Micronucleus Formation in Preimplanted Mouse

TNT . J . RADTAT . BIOL .,

1981,

VOL .

39,

NO .

3, 307-314

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Micronucleus formation in preimplanted mouse embryos cultured in vitro after irradiation with X-rays and neutrons M . MOLLS, C . STREFFER and N . ZAMBOGLOU Institut fur Medizinische Strahlenphysik and Strahlenbiologie, Universitatsklinikum Essen, Hufelandstr . 55, D-4300 Essen 1, F .R .G . (Received 20th March 1980 ; accepted 28th October 1980)

Preimplanted mouse embryos cultured in vitro were irradiated with X-rays and neutrons in the late G 2 -phase of the 2-cell stage . Both radiation qualities induced micronuclei at very low doses . The kinetics of micronucleus formation during the first and second cell cycles after X-irradiation depended on the radiation dose and on the extent of the division delay . New micronuclei appeared to be formed even after the third and later post-irradiation mitoses . The shape of the various doseeffect curves and the mechanism of micronucleus formation by the two radiation qualities are discussed .

1.

Introduction Mutagenesis by ionizing radiation is undoubtedly important . With respect to radioprotection the effects at low doses are of particular interest (Fliedner, Andrews, Cronkite and Bond 1964) . The micronucleus test described by Heddle (1973) and Schmid (1975) is being used increasingly especially in the field of chemical mutagenesis . This test which is a useful cytological indicator of genetic damage (Evans 1976) and which in human lymphocytes reflects the chromosomal aberration frequencies allows a rapid assessment of chromosomal damage (Countryman and Heddle 1976, Hollstein and McCann 1979) . However, not all types of chromosomal aberrations lead to the formation of micronuclei which are generally thought to derive from acentric chromosomal fragments which after mitosis are not incorporated into the daughter nuclei (Carrano and Heddle 1973) . The occurrence of micronuclei has also been observed after X-irradiation of preimplanted mouse embryos in vivo . However, no detailed studies have been undertaken (Russell 1965) . In this presentation the formation of micronuclei is reported after irradiation with fast neutrons and X-rays at low doses . The dose and time dependence of this effect are investigated in preimplanted mouse embryos cultured in vitro . In this fast proliferating in vitro system there is good agreement between the normal physiological conditions in vivo and its culture in vitro (Streffer, van Beuningen, Molls, Zamboglou and Schulz 1980) . We irradiated in our experiments 2-cell embryos in late G 2 -phase of the cell cycle . This is in contrast to previous in vitro studies concerning the fate of micronuclei or acentric chromosomal fragments in which G 1 -cells were irradiated (Grote and Revell 1972, Carrano 1973, Carrano and Heddle 1973) . 2.

Materials and methods Preimplanted 2-cell mouse embryos were incubated in culture medium BMOC2 (Brinster 1969) in vitro starting about 32 hours post conception (p .c .) . The details 0020-7616/81/3903 0307 S02-00,c 1981 Taylor & Francis Ltd

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of the culture system are given in Streffer et al . (1980) . The 2-cell embryos were irradiated at 33 hours p .c . either with 240 kV X-rays (Stabilipan, Siemens ; dose rate : 100 R/min) or with neutrons (D-*Be, average energy : 7 MeV) produced by a compact cyclotron unit (TCC, Berkeley, California, U .S .A .) (Rassow 1978) . At the time of irradiation the two cells of the embryos were in the late G,-phase of the cell cycle . This was ascertained by cytofluorometric determination of the DNA-content (Streffer et al . 1980) . Furthermore at the time of irradiation no mitotic figures were seen when a preparation of cell nuclei was checked according to the method of Tarkowski (1966) . Four-cell embryos were not present in the cultures immediately after irradiation . For the determination of micronuclei and cell numbers per embryo the nuclei of each individual embryo were prepared on a slide after hypotonic treatment (Tarkowski 1966) . After fixation with glacial acetic acid/ethanol (1 :3) and drying in air the nuclei and micronuclei were stained with the fluorescent dye ethidium bromide (Streffer et al. 1980) . The micronuclei present in the neighbourhood of the cell nuclei were counted at a microscope magnification of x 400 . Staining with ethidium bromide has the advantage that the micronuclei can be identified very easily as small chromatin-positive particles which have a diameter of less than 1/3 of the main nucleus . After X-irradiation the determinations were performed 48, 72, and 96 hours p .c . which is 15, 39, and 63 hours post radiation (p .r .) . At these times, the average control embryo consisted of 5 . 7, 21 . 5, and 65 cells respectively (Streffer et al. 1980) . The measurements after neutron irradiation were performed 39 hours p .r . 3.

Results Two different dose-effect curves for micronucleus formation at 39 hours p .r . are given in figure 1 . A linear relationship was found after X-irradiation . The effect after low neutron doses was much greater than after X-irradiation . The dose-effect curve was not linear, being steeper in the lower dose range than in the higher dose range .

2 .0 1 .0 1 .5 Dose (Gy ) Figure 1 . The number of micronuclei per 100 cells 39 hours post radiation (72 hours p .c .) in x ; neutrons : • relation to radiation dose . X-rays : x • . Each point is an average value for at least 20 embryos (about 400 cells) . 0.5

Micronucleus formation in preimplanted mouse embryos

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28 - 15 hours p . r. 24 20 /x 16 12 ~x x 8 - / /x 4 y

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• 24

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28 - 63 hours p . r. 24 20 16 12 8 4x 0.25

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0.5

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x

1 .0 1 .5 X - ray dose (Gy )

2 .0

Figure 2 . The number of micronuclei per 100 cells 15, 39 and 63 hours post radiation (48, 72 and 96 hours p .c .) in relation to X-ray dose . At 15 hours 30 embryos per dose were examined but less at 39 (at least 20 embryos) and 63 hours . A minimum of 500 cells was scored at each dose .

100 H

mU 80 0 0

60 0 40 •0

20 0 4 Figure 3 .

5

i 6 7 cells / embryo

8

The number of micronuclei per 100 cells in 4-,5-, 6-, 7- and 8-cell embryos 15 0 ; 0 .94 Gy : x - x ;

hours after X-irradiation (48 hours p .c .) . 1 .88 Gy : 0 0. 47 Gy : Q A ; 0 . 24 Gy : * • ; 0 . 12 Gy : 0 7.

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Furthermore figure 1 shows that remarkably low doses of a few rad caused an increase in micronuclei after both X- and neutron irradiation . In the controls 0 . 4 per cent of the cells showed one micronucleus on the average . An X-ray dose of 0 . 06 Gv doubled this value ; application of 0 . 03 Gy neutrons led to a 6-fold increase . In the high dose range we found very high values which exceeded the control value about 30 times for both radiation qualities . In another experiment the occurrence of micronuclei as a function of time after X-irradiation was studied (figure 2) . The dose-effect curve at 15 hours p .r . differed significantly from the curves at 39 hours and 63 hours p .r . At 15 hours p .r . the frequency of micronuclei (calculated per Gy) was relatively greater after low doses than after higher doses . This was in contrast to the results at 39 and 63 hours p .r . when a linear dose-effect relationship was found . At 63 hours p .r . micronuclei in the control had increased . Figure 3 shows micronucleus formation at 15 hours p .r . in relation to embryos of different cell number . As described, micronuclei were determined individually for each embryo, so that the average number of micronuclei could be calculated separately for the groups of 4-, 5-, 6-, 7- and 8-cell embryos . In 4-cell embryos which had undergone one mitosis after irradiation with 1 . 88 Gy we found 0 . 08 micronuclei per cell . In 8-cell embryos which were as old as the 4-cell embryos (48 hours p .c .) this value increased dramatically to 0 . 88 . The increase was dose dependent and was observed even after 0 . 12 Gy . Figure 4 shows the average number of micronuclei per embryo in relation to time . The values were calculated from the number of micronuclei per cell (figure 2) and the mean cell number per embryo given in the table . With increasing time the number of micronuclei per embryo also increased . This was found in the controls as well as in the irradiated embryos . However, after X-irradiation the effect was much more 8 0

7 6 0 T

E 5 w 4 U 7

-

0 3 u -

1 0 15

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39 hours p .r .

63

The number of micronuclei per embryo 15, 39 and 63 hours after X-irradiation (48, and 96 hours p .c .) . 1 . 88 Gy : 0 0 ; 0. 94 Gy : x x ; 0. 47 Gy : A A ; control : • • .

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Figure 5 . The number of micronuclei per 100 cells in those embryos which have already developed to the 8-cell stage 15 hours after X-irradiation (48 hours p .c .) .

Dose (Gy)

48 h .p .c .

72 h .p .c .

96 h .p .c .

Neutronirradiation

X-irradiation 0 . 00 0 .06 0 . 12 0 .24 0. 47 0. 94 1 . 88

72 h .p .c .

5.7

21 . 5

67

5.5 5.5 4.9 4.6 4.4

22 . 2 20 . 8 19 .9 18 . 7 16 . 6

65 59 48 34

21 . 5 21 . 9 20 . 2 18 . 5 17 . 4 16 . 5

The average cell number per embryo 15, 39 and 63 hours after X- and neutron irradiation (48, 72 and 96 hours p .c .) .

pronounced especially in the time interval between 39 and 63 hours p .r . The number of micronuclei per embryo increased from about 0 . 1 to 1 . 5 micronuclei in the controls but from 2 . 5 to 7 . 5 micronuclei after 1 . 88 Gy .

4.

Discussion It is well established that the induction of micronuclei and chromosomal aberrations in metaphase is closely correlated (Boller and Schmid 1970, Heddle 1973, Miller 1973) . Micronuclei consist of chromosomal fragments or chromosomes which have not participated in the normal distribution during anaphase (Carrano and Heddle 1973, Countryman and Heddle 1976, Hollstein and McCann 1979) . It is generally accepted that mitotic divisions are necessary for the appearance of micronuclei (Jenssen and Ramel 1976) .


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The production of chromosomal aberrations depends on the phase of the cell cycle in which cells are irradiated (Bender, Griggs and Bedford 1974) . The cells of the 2-cell embryos have been irradiated in the G 2 -phase . Although the direct connection between DNA strand breaks and chromatid breaks has not yet been proven, it may be assumed that chromatid breaks, which are directly induced by radiation and probably caused by double-strand breaks of DNA, lead to micronuclei after the first mitosis post radiation . These micronuclei will be observed in the cells of the 4-cell embryos . After X-irradiation double-strand breaks are comparatively rare (Streffer 1969), therefore few micronuclei are found after the first mitosis (figure 3) . However, after neutron irradiation the number of double-strand breaks of DNA is considerably higher, hence more micronuclei (37 and 14 micronuclei per 100 cells after 1 . 0 Gy and 0. 5 Gy respectively) appear at this developmental stage (unpublished data) . From these data it cannot be concluded whether and to what extent micronuclei might be derived from whole chromosomes brought about by radiationinduced non-disjunction . This has to be investigated . After the first mitosis when the cells progress through the next cell cycle with complete DNA replication in S-phase, unrepaired single strand breaks of DNA induced by X-irradiation of G 2 -cells in the 2-cell stage would also develop into chromatid breaks (Bender et al . 1974) . These chromatid breaks should yield micronuclei after the second post-irradiation mitosis . On the basis of these assumptions it is very plausible that the number of micronuclei is highest 15 hours p.r . in those embryos in which all four cells have already passed through the second mitosis and have reached 8 cells per embryo (figure 3) . The advantage of this biological system is that each embryo can be followed independently and the fate of the two `stem cells' can be analysed in this `closed' system . At present we cannot exclude the possibility that other mechanisms are involved in the kinetics of micronuclei formation . One could assume that some fragments present at the first mitosis only become visible as micronuclei after later cell division . However, the present data demonstrate that new micronuclei are apparently formed even after the third and later post-irradiation mitoses . This is most clearly seen in the embryos 63 hours p .r . (figures 2 and 4) . At this developmental stage the embryos consist of between 65 cells (unirradiated) and about 40 cells per embryo (1 . 88 Gy Xrays) (Streffer et al . 1980 and unpublished data) . At this stage 4-5 mitoses have taken place after irradiation and 1-2 mitoses from 39 to 63 hours p .r . The mechanisms of this effect will be further investigated . As the cell proliferation changes from an exponential growth to a plateau phase in which cell loss occurs, a general labilization of the genome might contribute to this effect . We already pointed out that in our investigations the cells were irradiated in G 2-phase. This is in contrast to the investigations of other authors in which irradiation was performed in G 1 (Grote and Revell 1972, Carrano 1973, Carrano and Heddle 1973) and may explain differences in the results . We are aware that further experimental evidence is necessary for clarification of our assumptions . The phenomena described above may also explain the shape of the dose-effect curve 15 hours after X-irradiation at 48 hours p .c . (figure 2) . The curve is the result of two major effects : (1) chromosomal damage which yields micronuclei after cell proliferation ; (2) division delay . Both effects increase with radiation dose, but the second effect inhibits the expression of the first effect . As no or a very little division delay generally occurs after low X-ray doses, the occurrence of micronuclei appears faster under these conditions and the relative radiation effect is higher . At the later



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periods (39-63 hours p .r .) the division delay of some hours is less important . As the proliferation rate is only slightly altered by these X-ray doses (0 to 1 . 88 Gy), the dose-effect curve becomes linear 39 hours p .r . and remains linear at least until 63 hours p .r . (figure 2) . This finding is underlined by the observed data in those cells which have divided comparatively soon after irradiation and which have already reached the 8-cell stage 15 hours p .r . If the number of micronuclei of these cells is plotted against the X-ray dose, the shape of the dose-effect curve is different from that which is obtained for all embryos (4-cell to 8-cell embryos, 15 hours p .r .) . It appears to be linear for each class of embryos, even 15 hours p .r . (figure 5) . The extremely high number of micronuclei is interesting especially in the 8-cell embryos and supports the proposed mechanism of micronucleus formation . Experiments are in progress in which the embryos with different cell numbers are cultured separately in order to obtain further insight into the biological relevance of micronucleus formation . At present the effect of these processes on the development of the embryos is not clear . In contrast to X-irradiation the shape of the dose-effect curve does not become linear after neutron irradiation . Therefore the RBE-value increases from 1B for a neutron dose of 1 . 00 Gy to 5 . 5 at 0. 1 Gy and 7 .4 at 0 . 05 Gy . The number of cells per embryo, from which the proliferation rate can be estimated, is not very different at the comparable X-ray and neutron doses in the low dose range (see table) . Therefore different proliferation rates appear not to be responsible for this effect . However the time course and other parameters must be further studied after neutron irradiation . The reported investigations show the high radiosensitivity of mammalian preimplanted embryos . The formation of micronuclei appears as a very useful method not only in studying cytogenetic damage but also in evaluating the mechanism of radiation effects .

Acknowledgments The expert technical assistance of Miss M .-L . Steimann and Mrs . U . Kaiser is gratefully acknowledged . We thank Messrs . ing . grad . G . Hiidepohl, ing . grad . T . TeBler, S . Schonteich (Institdt fur Medizinische Strahlenphysik and Strahlenbiologie, Abteilung fur Medizinische Strahlenphysik) for assistance with the cyclotron unit . This work was supported by grants from the Bundesministerium fur Forschung and Technologic and the Bundesministerium des Innern . Des embryons de souris constitues de 2 cellules pat -venues en fin de G2 ont etc irradies in vitro avec des rayons X ainsi qu'avec des neutrons . Meme avec de tres petites doses de ces

deux types de radiations, on a pu constater l'apparition de micronoyaux . Le rythme d'apparition de ces micronoyaux au cours des deux cycles cellulaires succedant a l'irradiation s'est revele dependant de la dose de radiations ainsi que du retard a la mitose . On a observe a nouveau 1'apparition de micronoyaux apres la troisieme et la quatrieme mitose . L'aspect des differentes courbes traduisant la relation dose-effet ainsi que le mecanisme de formation des micronoyaux par les deux types de radiations sont discutes . In vitro geziichtete praimplantierte Mauseembryonen wurden in der spaten G 2 -Phase des 2-Zellstadiums mit Rontgenstrahlen and Neutronen bestrahlt . Beide Strahlenqualitaten induzierten in einem sehr niedrigen Dosisbereich Mikronuklei .- Die Kinetik der Mikronukleusbildung wahrend des ersten and zweiten Zellzyklus nach Rontgenbestrahlung hing von der Strahlendosis and vom AusmaB der Teilungsverzogerung ab . Weiter

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beobachteten wir, daB neue Mikronuklei nach der dritten and weiteren Mitosen nach Bestrahlung gebildet wurden . Der Verlauf der verschiedenen Dosiseffektkurven and der Mechanismus der Mikronukleusbildung nach Anwendung der beiden Strahlenqualitaten werden diskutiert .


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