Harumi Ohyama

Evidence for the Internucleosomal Breakage of Chromatin in Rat Thymocytes Irradiated in Vitro

Low molecular-weight (free) DNA increases in lymphatic cells in association with radiation-induced interphase death. In this report, the size of the free DNA from rat thymocytes irradiated in vitro with 1-kR x rays was analyzed by agarose gel electrophoresis and compared with that of the DNA fragments extracted from micrococcal nuclease-digested thymocyte nuclei and with that of HaeIII-digested phi X-174 RF DNA. Results demonstrated clearly that the free DNA consisted of a series of discretely sized DNA fragments with lengths that were integral multiples of a nucleosomal DNA (180 base pairs), suggesting that internucleosomal chromatin breaks occurred in rat thymocytes after in vitro irradiation. Additional evidence was obtained from an electron microscopic observation on the breakdown of chromatin strands into particle form in the irradiated cells.

Cell Volume Reduction Associated with Interphase Death in Rat Thymocytes

Correlation of interphase death with cell volume reduction in rat thymocytes was examined using an automated cell size distribution analyzer. After x irradiation from 50 to 2000 r in vitro, cell suspension were incubated at 37/sup 0/C for 4 h. Measurement of cell size distribution during and after incubation demonstrated that the irradiated thymocyte suspension consisted of two discrete subpopulations, one of normal size and the other of smaller size. The number of smaller cells increased parallel to a rise in dead(erythrosine B stained) cell counts with incubation time and radiation dose, suggesting that smaller cells might correspond to dead thymocytes. This was confirmed by examining a centrifugally separated dead-cell population, which had a size distribution curve similar to that of the smaller cells. The possibility that small-cell counts could be used as a new criterion for interphase death is discussed.

Radiation-Induced Formation of Apoptotic Bodies in Rat Thymus

The process of interphase death of thymocytes in whole-body X-irradiated rats were studied. Cell size distribution analysis indicates that cell fragments (= apoptotic bodies) appeared in the thymus and increased in number depending on dose (200-1000 R) and time (2-6 hr) after irradiation with corresponding decrease in normal-size thymocytes. Occurrence of nuclear fragmentation in association with the cellular fragmentation was proved with cytofluorometric determination of DNA content in individual cells. Scanning electron microscopic observations also revealed extensive fragmentation of cells in the irradiated rat thymus. The results show clearly that cells as well as nuclei fragment rapidly into smaller pieces of various sizes in the irradiated rat thymus as commonly observed with apoptosis.

Adaptive response in embryogenesis : I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis

An adaptive response was demonstrated during embryogenesis in mice. Whole-body irradiation at a dose of 0-50 cGy was given to condition pregnant ICR mice on day 9 to day 11 of gestation. Then their whole bodies were exposed to a challenging dose of 5 Gy on the next day. The numbers of living fetuses, prenatal deaths and living fetuses with external gross malformations were determined on day 19. A conditioning dose of 30 cGy on day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations induced by a 5-Gy dose on day 12. This indicates the existence of a critical dose and timing for administering a conditioning dose for radioadaptation during the late period of organogenesis in mice. The possible mechanisms involved are discussed.

Radiosensitivity of CD45RO+ memory and CD45RO- naive T cells in culture.

Radiosensitivities of various human T-cell subsets were investigated by a proliferation assay and by a single-cell gel electrophoresis assay. Each T-cell subset was purified using a cell sorter and was induced to proliferate by ionomycin and interleukin 2. Unsorted T cells showed biphasic dose-survival curves, indicating the heterogeneity of T cells in terms of radiosensitivity. Purified CD4+ helper and CD8+ killer T cells showed similar biphasic dose-survival curves. Hence both T-cell subsets were composed of cells of different radiosensitivity. The T-cell subsets belonging to different activation stages such as CD45RO+ memory and CD45RO- naive T cells had different dose-survival curves. The former was more radiosensitive than the latter. The high radiosensitivity of CD45RO+ cells was also demonstrated by single-cell gel electrophoresis after irradiation. This is the first demonstration that a particular cell surface marker on T cells is correlated with greater radiosensitivity.

Adaptive Response in Embryogenesis. III. Relationship to Radiation-Induced Apoptosis and Trp53 Gene Status

Abstract Wang, B., Ohyama, H., Haginoya, K., Odaka, T., Itsukaichi, H., Yukawa, O., Yamada, T. and Hayata, I. Adaptive Response in Embryogenesis. III. Relationship to Radiation-Induced Apoptosis and Trp53 Gene Status. We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53+/+) and Trp53 heterozygous (Trp53+/–) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53+/+ embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53+/+ embryonic mice, while no such effect was found in the Trp53+/– embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.

Adaptive Response in Embryogenesis: V. Existence of Two Efficient Dose-Rate Ranges for 0.3 Gy of Priming Irradiation to Adapt Mouse Fetuses

Abstract Wang, B., Ohyama, H., Shang, Y., Tanaka, K., Aizawa, S., Yukawa, O. and Hayata, I. Adaptive Response in Embryogenesis: V. Existence of Two Efficient Dose-Rate Ranges for 0.3 Gy of Priming Irradiation to Adapt Mouse Fetuses. Radiat. Res. 161, 264–272 (2004). The adaptive response is an important phenomenon in radiobiology. A study of the conditions essential for the induction of an adaptive response is of critical importance to understanding the novel biological defense mechanisms against the hazardous effects of radiation. In our previous studies, the specific dose and timing of radiation for induction of an adaptive response were studied in ICR mouse fetuses. We found that exposure of the fetuses on embryonic day 11 to a priming dose of 0.3 Gy significantly suppressed prenatal death and malformation induced by a challenging dose of radiation on embryonic day 12. Since a significant dose-rate effect has been observed in a variety of radiobiological phenomena, the effect of dose rate on the effectiveness of induction of an adaptive response by a priming dose of 0.3 Gy administered to fetuses on embryonic day 11 was investigated over the range from 0.06 to 5.0 Gy/min. The occurrence of apoptosis in limb buds, incidences of prenatal death and digital defects, and postnatal mortality induced by a challenging dose of 3.5 Gy given at 1.8 Gy/min to the fetuses on embryonic day 12 were the biological end points examined. Unexpectedly, effective induction of an adaptive response was observed within two dose-rate ranges for the same dose of priming radiation, from 0.18 to 0.98 Gy/ min and from 3.5 to 4.6 Gy/min, for reduction of the detrimental effect induced by a challenging dose of 3.5 Gy. In contrast, when the priming irradiation was delivered at a dose rate outside these two ranges, no protective effect was observed, and at some dose rates elevation of detrimental effects was observed. In general, neither a normal nor a reverse dose- rate effect was found in the dose-rate range tested. These results clearly indicated that the dose rate at which the priming irradiation was delivered played a crucial role in the induction of an adaptive response. This paper provides the first evidence for the existence of two dose-rate ranges for the same dose of priming radiation to successfully induce an adaptive response in mouse fetuses.

The effect of X-irradiation on glycolysis of rat thymocytes

SummaryThe production of aerobic lactate by suspensions of rat thymocytes incubated at 37° was considerably increased after x-irradiation. Intracellular concentrations of intermediates and cofactors of glycolysis were measured at different incubation times. Fructose-1,6-diphosphate showed a remarkable increase during incubation. Crossover plot of changes in the levels of intermediates and changes in mass-action ratios for glycolytic reactions have clearly demonstrated the enhanced activity of phosphofructokinase after irradiation. The possible correlation between the activated phosphofructokinase and lactate production was discussed.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

Abstract Wang, B., Ohyama, H., Shang, Y., Fujita, K., Tanaka, K., Nakajima, T., Aizawa, S., Yukawa, O. and Hayata, I. Adaptive Response in Embryogenesis: IV. Protective and Detrimental Bystander Effects Induced by X Radiation in Cultured Limb Bud Cells of Fetal Mice. Radiat. Res. 161, 9–16 (2004). The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

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

Abstract Nose, M., Wang, B., Itsukaichi, H., Yukawa, O., Hayata, I., Yamada, T. and Ohyama, H. 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. Radiat. Res. 156, 195–204 (2001). 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 the survival-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.