OK-432 reduces mortality and bacterial translocation in irradiated and granulocyte-colony stimulating factor (G-CSF)-treated mice.

Bacterial translocation/Acute radiation syndrome/Endotoxin/G-CSF/OK-432 Acute radiation induces bacterial translocation from the gut,followed by systemic infection and sepsis. In order to reduce the mortality after acute whole body irradiation, it is essential to control bacterialtranslocation. In this study, we established a bacterial translocation assay as a sensitive method to detect minor mucosal injury by radiation. By utilizing this assay, we evaluated the adverse effects, if any, of hematopoietic reagents on the mucosal integrity in the respiratory and gastro-intestinal tracts. Bacterial translocation to the liver and spleen occurred after whole-body irradiation if the dose exceeded 6 Gy. The administration of G-CSF unexpectedly increased the bacterial translocation in 8 Gy-irradiated mice. The pharmaceutical preparation oflow-virulent Streptococcus pyogenes, OK-432, significantly reduced the endotoxin levels in peripheral blood without any reduction of bacterialtranslocation. A combined treatment with G-CSF and OK-432 decreased bacterial translocation and prevented death. This result indicates that the early administration of G-CSF has an adverse effect on bacterial translocation, and that a combined treatment of G-CSF and OK-432 attenuates the adverse effect of G-CSF and improves the survival rate after acute irradiation

Effects of Pre-exposure on the Survival and Hematological Changes in the Lethally Irradiated C57BL Mice

Exposing mice to 0.5 Gy 2 weeks before lethal (around LD50/30) whole-body irradiation has been reported to induce marked radio-resistance and to rescue them from "bone marrow death." It is widely accepted that cause of the death after dose around LD50/30 is mainly mediated by hematological failure. In order to elucidate the mechanism underlying the adaptive response, we examined effects of 0.5 Gy pre-exposure on the survival and hematological changes in C57BL mice irradiated with 6.5 Gy X-rays. The pre-exposure 2 weeks before the challenging dose enhanced survival to 77% at day 30, whereas without exposure, the survival decreased to 20 % at day 20 and 0% at day 26 after 6.5 Gy. Hematopoietic progenitor CFU-GM in the pre-irradiated mice began to recover around day 20 and then increased markedly. However, peripheral blood cell counts depleted to reach a nadir at day 20, regardless of the pre-irradiation, in spite of marked difference in the survival between the pre-irradiated and non-pre-irradiated mice. These cell counts in the pre-exposed mice recovered at day 30. We found that OK432, a bio-response modifier, could further enhance the survival of pre-exposed mice to 97%, when administrated 2 days before 0.5 Gy. The OK432 administrated mice also survived without recovery of the peripheral blood cell counts at day 20. These results manifest that the lethally irradiated mice are rescued by pre-exposure without recovery of hematological failure at least by day 20. Furthermore, we have observed long-term effect of pre-exposure for 1 year. More than half of the survivor of the pre-irradiated mice and the OK432 injected mice survived 1 year, even though they showed a variety of abnormalities.低線量放射線の生物影響に関する国際シンポジウ

Radioactive Response and radiation-Induced Teratogenesis in the Late Period of Organogenesis in Mice: Involvement of p53-Dependent Apoptosis

In the past 5 years, a series of study was done at our institute toinvestigate radiation effects on the embryogenesis in mice with an emphasison mechanisms involved in the radiation-induced adaptive response and therole of radiation-induced apoptosis played in teratogenesis in the lateperiod of organogenesis. Using the limb bud system, we first found thatradiation-induced apoptosis is involved in malformations, namely,radiation-induced apoptosis in the predigital regions of embryonic limb budsis responsible for digital defects in ICR mice. Examination of embryonicC57BL/6J mice with different p53 status led to further finding thatsusceptibility to the radiation-induced apoptosis and digital defectsdepends on both the p53 status and the radiation dose. p53 wild-type miceappeared to be the most sensitive, while p53 knockout mice were the mostresistant. These results indicate that p53-dependent apoptosis mediatesradiation-induced digital defects. The existence of a radioadaptive responsein fetuses, i.e., the priming dose significantly decreases the apoptosisinduction, prenatal death, and digital defects in the living fetuses inducedby the challenging dose, was found first in ICR strain mice and laterconfirmed again in C57BL/6J mice. p53 heterozygous embryos did not show theradioadaptive response, indicating the involvement of p53 in theradioadaptive response

Radioadaptive Response and Radiation-Induced Teratogenesis in the Late Period of Organogenesis in Mice: Involvement of p53-Dependent Apoptosis.

GENERAL INTRODUCTION In the past 5 years, a series of study was done at National Institute of Radiological Sciences to investigate the effects of radiation on the embryogenesis in mice with an emphasis on mechanisms involved in the radiation-induced adaptive response and the role of radiation-induced apoptosis played in teratogenesis in the late period of organogenesis. \nMATERIALS AND METHODS Animals: The pregnant ICR mice bearing p53(+/+) fetuses and pregnant C57BL/6 mice bearing p53(+/+), p53(+/-) and p53(-/-) fetuses were used. X-Irradiation: The animals were exposed to whole-body irradiation at room temperature with an X-ray machine, operated at 200 kVp and 20 mA, using a 0.5 mm Al + 0.5 mm Cu filter. For investigation on induction of limb defects, 1-5 Gy at 1.8 Gy/min was given on E11. For induction of radioadaptation, the priming dose of 0.05-0.50 Gy and challenging dose of 3 or 5 Gy were given on E11 and E12, respectively. The priming and challenging irradiation dose rates were 34 cGy/min and 1.8 Gy/min, respectively. Biological Endpoints: In both limb teratogenesis and induction of radioadaptation study, the dams were sacrificed by cervical dislocation. Fetuses were removed by cesarean section. The limb buds were obtained 4-6 h after irradiation, and apoptotic cells in the predigital regions were identified by the routine hematoxylin eosin staining method and quantified microscopically. The number of living fetuses and the percentage of living malformed fetuses in total living fetuses were scored on E18. \nRESULTS Radiation, via induction of apoptosis in a dose-dependent way, selectively killed the cells in the predigital regions of limb buds where the cells were undergoing differentiation. Radiation-induced apoptosis hardly occurred in the cells in the preinterdigital regions where cells had stopped DNA synthesis and were scheduled to go spontaneous apoptosis. Radiation-inducted apoptosis was responsible for both digital defects and the severity of limb teratogenesis in ICR p53(+/+) mice [1]. In C57BL/6 mice, susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both p53 gene dose and radiation dosage, namely, p53 wild-type (p53(+/+)) mice appeared the most sensitive, p53 heterozygous type (p53(+/-)) mice were intermediate, and p53 knockout (p53(-/-)) mice showed the most resistant. Therefore, it was concluded that prenatal radiation-induced limb defects were mediated by p53-dependent apoptosis [2]. In ICR p53(+/+) mice, prior to a challenging dose of 5.0 Gy on E12, a priming dose of 0.30 Gy on E11 significantly increased the number of the living fetuses per dam and decreased that of the external gross malformations among living fetuses on E19. The adapted dams could give some living births unexpectively, but the pups showed a high postnatal mortality, and the survivors suffered from various detrimental effects such as growth retardation and behavioral alterations. These indicated the existence of a radiation-induced adaptive response reducing prenatal death and limb defects induced by a challenging dose radiation in the late period of embryogenesis in mice [3,4]. For C57BL/6 mice, prior to a challenging dose of 3.0 Gy, the adaptive response could be induced with a priming dose at 0.05 or 0.30 Gy on E11, but this was only among the animals with p53(+/+) gene status. From these results, it was demonstrated that the adaptive response in embryogenesis was related to radiation-induced apoptosis and that induction of the radioadaptation required the involvement of p53 gene [5,6].\nREFERENCES1. B. Wang, K. Fujita, K. Watanabe, C. Ohhira, T. Odaka, I. Hayata , H. Mitani, H. Ohyama, T. Yamada and A. Shima, Radiation-induced apoptosis and limb bud teratogenesis in embryonic mice. Radiat. Res. 151, 63-68(1999).2. B. Wang, H. Ohyama, K. Haginoya, T. Odaka, T. Yamada and I. Hayata, Prenatal radiation-induced limb defects mediated by Trp53-dependent apoptosis in mice. Radiat. Res. 154, 673-679(2000).3. B. Wang, H. Ohyama, M. Nose, H. Itsukaichi, T. Nakajima , O. Yukawa, T. Odaka , K. Tanaka, E. Kojima, T. Yamada and I. Hayata, Adaptive response in embryogenesis: I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis. Radiat. Res. 150, 120-122(1998).4. B. Wang, K. Haginoya, H. Ohyama , M. Nose, H. Itsukaichi, T. Nakajima , O. Yukawa, T. Odaka , T. Yamada and I. Hayata, Adaptive response in embryogenesis: II. Postnatal developmental retardation in the prenatally irradiated mice. Radiat. Res. 152, 119-123(1999).5. B. Wang, H. Ohyama , K. Haginoya, T. Odaka , H. Itsukaichi, O. Yukawa, T. Yamada and I. Hayata, Adaptive response in embryogenesis: III. Relationship to radiation-induced apoptosis and Trp53 gene status. Radiat. Res. 154, 277-282(2000).6. B. Wang, Involvement of p53-dependent apoptosis in radiation teratogenesis and in the radioadaptive response in the late period of organogenesis of mice. J. Radiat. Res. 42, 1-10(2001).International Symposium on Biological Effects of Low Dose Radiation: Molecular Mechanisms for Radiation-induced Cellular Response and Cancer Development

Rescue of lethally irradiated mice from hematopoietic death by pre-exposure to 0.5 Gy X rays without recovery from peripheral blood cell depletion and its modification by OK432.

Exposing mice to 0.5 Gy X rays 2 weeks before lethal irradiation has been reported to induce marked radioresistance and to rescue them from hematopoietic death. Here we examined effects of the 0.5-Gy pre-exposure on hematological changes in C57BL mice that were lethally irradiated with 6.5 Gy X rays. Approximately 77% of pre-exposed mice survived 30 days after this irradiation, whereas 80% of mice that did not receive this pre-exposure died by day 20. However, regardless of the pre-exposure, peripheral blood cell counts decreased markedly by day 3 and reached a nadir at day 20. CFU-S in femur and CFU-GM in spleen had started to recover at day 10 and 14, respectively, but recovery of functional peripheral blood cells occurred later. The effect of pre-exposure on survival was altered by OK432, a bioresponse modifier; the effect　depended on the timing of its administration. OK432 given 2 days before 0.5 Gy enhanced the protective effect of pre-exposure, resulting in the survival of 97% of the mice. In contrast, injection of OK432 1 day before or 2 days after pre-exposure led to 100% mortality. Thus thesurvival-promoting effect of 0.5 Gy could be altered by OK432. The OK432-induced changes in the survival of mice could not be attributed solely to hematological changes, as shown by blood cell counts and progenitor cell contents. These results suggest that radioresistance induced by pre-exposure to 0.5 Gy X rays is not stable, but rather varies with the physiological conditions, and can be modulated by factors such as OK432