 60 Co ␥-ray (䊉) in immature oocytes of mice. Data represent mean ± S.D., n = 7.
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H. Zhang et al. / Mutation Research 595 (2006) 37–41
the frequencies of aberrations increased with increasing doses, especially for 12 C6+ ion irradiation, which confirmed the heavy ion with high LET and high RBE is significantly more deleterious on germ cells than low LET ionizing irradiation. Our results also showed that the average number of oocytes ovulated per female was reduced by exposures with 12 C6+ ion and 60 Co ␥-ray (Table 1). The effect was dose-dependent and more severe in the group exposed with 12 C6+ ion (Table 1). The mechanism of reduction in mean number of oocytes may be through the follicular degeneration induced by irradiation [9,24,25]. In conclusion, 12 C6+ ion irradiation significantly increased the frequencies of chromosomal aberrations in immature oocytes of mice, even at low dose of 0.5 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 [26]. 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 in order to better clarify genetic risk associated with the exposure of women in childhood to irradiation. Acknowledgements We express our thanks to the accelerator crew at the HIRFL, National Laboratory of Heavy Ion Accelerator in Lanzhou. References [1] W.H. Wallace, S.M. Shalet, J.H. Hendry, P.H. Morris-Jones, H.R. Gattamaneni, Ovarian failure following abdominal irradiation in childhood: the radiosensitivity of the human oocyte, Br. J. Radiol. 62 (1989) 995–998. [2] D. Meirow, D. Nugent, The effects of radiotherapy and chemotherapy on female reproduction, Hum. Reprod. Update 7 (2001) 535–543. [3] W. Reichert, I. Hansmann, G. Rohrborn, Chromosome anomalies in mouse oocytes after irradiation, Humangenetik 28 (1975) 25–38. [4] C.S. Griffin, C. Tease, Gamma-ray-induced numerical and structural chromosome anomalies in mouse immature oocytes, Mutat. Res. 202 (1988) 209–213. [5] C.S. Griffin, C. Tease, G. Fisher, The effect of low-dose Xirradiation on numerical and structural chromosome anomaly induction in mouse immature oocytes, Mutat. Res. 231 (1990) 137–142. [6] C. Tease, G. Fisher, The influence of maternal age on radiationinduced chromosome aberrations in mouse oocytes, Mutat. Res. 262 (1991) 57–62.
[7] C. Tease, G. Fisher, Cytogenetic and genetic studies of radiationinduced chromosome damage in mouse oocytes. I. Numerical and structural chromosome anomalies in metaphase II oocytes, pre- and post-implantation embryos, Mutat. Res. 349 (1996) 145– 153. [8] P. Jacquet, L. de Saint-Georges, J. Buset, S. Baatout, J. Vankerkom, L. Baugnet-Mahieu, Cytogenetic effects of X-rays in the guinea pig female germ cells. I. The immature oocyte, Mutat. Res. 391 (1997) 189–192. [9] P. Jacquet, L. de Saint-Georges, J. Buset, S. Baatout, J. Vankerkom, L. Baugnet-Mahieu, Cytogenetic effects of X-rays in the guinea pig female germ cells. II. The maturing oocyte, Mutat. Res. 391 (1997) 193–199. [10] M.A. Ritter, J.E. Cleaver, C.A. Tobias, High-LET radiations induce a large proportion of non-rejoining DNA breaks, Nature 266 (1977) 653–655. [11] 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. [12] 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. [13] C.R. Geard, Charged particle cytogentics: effects of LET, fluence, and particle separation on chromosome aberrations, Radiat. Res. 104 (1985) S112–S121. [14] G. Kraft, Radiological effects of very heavy ions: inactivation, induction of chromosome aberrations and strand breaks, Nucl. Sci. Appl. 3 (1987) 1–28. [15] T. Nomura, H. Nakajima, H. Ryo, L.Y. Li, Y. Fukudome, S. Adachi, H. Gotoh, H. Tanaka, Transgenerational transmission of radiation and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes, Cytogenet. Genome Res. 104 (2004) 252–260. [16] T. Straume, T.C. Kwan, L.S. Goldstein, R.L. Dobson, Measurement of neutron-induced genetic damage in mouse immature oocytes, Mutat. Res. 248 (1991) 123–133. [17] H. Peters, The effect of radiation in early life on the morphology and reproductive function of the mouse ovary, in: A. McLaren (Ed.), Advances in Reproductive Physiology, vol.4, Logos Press, London, UK, 1969, pp. 149–185. [18] T.G. Baker, Radiosensitivity of mammalian oocytes with particular reference to the human female, Am. J. Obstet. Gynecol. 110 (1971) 746–761. [19] A.K. Tarkowski, An air-drying method for chromosome preparations from mouse eggs, Cytogenetics 5 (1966) 394–400. [20] W.L. Russell, Effect of the interval between irradiation and conception on mutation frequency in female mice, Proc. Natl. Acad. Sci. U.S.A. 54 (1965) 1552–1557. [21] W.L. Russell, Mutation frequencies in female mice and the estimation of genetic hazards of radiation in women, Proc. Natl. Acad. Sci. U.S.A. 74 (1977) 3523–3527. [22] P. Jacquet, J. Buset, J. Vankerkom, S. Baatout, L. de SaintGeorges, L. Baugnet-Mahieu, C. Desaintes, Radiation-induced chromosome aberrations in guinea-pig growing oocytes, and their relation to follicular atresia, Mutat. Res. 473 (2001) 249– 254. [23] P. Jacquet, I. Adriaens, J. Buset, M. Neefs, J. Vankerkom, Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system, Mutat. Res. 583 (2005) 168–177.
H. Zhang et al. / Mutation Research 595 (2006) 37–41 [24] Y. Morita, G.I. Perez, F. Paris, S.R. Miranda, D. Ehleiter, A. Haimovitz-Friedman, Z. Fuks, Z. Xie, J.C. Reed, E.H. Schuchman, R.N. Kolesnick, J.L. Tilly, Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy, Nat. Med. 6 (2000) 1109– 1114.
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[25] Y.D. Yoon, J.H. Kim, K.H. Kim, J.K. Lee, Amifostine has an inhibitory effect on the radiation-induced p53-branched cascade in the immature mouse ovary, In Vivo 19 (2005) 509–514. [26] Y. Kamiguchi, H. Tateno, Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa, Mutat Res. 504 (2002) 183–191.
Chromosomal aberrations induced by 12C6+ ions and 60 Co ␥-rays in mouse immature oocytes Hong Zhang ∗ , Xin Duan, Zhigang Yuan, Wenjian Li, Guangming Zhou, Qingming Zhou, Liu Bing, Fengling Min, Xiaoda Li, Yi Xie Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China Received 26 July 2005; received in revised form 23 September 2005; accepted 10 October 2005 Available online 7 December 2005
Abstract The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of 12 C6+ ion or 60 Co ␥-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of 12 6+ C ion was calculated with respect to 60 Co ␥-ray for the induction of chromosomal aberrations. The 12 C6+ ion and 60 Co ␥-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for 12 C6+ ions relative to 60 Co ␥-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for 12 C6+ ion and 60 Co ␥-ray irradiations. The dose–response relationships for 12 C6+ ion and 60 Co ␥-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation. © 2005 Elsevier B.V. All rights reserved. Keywords:
12 C6+
heavy ion; Chromosomal aberration; Oocytes; Mouse
1. Introduction The relatively high sensitivity of oocytes to irradiation is manifested by ovarian failure and infertility following pelvic radiotherapy [1,2]. There is abundant evidence from the animals that exposure of female germ cells to ionizing radiation causes dose-related increases in the incidences of numerical and structural chromosome anomalies [3–9]. Most of these studies were performed with X-ray or gamma (␥)-ray irradiation. Comparing with X-ray or ␥-ray, the track of a heavy ion is complex, ∗
Corresponding author. Tel.: +86 931 4969344; fax: +86 931 8272100. E-mail address: [email protected] (H. Zhang). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.10.010
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 (LET) and high relative biological effectiveness (RBE) is also significantly more deleterious on the cellular or molecular level than low LET ionizing irradiation, such as X-ray or ␥-ray. For example, heavy ion irradiation induces irreparable breaks in DNA more readily than low-LET irradiation [10]. It has been repeatedly demonstrated that heavy ion irradiations induce chromosomal aberrations of somatic cells [11–14]. However, 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
38
H. Zhang et al. / Mutation Research 595 (2006) 37–41
offspring and result in genetic effects, such as abnormalities, sterility and malignant diseases [15]. Moreover, the ovary is the radiosensitive organs of the body and is the critical organ affected in the radiotherapy of Hodgkin’s disease, cervical and rectal cancers [2]. 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 women 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 immature oocyte is the highly radiosensitive germ cell in female mammals [16–18]. Moreover, radiation-induced chromosomal aberrations of immature oocytes may be transmitted into mature oocytes, and these chromosomally abnormal eggs may also be fertilized. Hence, the aim of the present study is to investigate the frequency and characteristic of chromosomal aberrations induced by 12 C6+ ion and 60 Co ␥-rays in immature oocytes of mice. 2. Materials and methods 2.1. Animals Female outbreed Kun-Ming mice (3 weeks) provided by Lanzhou Medical College (Lanzhou, China) were used under identical breeding conditions. They were randomly divided with 7 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 (HIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China). The lower abdomen of the mouse was radiated with 12 C6+ ion beam at energy 80 MeV/u and LET 31.3 keV/m in the water generated from HIRFL, with a dose rate of 0.5 Gy/min. The remainder of the body was shielded with lead plate. The acquisition of data (preset numbers converted by doses of irradiation) was automatically accomplished using a microcomputer during irradiation. Doses of the beams were determined with air ionization chamber. Animals irradiated by 60 Co ␥-ray were similarly given lower abdomen irradiation by a FTC-50H model 60 Co teletherapy machine (Shanghai Nuclear Equipment Factory, China) at a source to surface distance (SSD) of 75 cm, with a dose rate 0.4 Gy/min. The doses used for each kind of irradiation (12 C6+ ion or 60 Co ␥-ray) were 0, 0.5, 1.0, 2.0, 4.0 or 6.0 Gy.
2.3. Preparation and analysis of chromosome Preparation and analysis of chromosome were done according to the air-drying method of Tarkowski [19]. The mice were injected (i.p.) with 5 IU of pregnant mare’s serum gonadotrophin (PMSG) followed 48 h later by 5 IU of human chorionic gonadotrophin (HCG) at 7 weeks after irradiation, which interval was used to observe the effects of exposure of mouse immature oocytes with irradiations on chromosomal aberrations after sexual maturity. At 16 h after HCG, the mice were injected intraperitoneally with colcemid (0.002 mg/kg) and 2 h later they were killed by cervical dislocation. The oocytes were collected from the oviducts and treated with hypotonic solution (1% sodium citrate) for 5–10 min at room temperature. The cells were fixed with 1:3 of methanol: glacial acetic acid. Three to four droplets of the final suspension were on a slide and dried by air. Slides were stained with lactic–acetic–orcein. Intact and well-spread metaphase II oocytes were analyzed. Abnormal chromosomes were classified into gap, chromatid break and chromosome break (including fragments) [6,16].
3. Results and discussion The frequencies and distributions of chromosomal aberrations observed in mouse immature oocytes induced by 12 C6+ ion and 60 Co ␥-ray are shown in Table 1. The total number of aberrations in the 0.5 60 Co ␥ group did not differ statistically (p > 0.05) from the controls (see Table 1). There was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. Despite there were reports that immature oocytes were essentially refractory to radiation-induced to specific-locus mutations in mouse [20,21] and guinea-pig [22], preovulatory phase of mouse oogenesis was more radiosensitive than earlier stages [3,23] and the rate of chromosomal anomalies in mouse oocytes induced by irradiation was larger in old than young females [6], our data indicated both 12 C6+ ion and 60 Co ␥-ray induced significant increases in chromosomal aberrations in mouse immature oocytes. This data are consistent with earlier results from some researchers [4,5] and provided support for the conclusion that follicles in primordial stages are highly radiosensitive [16–18]. Moreover, chromosomal aberrations induced by 12 C6+ were higher than that induced by 60 Co ␥-ray (Table 1), for example there were 55.3% chromosomal aberrations exposed to 6 Gy of 12 C6+ ion and only 41.8% in the oocytes which received 6 Gy of 60 Co ␥-ray. The RBE of 12 C6+ ion was calculated with respect to 60 Co ␥ray for induction of chromosomal aberrations. The RBE values were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses
H. Zhang et al. / Mutation Research 595 (2006) 37–41
39
Table 1 Mean numbers of oocytes and chromosome aberrations induced by 12 C6+ ion or 60 Co ␥-ray in immature oocytes of mice Group
Number of aberrations per cell (%)
Control 0 Gy 12 C6+
Gap
Chromatid break
1 (0.7 ± 0.80)
0
Total number of aberrations per cell (%) Chromosome breaks (including fragments) 0
Number of oocytes ovulated/collected per mouse
Total number of cells analyzed
1 (0.7 ± 0.80)
20.7 ± 4.15
145
(Gy) 1 (0.8 ± 0.91) 4 (3.4 ± 1.09) 4 (4.0 ± 2.17) 6 (8.0 ± 3.25) 4 (10.5 ± 4.20)
4 (3.0 ± 1.81) 3 (2.5 ± 1.44) 5 (5.0 ± 2.96) 6 (8.0 ± 3.25) 6 (15.8 ± 3.25)
2 (1.5 ± 1.10) 5 (4.2 ± 3.23) 12 (11.9 ± 4.14) 14 (18.4 ± 4.68) 11 (29.0 ± 7.10)
7 (5.3 ± 2.05)b 12 (10.1 ± 2.15)c 21 (20.8 ± 4.12)c 26 (34.2 ± 5.01)c 21 (55.3 ± 6.83)c
19.0 ± 4.40 17.0 ± 4.16a 14.4 ± 3.51a 10.9 ± 2.67b 5.4 ± 2.30c
133 119 101 76 38
(Gy) 0.5 1 (0.7 ± 1.03) 1.0 3 (2.2 ± 1.34) 2.0 7 (5.8 ± 2.84) 4.0 7 (7.7 ± 2.39) 6.0 8 (14.6 ± 4.01)
2 (1.4 ± 1.64) 3 (2.2 ± 1.34) 6 (5.0 ± 2.62) 9 (9. 9 ± 4.69) 8 (14.6 ± 4.01)
0 0 3 (2.5 ± 1.09) 6 (6.6 ± 3.81) 6 (10.9 ± 4.25)
3 (2.1 ± 1.21) 6 (4.4 ± 3.21)a 16 (13.2 ± 5.07)c 22 (24.2 ± 6.17)c 23 (41.8 ± 8.14)c
20.3 ± 4.82 19.4 ± 3.87 17.3 ± 3.15 13.0 ± 4.24b 7.9 ± 2.85c
142 136 121 91 55
0.5 1.0 2.0 4.0 6.0 60 Co
Data represent mean ± S.D. 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.
of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, and showed decrease with increase of radiation doses. It may be possible that at high doses, there is an interaction between the chromosomal damage and a block in cell cycle, especially in irradiation of 12 C6+ ion which induces more unrepairable breaks in DNA than 60 Co ␥-ray. The majority of heavily damaged cells was arrested in the G2 phase and cannot proceed to mitosis or meiosis. This interference between cell cycle effects and expression of chromosomal damage should be minimal at low doses [13]. The majority of aberrations observed were chromatid break and chromosome break (including fragments). A different distribution of the various types of aberrations has been found for 12 C6+ ion and 60 Co ␥-ray irradiations (Table 1): in 60 Co ␥-ray irradiation, the distribution was dominated by the chromatid break; whereas in 12 C6+ 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 [13]. The 12 C6+ ion and 60 Co ␥-ray dose–response relationships for chromosomal aberrations were plotted by linear quadratic models in Fig. 1. The equations were Y = 1.711 + 8.253 D + 0.095 D2 in 12 C6+ ion exposure and Y = 0.567 + 4.658 D + 0.364 D2 in 60 Co ␥-ray exposure (Y is the chromosomal aberrations per cell and D
is the irradiation dose in Gy). And the curvature (D2 ) of dose–response relationship was less for 12 C6+ ion irradiation than for 60 Co ␥-ray irradiation. This may be interpreted that DNA lesions induced by low LET irradiation are repaired partly by cell and lesions induced by high LET irradiation, however, are unrepairiable [14]. As shown in Fig. 1, the dose–response relationships exhibited positive correlations (r2 = 0.995 for 12 C6+ ion exposure and r2 = 0.990 for 60 Co ␥-ray exposure), and
Fig. 1. Dose–response relationships of chromosomal aberrations induced by 12 C6+ ion () and 60 Co ␥-ray (䊉) in immature oocytes of mice. Data represent mean ± S.D., n = 7.
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the frequencies of aberrations increased with increasing doses, especially for 12 C6+ ion irradiation, which confirmed the heavy ion with high LET and high RBE is significantly more deleterious on germ cells than low LET ionizing irradiation. Our results also showed that the average number of oocytes ovulated per female was reduced by exposures with 12 C6+ ion and 60 Co ␥-ray (Table 1). The effect was dose-dependent and more severe in the group exposed with 12 C6+ ion (Table 1). The mechanism of reduction in mean number of oocytes may be through the follicular degeneration induced by irradiation [9,24,25]. In conclusion, 12 C6+ ion irradiation significantly increased the frequencies of chromosomal aberrations in immature oocytes of mice, even at low dose of 0.5 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 [26]. 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 in order to better clarify genetic risk associated with the exposure of women in childhood to irradiation. Acknowledgements We express our thanks to the accelerator crew at the HIRFL, National Laboratory of Heavy Ion Accelerator in Lanzhou. References [1] W.H. Wallace, S.M. Shalet, J.H. Hendry, P.H. Morris-Jones, H.R. Gattamaneni, Ovarian failure following abdominal irradiation in childhood: the radiosensitivity of the human oocyte, Br. J. Radiol. 62 (1989) 995–998. [2] D. Meirow, D. Nugent, The effects of radiotherapy and chemotherapy on female reproduction, Hum. Reprod. Update 7 (2001) 535–543. [3] W. Reichert, I. Hansmann, G. Rohrborn, Chromosome anomalies in mouse oocytes after irradiation, Humangenetik 28 (1975) 25–38. [4] C.S. Griffin, C. Tease, Gamma-ray-induced numerical and structural chromosome anomalies in mouse immature oocytes, Mutat. Res. 202 (1988) 209–213. [5] C.S. Griffin, C. Tease, G. Fisher, The effect of low-dose Xirradiation on numerical and structural chromosome anomaly induction in mouse immature oocytes, Mutat. Res. 231 (1990) 137–142. [6] C. Tease, G. Fisher, The influence of maternal age on radiationinduced chromosome aberrations in mouse oocytes, Mutat. Res. 262 (1991) 57–62.
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