Dose Rate Effectiveness in Radiation-induced Teratogenesis in Mice

To investigate the role of p53 gene in tissue repair of teratogenic injury, we compared incidence of radiationinduced malformations in homozygous p53(-/-) mice, heterozygous p53(+/-) mice and wild-type p53(+/+) mice. After X-irradiation with 2 Gy at high dose rate on 9.5 days of gestation, p53(-/-) mice showed higher incidences of anomalies and higher resistance to prenatal deaths than p53(+/+) mice. This reciprocal relationship of radiosensitivity to anomalies and deaths supports the notion that embryos or fetuses have a p53-dependent 'guardian' that aborts cells bearing radiation-induced teratogenic DNA damage. In fact, after X-irradiation, the number of apoptotic cells was greatly increased in p53(+/+) fetuses but not in p53(-/-) fetuses. The same dose of γ-ray exposure at low dose rate on 9.5-10.5 day of gestation produced significant reduction of radiationinduced malformation in p53(+/+) and p53(+/-) mice, remained teratogenic for p53(-/-) mice. These results suggest that complete elimination of teratogenic damage from irradiated tissues requires the concerted cooperation of two mechanisms; proficient DNA repair and the p53-dependent apoptotic tissue repair. When concerted DNA repair and apoptosis functions efficiently, there is a threshold dose-rate for radiation-induced malformations. INTRODUCTION Irradiation of the mammalian fetus produces various kinds of congenital anomaly depending on the dose and the specific gestational phase of irradiation. Early-stage embryos of wild-type mice are radiosensitive to killing, but the survivors are born without malformations. However, fetuses at mid-gestational stages are highly susceptible to malformation at high doses of radiation but resistant at low doses (1-3). In previous experiment, we proposed that mouse embryonic or fetal tissues have a p53-dependent ‘guardian’ that aborts cells with radiation-induced teratogenic damage, on the basis of the observations that 60% of the p53(+/+) mouse fetuses died after X-irradiation with 2 Gy on day 9.5 of gestation, but few of the survivors showed anomalies, whereas few of p53(ー/ー) mouse fetuses died, but 76% of the survivors showed anomalies (4). Dose and dose rate effectiveness is one of the most important subject in estimating the risks of radiation. In general, the dose rate effectiveness of radiation is closely related to the repairing ability of induced DNA damage. However, DNA repair is not perfect. Therefore, defense mechanisms other than DNA repair must eliminate badly damaged cells that fail to fully repair their DNA damage. To elucidate the mechanisms of tissue repair of teratogenic injury caused by radiation, we compared incidence of radiation-induced malformations and abortions in homozygous p53(-/-) mice, heterozygous p53(+/-) mice and wild-type p53 (+/+) mice. MATERIALS & METHODS Mice carrying a disrupted, nonfunctional p53 gene, p53(-/-), were derived by homologous recombination in an ES cell line from 129/SvJ mice (5). MCH:ICR/jcl mice were used as p53(+/+) mice. Fertilized eggs at two-cell stage were transplanted into pseudopregnant recipient mice. The resultant pregnant mice were exposed to 2 Gy of X-rays at high dose rate (450-1100 mGy/min) or low dose rate (0.7-1.2 mGy/min) on day 9.5 of gestation. On day 18.5 of gestation, fetuses were removed by cesarean operation, and the numbers of implants, deaths (deaths after day 10), live fetuses with congenital anomalies recorded. To assess the extent of apoptosis in p53(+/+) and p53(+/-) fetuses, pregnant mice were exposed to 0-3 Gy of X-rays (250 kVp; dose rate, 0.45 Gy/min) on day 9.5 of gestation and killed after irradiation; fetuses were then fixed and paraffin-embedded, and 4-um sections were stained using the ApopTag in situ apoptosis detection kit (Oncor, Inc., USA; a modified TUNEL method), which allows direct immunoperoxidase detection of digoxigeninlabeled DNA fragments in apoptotic cells.