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Chromosomal damage in preimplantation mouse embryos and its development through the cell cycle
Mutation Research, 299 (1993) 313-315 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
313
MUTGEN 00018
Chromosomal damage in preimplantation mouse embryos and its development through the cell cycle Christian Streffer Institut flir Medizinische Strahlenbiologie, Universitiitsklinikum Essen, Essen, Germany (Received 24 June 1992) (Accepted 11 September 1992)
Keywords: Preimplantation mouse embryo; Ionising radiation; Cell cycle; Chromosomal damage
Summary
Cytogenetic damage is usually studied in the first metaphase after exposure to ionizing radiation although it is well known that further chromosomal abberations are also expressed during later mitotic divisions (Bauchinger et al., 1986; Lloyd et al., 1992). For such investigations the preimplantation mouse embryo is very well suited. If irradiation takes place in the 1-cell stage it can easily be followed whether the cells of the embryo have reached the first, second or third mitosis after the exposure. Furthermore, the duration of the various cell cycle phases is well known and therefore radiation effects on the cell cycle, e.g., the G 2 block, can be investigated (Streffer and Molls, 1987). Studies on these two phenomena will be reported here.
Studies on the G 2 block
The G 2 phase of the second cell cycle is comparatively long (12 h) in preimplantation mouse embryos. X-Irradiation in the early G 2 phase causes less cytogenetic damage than exposure in the late G 2 phase (Molls and Streffer, 1984). In both cases a G 2 block is provoked. It is supposed that this G 2 block allows a better repair of radiation damage and this effect is apparently regulated through the phosphorylation of proteins (Jung and Streffer, 1991). Thus it was found by
Correspondence: Dr. C. Streffer, Institut fiir Medizinisehe Strahlenbiologie, Universit~itsklinikum Essen, Hufelandstr. 55, 4300 Essen 1, Germany.
two-dimensional gel electrophoresis that the phosphorylation of certain proteins induces the start of mitosis. After an X-ray exposure of 2-4 Gy the phosphorylation of these proteins is delayed. These processes start at a time when the G 2 block comes to an end. Chromosomal damage at successive mitoses after irradiation
For these investigations chromosomal aberrations were studied at mitotic division from the 1to the 2-cell stage, from the 2- to the 4-cell stage and from the 4- to the 8-cell stage. The irradiation with X-rays or fast neutrons took place in all cases before the start of the S phase during the 1-cell stage (Weissenborn and Streffer, 1988a).
314 TABLE 1
TABLE 3
DISTRIBUTION OF CHROMOSOME ABERRATIONS PER EMBRYO IN THE FIRST TO THIRD MITOSES (%) AFTER X-rays (0.94-1.88 Gy) AT THE I-CELL STAGE (3 h p.c.)
CHROMOSOME ABERRATIONSIN EMBRYOS AFTER 0.94 Gy X-RAYS AT VARIOUS TIMES AT THE 1-CELL STAGE (FIRST AND THIRD MITOSES)
Total ChromosomeChromatid aberrations breaks breaks First mitosis p.r. 26 Second mitosis p.r. 34 Third mitosis p.r. 40
43 26 31
11 42 47
p.r., post radiation.
As expected, the majority of the chromosome aberrations were chromosome breaks in the first mitosis after exposure to X-rays as well as to neutrons. However, in the second and third postradiation mitoses the number of chromatid breaks was higher than that of chromosome breaks after X-irradiation with 0.94-1.88 Gy. The same phenomenon was observed for neutron irradiation with 0.25-0.75 Gy at the third mitosis. When the number of chromosome aberrations was calculated per embryo 40% of all aberrations, 31% of all chromosome breaks and 47% of all chromatid breaks were observed after X-irradiation in the third mitosis (Table 1), after neutrons these numbers were 54, 51 and 68%, respectively (Table 2). The numbers of chromatid breaks increased steadily from the first to the third mitosis after X-irradiation and this increase was even more pronounced after exposure to neutrons. In further experiments the number of chromosomal aberrations was studied after X-irradiation with 0.94 Gy at various phases of the first embry-
TABLE 2 DISTRIBUTION OF CHROMOSOME ABERRATIONS PER EMBRYO IN THE FIRST TO THIRD MITOSES (8B/) AFTER NEUTRONS (0.25-0.75 G-y)AT THE 1-CELL STAGE (3 h p.o.) Total ChromosomeChromatid aberrations breaks breaks First mitosis p.r. 19 Second mitosis p.r. 27 Third mitosis p.r. 54
33 16 51
6 26 68
Irradiation time (h p . c . )
Total aberrations/ metaphase
C h r o m o s o m e Chromatid breaks breaks (%) (%)
1 3 6 9
0.201 0.433 0.313 0.280
75 42 29 14
17 35 45 50
1 3 6 9
0.186 0.405 0.352 0.288
31 23 32 32
54 66 56 63
onic cell cycle. Irradiation was done directly post conception (1 h p.c.), shortly before S phase started (3 h p.c.), during S phase (6 h p.c.) and in early G2 phase (9 h p.c.). As can be seen from Table 3, the number of total chromosomal aberrations varied considerably in the various phases of the cell cycle. This variation was about the same in the first and the third post-radiation mitosis (Weissenborn and Streffer, 1988b). In the first mitosis the distribution of the aberrations between chromosome and chromatid breaks was such that the chromosome breaks were more frequent than the chromatid breaks before S phase. At the later periods the relative number of chromosome breaks decreased. However, no consistent changes were observed at the third postradiation mitosis with respect to the cell cycle. At all investigated periods the number of chromatid breaks was higher than the number of chromosome breaks (Table 3). The data clearly demonstrated that only a small part of the radiation-induced chromosomal damage is expressed as chromosome aberration at the first mitosis after exposure. Further damage is processed through the following cell cycles and expressed in the form of aberrations at later mitotic divisions. It is remarkable that this effect is even more pronounced with high-LET radiation, such as neutrons, than with X-rays. One would have expected that the higher ratio of D N A double-strand breaks over D N A single-
315 strand breaks after n e u r o n would have led to an earlier expression o f c h r o m o s o m e aberration. It may be possible that radiation-induced D N A base d a m a g e is processed t h r o u g h D N A strand breaks to chromatid breaks. In some embryos it was possible to study all four m e t a p h a s e s while the embryo developed f r o m the 4-cell stage to the 8-cell stage. These four m e t a p h a s e s originated from the same cell which was irradiated and they had r e a c h e d the third post-radiation mitotic division. A f t e r Xirradiation (3 h p.c.) nine out of 28 embryos had one m e t a p h a s e with one or several c h r o m o s o m e aberrations and only one out of the 28 embryos had two m e t a p h a s e s with c h r o m o s o m e aberrations. T h e aberrations differed in such a way that they could not have developed from the same d a m a g e in the irradiated genome. A f t e r neutrons (3 h p.c.) 13 out of 31 embryos had one m e t a p h a s e with o n e or several aberrations and six out of the 31 embryos had two m e t a p h a s e s with one or several aberrations. Only in one embryo the aberration type in the two m e t a p h a s e s was identical (in b o t h cases a chromatid break). These findings d e m o n s t r a t e that some radiation d a m a g e which is induced during exposure remains u n r e p a i r e d or misrepaired and develops during the following cell cycles in such a way that it is expressed in the form of c h r o m o s o m e aberrations in later mitoses. T h e aberrations seen in the third post-radiation mitosis are new c h r o m o s o m e aberrations at least in part.
Acknowledgement T h e studies have b e e n s u p p o r t e d by the Bundesminister fiir Umwelt, Naturschutz und Reaktorsicherheit.
References Bauchinger, M., E. Schmid and H. Braselmann (1986) Cell survival and radiation induced chromosome aberrations. II. Experimental findings in human lymphocytes analysed in first and second post-irradiation metaphases, Radiat. Environ. Biophys., 25, 253-260. Jung, Th., and C. Streffer (1991) Association of protein phosphorylation and cell cycle progression after X-irradiation of two-cell mouse embryos, Int. J. Radiat. Biol., 60, 511523. Lloyd, D.C., A.A. Edwards, A. Leonard, G.L. Deknudt, L. Verschaeve, A.T. Natarajan, F. Darroudi, G. Obe, F. Palitti, C. Tanzarella and E.J. Tawn (1992) Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays, Int. J. Radiat. Biol., 61,335-343. Molls, M., and C. Streffer (1984) The influence of G 2 progression on X-ray sensitivity of two-cell mouse embyos, Int. J. Radiat. Biol., 46, 355-365. Streffer, C., and M. Molls (1987) Cultures of preimplantation mouse embryos: A model for radiobiological studies, Adv. Radiat. Biol., 13, 169-213. Weissenborn, U., and C. Streffer (1988a) Analysis of structural and numerical chromosomal anomalies at the first, second, and third mitosis after irradiation of one-cell mouse embryos with X-rays and neutrons, Int. J. Radiat. Biol., 54, 381-394. Weissenborn, U., and C. Streffer (1988b) The one-cell mouse embryo: Cell cycle-dependent radiosensitivity and development of chromosomal anomalies in postradiation cell cycles, Int. J. Radiat. Biol., 54, 659-674.
313
MUTGEN 00018
Chromosomal damage in preimplantation mouse embryos and its development through the cell cycle Christian Streffer Institut flir Medizinische Strahlenbiologie, Universitiitsklinikum Essen, Essen, Germany (Received 24 June 1992) (Accepted 11 September 1992)
Keywords: Preimplantation mouse embryo; Ionising radiation; Cell cycle; Chromosomal damage
Summary
Cytogenetic damage is usually studied in the first metaphase after exposure to ionizing radiation although it is well known that further chromosomal abberations are also expressed during later mitotic divisions (Bauchinger et al., 1986; Lloyd et al., 1992). For such investigations the preimplantation mouse embryo is very well suited. If irradiation takes place in the 1-cell stage it can easily be followed whether the cells of the embryo have reached the first, second or third mitosis after the exposure. Furthermore, the duration of the various cell cycle phases is well known and therefore radiation effects on the cell cycle, e.g., the G 2 block, can be investigated (Streffer and Molls, 1987). Studies on these two phenomena will be reported here.
Studies on the G 2 block
The G 2 phase of the second cell cycle is comparatively long (12 h) in preimplantation mouse embryos. X-Irradiation in the early G 2 phase causes less cytogenetic damage than exposure in the late G 2 phase (Molls and Streffer, 1984). In both cases a G 2 block is provoked. It is supposed that this G 2 block allows a better repair of radiation damage and this effect is apparently regulated through the phosphorylation of proteins (Jung and Streffer, 1991). Thus it was found by
Correspondence: Dr. C. Streffer, Institut fiir Medizinisehe Strahlenbiologie, Universit~itsklinikum Essen, Hufelandstr. 55, 4300 Essen 1, Germany.
two-dimensional gel electrophoresis that the phosphorylation of certain proteins induces the start of mitosis. After an X-ray exposure of 2-4 Gy the phosphorylation of these proteins is delayed. These processes start at a time when the G 2 block comes to an end. Chromosomal damage at successive mitoses after irradiation
For these investigations chromosomal aberrations were studied at mitotic division from the 1to the 2-cell stage, from the 2- to the 4-cell stage and from the 4- to the 8-cell stage. The irradiation with X-rays or fast neutrons took place in all cases before the start of the S phase during the 1-cell stage (Weissenborn and Streffer, 1988a).
314 TABLE 1
TABLE 3
DISTRIBUTION OF CHROMOSOME ABERRATIONS PER EMBRYO IN THE FIRST TO THIRD MITOSES (%) AFTER X-rays (0.94-1.88 Gy) AT THE I-CELL STAGE (3 h p.c.)
CHROMOSOME ABERRATIONSIN EMBRYOS AFTER 0.94 Gy X-RAYS AT VARIOUS TIMES AT THE 1-CELL STAGE (FIRST AND THIRD MITOSES)
Total ChromosomeChromatid aberrations breaks breaks First mitosis p.r. 26 Second mitosis p.r. 34 Third mitosis p.r. 40
43 26 31
11 42 47
p.r., post radiation.
As expected, the majority of the chromosome aberrations were chromosome breaks in the first mitosis after exposure to X-rays as well as to neutrons. However, in the second and third postradiation mitoses the number of chromatid breaks was higher than that of chromosome breaks after X-irradiation with 0.94-1.88 Gy. The same phenomenon was observed for neutron irradiation with 0.25-0.75 Gy at the third mitosis. When the number of chromosome aberrations was calculated per embryo 40% of all aberrations, 31% of all chromosome breaks and 47% of all chromatid breaks were observed after X-irradiation in the third mitosis (Table 1), after neutrons these numbers were 54, 51 and 68%, respectively (Table 2). The numbers of chromatid breaks increased steadily from the first to the third mitosis after X-irradiation and this increase was even more pronounced after exposure to neutrons. In further experiments the number of chromosomal aberrations was studied after X-irradiation with 0.94 Gy at various phases of the first embry-
TABLE 2 DISTRIBUTION OF CHROMOSOME ABERRATIONS PER EMBRYO IN THE FIRST TO THIRD MITOSES (8B/) AFTER NEUTRONS (0.25-0.75 G-y)AT THE 1-CELL STAGE (3 h p.o.) Total ChromosomeChromatid aberrations breaks breaks First mitosis p.r. 19 Second mitosis p.r. 27 Third mitosis p.r. 54
33 16 51
6 26 68
Irradiation time (h p . c . )
Total aberrations/ metaphase
C h r o m o s o m e Chromatid breaks breaks (%) (%)
1 3 6 9
0.201 0.433 0.313 0.280
75 42 29 14
17 35 45 50
1 3 6 9
0.186 0.405 0.352 0.288
31 23 32 32
54 66 56 63
onic cell cycle. Irradiation was done directly post conception (1 h p.c.), shortly before S phase started (3 h p.c.), during S phase (6 h p.c.) and in early G2 phase (9 h p.c.). As can be seen from Table 3, the number of total chromosomal aberrations varied considerably in the various phases of the cell cycle. This variation was about the same in the first and the third post-radiation mitosis (Weissenborn and Streffer, 1988b). In the first mitosis the distribution of the aberrations between chromosome and chromatid breaks was such that the chromosome breaks were more frequent than the chromatid breaks before S phase. At the later periods the relative number of chromosome breaks decreased. However, no consistent changes were observed at the third postradiation mitosis with respect to the cell cycle. At all investigated periods the number of chromatid breaks was higher than the number of chromosome breaks (Table 3). The data clearly demonstrated that only a small part of the radiation-induced chromosomal damage is expressed as chromosome aberration at the first mitosis after exposure. Further damage is processed through the following cell cycles and expressed in the form of aberrations at later mitotic divisions. It is remarkable that this effect is even more pronounced with high-LET radiation, such as neutrons, than with X-rays. One would have expected that the higher ratio of D N A double-strand breaks over D N A single-
315 strand breaks after n e u r o n would have led to an earlier expression o f c h r o m o s o m e aberration. It may be possible that radiation-induced D N A base d a m a g e is processed t h r o u g h D N A strand breaks to chromatid breaks. In some embryos it was possible to study all four m e t a p h a s e s while the embryo developed f r o m the 4-cell stage to the 8-cell stage. These four m e t a p h a s e s originated from the same cell which was irradiated and they had r e a c h e d the third post-radiation mitotic division. A f t e r Xirradiation (3 h p.c.) nine out of 28 embryos had one m e t a p h a s e with one or several c h r o m o s o m e aberrations and only one out of the 28 embryos had two m e t a p h a s e s with c h r o m o s o m e aberrations. T h e aberrations differed in such a way that they could not have developed from the same d a m a g e in the irradiated genome. A f t e r neutrons (3 h p.c.) 13 out of 31 embryos had one m e t a p h a s e with o n e or several aberrations and six out of the 31 embryos had two m e t a p h a s e s with one or several aberrations. Only in one embryo the aberration type in the two m e t a p h a s e s was identical (in b o t h cases a chromatid break). These findings d e m o n s t r a t e that some radiation d a m a g e which is induced during exposure remains u n r e p a i r e d or misrepaired and develops during the following cell cycles in such a way that it is expressed in the form of c h r o m o s o m e aberrations in later mitoses. T h e aberrations seen in the third post-radiation mitosis are new c h r o m o s o m e aberrations at least in part.
Acknowledgement T h e studies have b e e n s u p p o r t e d by the Bundesminister fiir Umwelt, Naturschutz und Reaktorsicherheit.
References Bauchinger, M., E. Schmid and H. Braselmann (1986) Cell survival and radiation induced chromosome aberrations. II. Experimental findings in human lymphocytes analysed in first and second post-irradiation metaphases, Radiat. Environ. Biophys., 25, 253-260. Jung, Th., and C. Streffer (1991) Association of protein phosphorylation and cell cycle progression after X-irradiation of two-cell mouse embryos, Int. J. Radiat. Biol., 60, 511523. Lloyd, D.C., A.A. Edwards, A. Leonard, G.L. Deknudt, L. Verschaeve, A.T. Natarajan, F. Darroudi, G. Obe, F. Palitti, C. Tanzarella and E.J. Tawn (1992) Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays, Int. J. Radiat. Biol., 61,335-343. Molls, M., and C. Streffer (1984) The influence of G 2 progression on X-ray sensitivity of two-cell mouse embyos, Int. J. Radiat. Biol., 46, 355-365. Streffer, C., and M. Molls (1987) Cultures of preimplantation mouse embryos: A model for radiobiological studies, Adv. Radiat. Biol., 13, 169-213. Weissenborn, U., and C. Streffer (1988a) Analysis of structural and numerical chromosomal anomalies at the first, second, and third mitosis after irradiation of one-cell mouse embryos with X-rays and neutrons, Int. J. Radiat. Biol., 54, 381-394. Weissenborn, U., and C. Streffer (1988b) The one-cell mouse embryo: Cell cycle-dependent radiosensitivity and development of chromosomal anomalies in postradiation cell cycles, Int. J. Radiat. Biol., 54, 659-674.