Radiation and Environmental Biophysics
Rad. and Environm. Biophys. 16, 299--302 (1979)
© Springer-Verlag 1979
Low-Dose Effects of X-Rays and Negative Pions on the Pronuclear Zygote Stage of Mouse Embryos C. Michel, H. Blattmann, I. Cordt-Riehle, and H. Fritz-Niggli Radiobiological Institute, University of Zurich, CH-8029 Zurich, Switzerland
Summary. Mouse embryos in the pronuclear-zygote stage (day 0 of gestation) were irradiated with 13.5 rad of 140 kV X-rays or negative pions. On day 13 the fetuses were examined for developmental anomalies such as intrauterine death, growth retardation and malformations. Significant decreases in the percentage of normal implantations were obtained with peak pions (high LET) and X-rays, whereas the effect of plateau pions was less obvious. Irradiation with peak pions was more effective than with X-rays by a factor of about 1.7. Introduction
It is generally accepted that early embryonic stages of mammalian development belong to the most radiosensitive biological systems. Doses in the order of 5 to 10 rad on the precleavage zygote in the mouse for example resulted in a significant decrease in the percentage of surviving implantations (Ohzu, 1965; Rugh et aI., 1969; Templier et al., 1978). Concerning possible teratogenic effects of irradiation during preimplantation stages there are still certain discrepancies in experimental results. Whereas some investigators could not find an increase of malformation rates, even not with high doses, other authors reported the appearance of anomalies in the central nervous system and skeletal system (see Unscear report, 1977). The induction of growth retardation in preimplantation embryos has also been observed, but it is not a response so obviously recognized as embryonic death. These facts may show that further information in this field is needed and may be valuable in regard to risk estimations for early stages of human development. The purpose of this study was a comparative evaluation of low-dose effects of qualitatively different radiations on mouse embryos during the pronuclear-zygote stage, which is characterized by the presence of a separate male and female nucleus. Materials and Methods
Female virgin NMRI mice, 10 to 12 weeks old and weighing 31 + 2 g were mated overnight with males of proven fertility. Between 3 and 4 p.m. on day 0 of gestation
0301-634X/79/0016/0299/$ 01.00
300
c. Michel et al.
individual females were whole-body irradiated with 13.5 rad. The following types of radiation were tested: 140 kV X-rays at a dose rate of 2.5 rad/min and negative pions at dose rates of approx. 2 rad/min in the Bragg peak and 1.6 to 3.3 rad/min in the plateau region. Details of animal housing, irradiation conditions and methods of dosimetry have been reported earlier (Michel and Fritz-Niggli, 1977). On day 13 of gestation the following end-points were studied: pre- and postimplantation loss of embryos, growth retardation, eye and brain defects and tail and limb anomalies. The data were analysed with Wilcoxon tests and Student t-tests.
Results
Stage of Development During Irradiation: In parallel experiments the oviducts from 11 untreated pregnant females were examined in serial sections. Of 104 eggs observed more than 89% were in the pronuclear-zygote stage at the time of irradiation, which is in very good agreement with the findings from Friedberg et al. (1973).
Embryonic Survival: The mean rates of implantations per female varied between 12.8 in the X-irradiated group and 14.1 in the control (Table 1). However the differences within the various groups were statistically not significant. This finding indicates that under the experimental conditions used the rate of preimplantation death was not enhanced compared to the control. Concerning postimplantation resorption, frequencies of 5.61% and 6.80% were found in the untreated and restrained control group, respectively. Irradiation with Xrays and peak pions significantly increased the postimplantation mortality, whereas with plateau pions the death rate remained on control level.
Growth Retardation and Malformath)ns: In the unrestrained control group 6.93% growth retarded and 2.23% malformed fetuses were observed among 404 live fetuses. A restraint of females for about 6 min in the irradiation container resulted in a small increase of abnormal fetuses, but the difference to the incidence in the untreated control group is not significant. The maximum effect on prenatal growth was seen with peak pions (22.73%), revealing a significant difference in frequency compared to the restrained control. The frequencies of gross structural malformations in day 13 fetuses after irradiation were not significantly different from those in the controls. Overall Induction of Developmental Defects: The percentages of anomalies, including postimplantation death, growth disorders and malformations are summarized in Table 2. After subtracting the so-called 'cage-effect' (control value) RBE values of approx. 0.4 and 1.7 for plateau and peak pions respectively, were calculated. The ratio of the net effects of the same dose in peak and plateau showed a value of about 4.
Effects of X-Rays and Pions on Pronuclear Zygotes 0 0
u~ 0
0
&
l
~
0
o
0 0
n~
0
.o 0
Z 0 0
r~o
"F: "a
~g ~~
o
~
~
~
~
o
301
302 Treatment
C. Michel et al. Anomalies (%)
RBE
Control restrained
19.82 _+2.55
-
140 kV X-rays
30.25 _+3.48a
1
Plateau pions
24.23 + 3.82
0.4
Peak pions
37.91 _+3.92b
1.7
Table 2. Tentative RBE values for pions concerning overall induction of developmental anomalies in mice
a Significant at the 2% level Significant at the 1% level Diseussion
Our results show that concerning prenatal mortality, preimplantation death was not obviously influenced b y irradiation with 13.5 tad, whereas postimplantation resorption was significantly enhanced b y X-irradiation and peak pions only. Secondly malformation incidence was low in all experimental groups and thirdly, interestingly growth retardation tended to be the major effect especially after pion irradiation. It is assumed that growth disorders m a y be related to a cleavage delay which by itself is p r o b a b l y due to radiation induced c h r o m o s o m a l aberrations. The principal finding of the presented d a t a is that peak pions as a high L E T radiation showed only a relatively slight increased effectiveness c o m p a r e d to low L E T X-rays. This is in striking contrast to the high R B E value o f 4.5 for fission neutrons with respect to 300 kV X - r a y s found b y Friedberg et al. (1973) in the same experimental system. Besides biological variables such as embryonic metabolism a n d / o r genetic factors also physical conditions m a y contribute to the inconsistent results. Thereby different L E T distribution, different mean lineal energy and some disagreements in dose rates m a y be the most decisive parameters. In conclusion more systematic investigations should be performed using various L E T spectra and different dose rates to provide information relevant to the assessment o f possible radiation effects in early pregnancy.
References
Friedberg, W., Hanneman, G. D., Faulkner, D. N.: Prenatal survival of mice irradiated with fission neutrons or 300 kVp X-rays during the pronuclear-zygote stage: survival curves, effect of dose fractionation. Int. J. Radiat. Biol. 24, 549-560 (1973) Michel, C., Fritz-Niggli, H., Blattmann, H., Cordt-Riehle, I.: Effects of low-dose irradiation with Xrays and pi-mesons on embryos of two different mouse strains. Strahlentherapie 153, 674-681 (1977) Ohzu, E.: Effects of low-dose X-irradiation on early mouse embryos. Radiat. Res. 26, 107-113 (1965) Rugh, R., Wohlfromm, M., Varma, A.: Low-dose X-ray effects on the Precleavage mammalian zygote. Radiat. Res. 37, 401--414 (1969) Templier, J., Maudouyt, M. A., Charpenteau, M., Bonnet, J: Effets de faibles doses de rayonnement gamma sur la gestation de la Souris. C.R. Soc. Biol. 172, 177--180 (1978) United Nations Report: Scientific Committee on the Effects of Atomic Radiation. Sources and Effects of Ionizing Radiation, pp. 655--725. New York: United Nations 1977 Received February 15, 1979
Rad. and Environm. Biophys. 16, 299--302 (1979)
© Springer-Verlag 1979
Low-Dose Effects of X-Rays and Negative Pions on the Pronuclear Zygote Stage of Mouse Embryos C. Michel, H. Blattmann, I. Cordt-Riehle, and H. Fritz-Niggli Radiobiological Institute, University of Zurich, CH-8029 Zurich, Switzerland
Summary. Mouse embryos in the pronuclear-zygote stage (day 0 of gestation) were irradiated with 13.5 rad of 140 kV X-rays or negative pions. On day 13 the fetuses were examined for developmental anomalies such as intrauterine death, growth retardation and malformations. Significant decreases in the percentage of normal implantations were obtained with peak pions (high LET) and X-rays, whereas the effect of plateau pions was less obvious. Irradiation with peak pions was more effective than with X-rays by a factor of about 1.7. Introduction
It is generally accepted that early embryonic stages of mammalian development belong to the most radiosensitive biological systems. Doses in the order of 5 to 10 rad on the precleavage zygote in the mouse for example resulted in a significant decrease in the percentage of surviving implantations (Ohzu, 1965; Rugh et aI., 1969; Templier et al., 1978). Concerning possible teratogenic effects of irradiation during preimplantation stages there are still certain discrepancies in experimental results. Whereas some investigators could not find an increase of malformation rates, even not with high doses, other authors reported the appearance of anomalies in the central nervous system and skeletal system (see Unscear report, 1977). The induction of growth retardation in preimplantation embryos has also been observed, but it is not a response so obviously recognized as embryonic death. These facts may show that further information in this field is needed and may be valuable in regard to risk estimations for early stages of human development. The purpose of this study was a comparative evaluation of low-dose effects of qualitatively different radiations on mouse embryos during the pronuclear-zygote stage, which is characterized by the presence of a separate male and female nucleus. Materials and Methods
Female virgin NMRI mice, 10 to 12 weeks old and weighing 31 + 2 g were mated overnight with males of proven fertility. Between 3 and 4 p.m. on day 0 of gestation
0301-634X/79/0016/0299/$ 01.00
300
c. Michel et al.
individual females were whole-body irradiated with 13.5 rad. The following types of radiation were tested: 140 kV X-rays at a dose rate of 2.5 rad/min and negative pions at dose rates of approx. 2 rad/min in the Bragg peak and 1.6 to 3.3 rad/min in the plateau region. Details of animal housing, irradiation conditions and methods of dosimetry have been reported earlier (Michel and Fritz-Niggli, 1977). On day 13 of gestation the following end-points were studied: pre- and postimplantation loss of embryos, growth retardation, eye and brain defects and tail and limb anomalies. The data were analysed with Wilcoxon tests and Student t-tests.
Results
Stage of Development During Irradiation: In parallel experiments the oviducts from 11 untreated pregnant females were examined in serial sections. Of 104 eggs observed more than 89% were in the pronuclear-zygote stage at the time of irradiation, which is in very good agreement with the findings from Friedberg et al. (1973).
Embryonic Survival: The mean rates of implantations per female varied between 12.8 in the X-irradiated group and 14.1 in the control (Table 1). However the differences within the various groups were statistically not significant. This finding indicates that under the experimental conditions used the rate of preimplantation death was not enhanced compared to the control. Concerning postimplantation resorption, frequencies of 5.61% and 6.80% were found in the untreated and restrained control group, respectively. Irradiation with Xrays and peak pions significantly increased the postimplantation mortality, whereas with plateau pions the death rate remained on control level.
Growth Retardation and Malformath)ns: In the unrestrained control group 6.93% growth retarded and 2.23% malformed fetuses were observed among 404 live fetuses. A restraint of females for about 6 min in the irradiation container resulted in a small increase of abnormal fetuses, but the difference to the incidence in the untreated control group is not significant. The maximum effect on prenatal growth was seen with peak pions (22.73%), revealing a significant difference in frequency compared to the restrained control. The frequencies of gross structural malformations in day 13 fetuses after irradiation were not significantly different from those in the controls. Overall Induction of Developmental Defects: The percentages of anomalies, including postimplantation death, growth disorders and malformations are summarized in Table 2. After subtracting the so-called 'cage-effect' (control value) RBE values of approx. 0.4 and 1.7 for plateau and peak pions respectively, were calculated. The ratio of the net effects of the same dose in peak and plateau showed a value of about 4.
Effects of X-Rays and Pions on Pronuclear Zygotes 0 0
u~ 0
0
&
l
~
0
o
0 0
n~
0
.o 0
Z 0 0
r~o
"F: "a
~g ~~
o
~
~
~
~
o
301
302 Treatment
C. Michel et al. Anomalies (%)
RBE
Control restrained
19.82 _+2.55
-
140 kV X-rays
30.25 _+3.48a
1
Plateau pions
24.23 + 3.82
0.4
Peak pions
37.91 _+3.92b
1.7
Table 2. Tentative RBE values for pions concerning overall induction of developmental anomalies in mice
a Significant at the 2% level Significant at the 1% level Diseussion
Our results show that concerning prenatal mortality, preimplantation death was not obviously influenced b y irradiation with 13.5 tad, whereas postimplantation resorption was significantly enhanced b y X-irradiation and peak pions only. Secondly malformation incidence was low in all experimental groups and thirdly, interestingly growth retardation tended to be the major effect especially after pion irradiation. It is assumed that growth disorders m a y be related to a cleavage delay which by itself is p r o b a b l y due to radiation induced c h r o m o s o m a l aberrations. The principal finding of the presented d a t a is that peak pions as a high L E T radiation showed only a relatively slight increased effectiveness c o m p a r e d to low L E T X-rays. This is in striking contrast to the high R B E value o f 4.5 for fission neutrons with respect to 300 kV X - r a y s found b y Friedberg et al. (1973) in the same experimental system. Besides biological variables such as embryonic metabolism a n d / o r genetic factors also physical conditions m a y contribute to the inconsistent results. Thereby different L E T distribution, different mean lineal energy and some disagreements in dose rates m a y be the most decisive parameters. In conclusion more systematic investigations should be performed using various L E T spectra and different dose rates to provide information relevant to the assessment o f possible radiation effects in early pregnancy.
References
Friedberg, W., Hanneman, G. D., Faulkner, D. N.: Prenatal survival of mice irradiated with fission neutrons or 300 kVp X-rays during the pronuclear-zygote stage: survival curves, effect of dose fractionation. Int. J. Radiat. Biol. 24, 549-560 (1973) Michel, C., Fritz-Niggli, H., Blattmann, H., Cordt-Riehle, I.: Effects of low-dose irradiation with Xrays and pi-mesons on embryos of two different mouse strains. Strahlentherapie 153, 674-681 (1977) Ohzu, E.: Effects of low-dose X-irradiation on early mouse embryos. Radiat. Res. 26, 107-113 (1965) Rugh, R., Wohlfromm, M., Varma, A.: Low-dose X-ray effects on the Precleavage mammalian zygote. Radiat. Res. 37, 401--414 (1969) Templier, J., Maudouyt, M. A., Charpenteau, M., Bonnet, J: Effets de faibles doses de rayonnement gamma sur la gestation de la Souris. C.R. Soc. Biol. 172, 177--180 (1978) United Nations Report: Scientific Committee on the Effects of Atomic Radiation. Sources and Effects of Ionizing Radiation, pp. 655--725. New York: United Nations 1977 Received February 15, 1979
