The project aims at a better understanding of radiation risk during early pregnancy, a period for which virtually nothing is known with regard to radiation risk for the human embryo. As development during the preimplantation stage is similar in almost all mammals (including man), one cell mouse embryos in vitro and in vivo will be exposed to different radiation qualities (neutrons, X-rays, beta rays) in different cell cycle stages.
These experiments underline previous results in which the high potential of exerting deleterious effects on early embryonic development has been shown for hydrogen-3 thymidine and, in particular, for hydrogen-3.
The major radiation risk during the preimplantation period is prenatal death. However, there are some reports in the literature pointing also to an enhanced teratogenic risk for the surviving foetuses. Our results support this conclusion. There was a marked dose dependent increase in the frequency of gross abnormalities after irradiation of 1-cell mouse embryos with either neutrons or X-rays. The most prominent malformation was gastroschisis, whereas exencephaly was observed at much lower frequency. The number of malformed foetuses increase with a quadratic function of neutro of X-ray dose, whereas foetal death was compatible with a simple exponential function.
The results of the teratogenic experiments emphasize that one should not look at the preimplantation period only as a period of either embryonic death or unimparied development after irradiation. Obviously, there exist conditions under which those embryos that survive irradiation are at an increased teratogenic risk.
The possibility of the induction of malformations after radiation exposure during pregnancy is a serious radiation hazard. Studies have shown that teratogenic effects are primarily induced when radiation exposure has taken place during organogenesis, and, there have been reports of increased frequency of abnormal foetuses after exposure during the preimplantation stage. This result is strongly dependent on the mouse strain. From the point of view of radiation risk, it may be even more important to look for a potential teratogenic risk after radiation exposure or germ cells, because they are at risk over the whole reproductive life time.
Studies in our institute have shown that mouse strain Heiligenberger (NMRI like) does respond with a higher frequency of malformations after r adiation exposure during the preimplantation stage. The fact that malformations, which are observed on day 19, are inducible even in the 1-cell stage, suggests that there is an effect on the genetic material and that this effect is inherited over a number of cell generations. It is conceivable that such an effect on the genetic material can also result from the irradiation of germ cells.
In order to investigate these observations the germ cell stages were exposed to different radiation qualitites (X-rays, gamma-rays or beta-rays) and different dose rates (1 Gy/min and below 0.01 Gy/min in order to avoid lethal effects on the sensitive immature female germ cells). Female mice were irradiated either with X-rays (dose rate 1 Gy/min), gamma-rays (caesium-137; dose rate 3.25 mGy/min) or sham irradiated. Mating strated immediately after radiation exposure or, in some cases, after a delay of 2 weeks. 19 days after copulation (day of copulation equals day 1) the mice were killed by cervical dislocation and the uterinecontent checked for early resorptions, late resorptions, late foetal death, surviving, foetuses and foetuses with macroscopically visible malformations.
Even after the lowest dose (0.5 Gy) used in the experiments pregnant females were obtained only during the first 4 weeks after radiation exposure, irrespective of whether mating started immediately after radiation exposure or with a delay of 2 weeks. This result is in line with previous observations reported in the literature and reflects the high radiation sensitivity of oocytes after high dose rate exposures.
The total litter loss was very pronounced in the controls, possibly reflecting inbreeding of the strain. In previous years, colony bred mice were used and the total litter loss never exceeded 10%. Only after the highest dose (3 Gy) was a significant increase in litter loss observed. The number of early resorptions was enhanced after doses of 2 and 3 Gy, whereas late resorptions and late foetal d eaths were not affected by radiation exposure of oocytes. An increase in malformed foetuses was also found and gastroschises were seen almost exclusively. Radiation sensitivity of oogenesis was somewhat lower than that of 1-cell embryos, but comparable to preimplantation stages succeeding the 1-cell stage.
The following endpoints will be studied:
chromosomal aberrations in the first, second and third mitosis after exposure;
the number of micronuclei in the first, second and third interphase after exposure;
the type and frequency of teratogenic effects on day 19 of gestation.
Marked differences in radiation sensitivity at the various cell cycle phases of the one cell stage have been observed. After neutron or X-ray exposure, the number of chromosomal aberrations had increased by the first mitosis after radiation; this was different for radiation exposure due to beta rays from 3H-arginine or 3H-thymidine, where a rise in the number of aberrations was seen only at the second mitosis. A high number of complete chromosomes were lost in the second and third mitosis after neutron or X-ray exposure. The lost chromosomes contributed, besides acentric fragments and polycentric chromosomes, to the micronucleus frequency of the cells.
The mouse strain used in the experiments responded to radiation exposure of the one cell stage not only with lethal events, but also with malformed foetuses (mainly gastroschisis). The number of malformed foetuses increased with the square of radiation dose and without an indication of a threshold dose. Further experiments confirming and expanding these results will be made.