Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Comparison of biological measurement and physical estimates of space radiation in the International Space Station.

Nowadays, ordinary people can travel in space, and the possibility of extended durations in an environment such as moon of the Earth and Mars with higher space radiation exposures compared to past missions, is increasing. Until now, the physical doses of space radiation have been measured, but measurement of direct biological effects has been hampered by its low dose and low dose-rate effect. To assess the biological effects of space radiation, we launched and kept frozen mouse embryonic stem (ES) cells in minus eighty degree Celsius freezer in ISS (MELFI) on the International Space Station (ISS) for a maximum of 1,584 days. The passive dosimeter for life science experiments in space (PADLES) was attached on the surface of the sample case of the ES cells. The physical dosimeter measured the absorbed dose in water. After return, the frozen cells were thawed and cultured and their chromosome aberrations were analyzed. Comparative experiments with proton and iron ion irradiation were performed at particle accelerators on Earth. The wild-type ES cells showed no differences in chromosomal aberrations between the ground control and ISS exposures. However, we detected an increase of chromosome aberrations in radio-sensitized histone H2AX heterozygous-deficient mouse ES cells and found that the rate of increase against the absorbed dose was 1.54-fold of proton irradiation at an accelerator. On the other hand, we estimated the quality factor of space radiation as 1.48 ± 0.2. using formulas of International Commission of Radiation Protection (ICRP) 60. The relative biological effectiveness (RBE) observed from our experiments (1.54-fold of proton) was almost equal (1.04-fold) to the physical estimation (1.48 ± 0.2). It should be important to clarify the relation between biological effect and physical estimates of space radiation. This comparative study paves a way to reveal the complex radiation environments to reduce the uncertainty for risk assessment of human stay in space.

Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect).

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

Relieved residual damage in the hematopoietic system of mice rescued by radiation-induced adaptive response (Yonezawa Effect).

Existence of adaptive response (AR) was previously demonstrated in C57BL/6J mice. Irradiations were performed by delivering a priming low dose of X-rays (0.50 Gy) in combination with a challenge high dose of accelerated carbon or neon ion particles. AR was characterized by significantly decreased mortality in the 30-day survival test. This mouse AR model ('Yonezawa Effect') was originally established by using X-rays as both the priming and challenge irradiations. The underlying mechanism was due to radio-resistance occurring in blood-forming tissues. In this study, we verified the existence of AR and further investigated residual damage in the hematopoietic system in surviving animals. Results showed that the priming low dose of X-rays could relieve the detrimental effects on the hematopoietic system. We observed both an improvement in the blood platelet count and the ratio of polychromatic erythrocytes (PCEs) to the sum of PCEs and normochromatic erythrocytes (NCEs) and a marked reduction of the incidences of micronucleated PCEs and micronucleated NCEs. These findings suggest that the priming low dose of low linear energy transfer (LET) X-rays induced a protective effect on the hematopoietic system, which may play an important role in both rescue from acute lethal damage (mouse killing) and prevention of late detrimental consequences (residual anhematopoiesis and delayed genotoxic effects) caused by exposure to a high challenge dose from low-LET (X-ray) or high-LET (carbon and neon ion) irradiations. These findings provide new knowledge of the characterization of the Yonezawa Effect by providing new insight into the mechanistic study of AR in vivo.

Effects of low-dose heavy ions on embryonic development in mice and on melanocyte differentiation in the epidermis and hair bulb.

The effects of prenatal low-dose irradiation with heavy ions on embryonic development in mice and on melanocyte differentiation are not well understood. We performed whole-body irradiation of pregnant C57BL/10J mice at embryonic Day 9 (E9) with a single dose of γ-rays, silicon, argon or iron ions. The number of living embryos and embryonic body weight at E18 decreased after exposure to heavy ions at high doses. Malformations such as small eyes and limb anomalies were observed in heavy-ion-treated embryos, but not in γ-ray-treated embryos. The frequency of abnormally curved tails was increased by exposure to γ-rays and argon and iron ions even at a dose of 0.1 Gy (P < 0.05). In contrast, a dose-dependent decrease in the number of epidermal melanoblasts/melanocytes and hair bulb melanocytes was observed after 0.1 Gy irradiation with γ-rays or heavy ions (P < 0.01). The decrease in the number of dorsal hair bulb melanocytes, dorsal and ventral epidermal melanoblasts/melanocytes and ventral hair bulb melanocytes was not necessarily correlated with the linear energy transfer of the radiation tested. Moreover, the effects of heavy ions were larger on the ventral skin than on the dorsal skin, indicating that the sensitivity of melanocytes to heavy ions differs between the dorsal and ventral skin. Taken together, these results suggest that the effects of the low-dose heavy ions differ between cell types and tissues, and the effects on the prenatal development of mice and melanocyte development are not necessarily greater than those of γ-rays.

Adaptive response of low linear energy transfer X-rays for protection against high linear energy transfer accelerated heavy ion-induced teratogenesis.

BACKGROUND: Adaptive response (AR) of low linear energy transfer (LET) irradiations for protection against teratogenesis induced by high LET irradiations is not well documented. In this study, induction of AR by X-rays against teratogenesis induced by accelerated heavy ions was examined in fetal mice. METHODS: Irradiations of pregnant C57BL/6J mice were performed by delivering a priming low dose from X-rays at 0.05 or 0.30 Gy on gestation day 11 followed one day later by a challenge high dose from either X-rays or accelerated heavy ions. Monoenergetic beams of carbon, neon, silicon, and iron with the LET values of about 15, 30, 55, and 200 keV/µm, respectively, were examined. Significant suppression of teratogenic effects (fetal death, malformation of live fetuses, or low body weight) was used as the endpoint for judgment of a successful AR induction. RESULTS: Existence of AR induced by low-LET X-rays against teratogenic effect induced by high-LET accelerated heavy ions was demonstrated. The priming low dose of X-rays significantly reduced the occurrence of prenatal fetal death, malformation, and/or low body weight induced by the challenge high dose from either X-rays or accelerated heavy ions of carbon, neon or silicon but not iron particles. CONCLUSIONS: Successful AR induction appears to be a radiation quality event, depending on the LET value and/or the particle species of the challenge irradiations. These findings would provide a new insight into the study on radiation-induced AR in utero.

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs.

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.

Effects of low-dose heavy ions on the postnatal development of mice and the yield of white spots in the mid-ventrum and tail-tips.

Effects of prenatal low-dose irradiations of heavy ions on the postnatal development of mice and of melanocytes have not been well studied. Pregnant females of C57BL/10J mice were irradiated whole-body at 9 days of gestation with a single acute dose of γ-rays, silicon (Si, 57 keV/µm), argon (Ar, 100 keV/µm) and iron (Fe, 220 keV/µm) ions. The effects were studied by scoring changes in the postnatal development of mice as well as in the pigmentation of cutaneous coats and tail-tips of their offspring 22 days after birth. The survival to day 22 decreased from the offspring exposed to 0.4 Gy of argon and iron ions and to 0.75 Gy of silicon ions. White spots were found in the mid-ventrum and tail-tips of irradiated offspring. The frequency and size of the white spots in the mid-ventrum in mice exposed to silicon, argon and iron ions were greater than those of γ-rays. Even in the low dose (0.1 Gy), γ-rays and heavy ions increased the frequency of the ventral spots. The RBE estimated by the frequency of the ventral spots was 2.3 (Si), 3.1 (Ar) and 4.5 (Fe). These results suggest that prenatal exposure to heavy ions possesses a greater effect on the postnatal development of mice as well as melanocyte development than does exposure to γ-rays.

X-ray-induced radioresistance against high-LET radiations from accelerated heavy ions in mice.

Induction of an adaptive response by priming X rays in combination with challenge irradiations from high-LET accelerated heavy ions was attempted in young adult female C57BL/6J Jms mice using 30-day survival after the challenge irradiations as an index. Three kinds of accelerated heavy ions from monoenergetic beams of carbon, silicon and iron ions with LETs of about 15, 55 and 200 keV/µm, respectively, were examined. A priming low dose of 0.50 Gy X rays in combination with a challenging dose of 7.50 Gy was used in the animals serving as a positive control group to confirm the successful induction of an adaptive response. The priming low dose of 0.50 Gy X rays was also used in combination with accelerated heavy ions. The priming low dose of X rays significantly reduced the mortality from the high challenge doses of carbon or silicon particles but not from iron particles. These results indicate that an adaptive response could be induced by priming low-LET X rays in combination with subsequent challenge high-LET irradiations from certain kinds of accelerated heavy ions, and successful induction of an adaptive response would possibly be an event related to the LET and/or the type of heavy ions. This is the first time that the existence of an adaptive response induced by low-LET X rays against high-LET whole-body irradiation in mice has been demonstrated. These findings would provide new insight into the radiation-induced adaptive response in vivo.

Two major factors involved in the reverse dose-rate effect for somatic mutation induction are the cell cycle position and LET value.

To study mechanisms which could be involved in the reverse dose rate effect observed during mutation induction after exposure to high LET radiation, synchronized mouse L5178Y cells were exposed to carbon 290 MeV/n beams with different LET values at the G2/M, G1, G1/S or S phases in the cell cycle. The frequency of Hprt-deficient (6-thioguanine-resistant) mutant induction was subsequently determined. The results showed that after exposure to high LET value radiation (50.8 and 76.5 keV/microm), maximum mutation frequencies were seen at the G2/M phase, but after exposure to lower LET radiation (13.3 keV/microm), the highest mutation frequencies were observed at the G1 phase. The higher LET beam always produced higher mutation frequencies in the G2/M phase than in the G1 phase, regardless of radiation dose. These results suggest that cells in the G2/M phase is hyper-sensitive for mutation induction from high LET radiation, but not to mutation induction from low LET radiation. Molecular analysis of mutation spectra showed that large deletions (which could include almost entire exons) of the mouse Hprt gene were most efficiently induced in G2/M cells irradiated with high LET radiation. These entire exon deletions were not as frequent in cells exposed to lower LET radiation. This suggests that inappropriate recombination repair might have occurred in response to condensed damage in condensed chromatin in the G2/M phase. In addition, by using a hyper-sensitive mutation detection system (GM06318-10 cells), a reverse dose-rate effect was clearly observed after exposure to carbon beams with higher LET values (66 keV/microm), but not after exposure to beams with lower LET values (13.3 keV/microm). Thus, G2/M sensitivity towards mutation induction, and the dependence on radiation LET values could both be major factors involved in the reverse dose rate effect produced by high LET radiation.

Effects of a 2-step culture with cytokine combinations on megakaryocytopoiesis and thrombopoiesis from carbon-ion beam-irradiated human hematopoietic stem/progenitor cells.

To evaluate whether the continuous treatment of two cytokine combinations is effective in megakaryocytopoiesis and thrombopoiesis in hematopoietic stem/progenitor cells exposed to heavy ion beams, the effects of a 2-step culture by a combination of recombinant human interleukin-3 (IL-3) + stem cell factor (SCF) + thrombopoietin (TPO), which just slightly protected against carbon-ion beam-induced damages, and a combination of IL-3 + TPO, which selectively stimulated the differentiation of the hematopoietic stem/progenitor cells to megakaryocytes and platelets, were examined. CD34(+)-hematopoietic stem/progenitor cells isolated from the human placental and umbilical cord blood were exposed to carbon-ion beams (LET = 50 keV/microm) at 2 Gy. These cells were cultured under three cytokine conditions. The number of megakaryocytes, platelets and hematopoietic progenitors were assessed using a flow cytometer and a clonogenic assay at 14 and 21 days after irradiation, respectively. However, the efficacy of each 2-step culture was equal or lower than that of using the IL-3 + SCF + TPO combination alone and the 2-step culture could not induce megakaryocytes and platelets from hematopoietic stem/progenitor cells exposed to high LET-radiation such as carbon-ion beams. Therefore, additional cytokines and/or hematopoietic promoting compounds might be required to overcome damage to hematopoietic cells by high LET radiation.

Severe damage of human megakaryocytopoiesis and thrombopoiesis by heavy-ion beam radiation.

Heavy ions have a unique efficacy for tumor control in radiotherapy. To clarify the effects of heavy-ion beams on hematopoietic stem/progenitor cells, the effects of carbon-ion beams on megakaryocytopoiesis and thrombopoiesis in CD34(+) cells derived from human placental and umbilical cord blood were investigated. The cells were exposed to carbon-ion beams (LET = 50 keV/microm) and then were treated with thrombopoietin (TPO) alone or TPO plus other cytokines. Megakaryocytic progenitor cells, such as megakaryocyte colony-forming units (CFU-Meg), were far more sensitive to carbon-ion beams than to X rays, and no restoration of carbon-ion beam-irradiated CFU-Meg by treatment with any cytokine combination was observed. However, total cell expansion in liquid culture was not different after either carbon-ion beam or X irradiation of CD34(+) cells. The activation of gamma-H2AX, a marker of DNA double strand-breaks (DSBs), was promoted by the cytokine treatment in X-irradiated CD34(+) cells but not in carbon-ion-irradiated cells. These results showed that carbon-ion beams inflicted severe damage on megakaryocytopoiesis and thrombopoiesis and that a better combination of cytokines and other agents may be needed to stimulate the recovery of hematopoietic cells and repair this damage.

Extremely low dose ionizing radiation up-regulates CXC chemokines in normal human fibroblasts.

Although the public today could be exposed to X-rays as high as 1 cGy due to diagnostic procedures, the biological effects of this low-dose range have not been well established. We searched through >23,000 transcripts in normal human fibroblasts, HFLIII, using a novel comprehensive expression analysis method. More than 200 genes were up-regulated transiently by 1 cGy of X-rays during the 1-hour period after irradiation. We determined the nucleotide sequence of 10 up-regulated transcripts with the greatest rate of increase in the irradiated HFLIII cells. Three of the 10 transcripts encoded CXC chemokines (CXCL1, CXCL2, and CXCL6). The rest included the transcripts of other secretory products (secretogranin II, thrombospondin type I domain containing 2, amphiregulin, and interleukin-6) and unknown genes. To test the involvement of CXC chemokines in cells irradiated with low doses, we irradiated HFLIII cells with 1 to 20 cGy X-rays and transferred the media from HFLIII culture to two melanoma cell lines characteristic of excessive numbers of the CXC chemokine-specific receptors. The growth of these melanoma lines were significantly stimulated by the medium from HFLIII irradiated at 1 to 5 cGy. Our results indicate that human cells respond to doses of radiation as low as 1 cGy, and mechanisms alternative to those involved in moderate/high-dose studies have to be considered in understanding the biological effects of diagnostic level radiation. In addition, our comprehensive approach using a novel expression profiling method is a powerful strategy to explore biological functions associated with very low levels of toxic agents.

Radiosensitivity of hypoxic and proliferating clonogen in a human lung cancer grown in nude mice.

Using cultured and nude mouse tumor cells (IA) derived from a human lung cancer, we studied their radiosensitivity by focusing attention on the dynamics of tumor clonogens. The movement of clonogens in the regrowing IA tumor after irradiation can be divided into three phases: first, the early and rapid survival recovery (PLD repair) phase; second, the delay phase involving a certain lag in survival change; and third, the repopulation phase consisting of two stages: the anoxic repopulation before angiogenesis and the hypoxic repopulation in the presence of a poorly developed vascular network. Clonogens in a regrowing tumor after irradiation were found to actively proliferate even in an anoxic environment before angiogenesis and under the hypoxic conditions prevailing after the formation of a tumor with a poorly developed vascular system. This re-grown tumor was found to be more radioresistant than a sham-treated control tumor. It is believed that these clonogens are genetically selected under hypoxic conditions throughout the process of tumor growth and regrowth, and may be primarily involved in tumor recurrence or accelerated repopulation in fractionated irradiation.

Effects of prenatal irradiation with an accelerated heavy-ion beam on postnatal development in rats: I. Neurophysiological alterations.

Effects on postnatal neurophysiological development in offspring were studied after exposure of pregnant Wistar rats to accelerated carbon-ion beams with an LET of about 13 keV/ mum at doses ranging from 0.1 Gy to 2.5 Gy on the 15th day of gestation. The age at which four physiological markers appeared and five reflexes were acquired was examined prior to weaning. Gain in body weight was monitored until the offspring were 3 months old. Male offspring were evaluated as young adults using two behavioral tests. The effects of X rays estimated for the same biological end points were studied for comparison. For most of the end points at early age, no significant alterations were observed in offspring that received prenatal irradiation with 0.1 Gy of either accelerated carbon ions or X rays compared to the offspring of sham-irradiated dams. However, all offspring whose dams received 2.5 Gy died prior to weaning. Offspring from dams irradiated with accelerated carbon ions generally showed higher incidences of prenatal death and preweaning mortality, markedly delayed accomplishment in their physiological markers and reflexes, and gain in body weight compared to those exposed to X rays at doses of 0.5 to 2 Gy. Significantly reduced ratios of main organ weight to body weight at the postnatal ages of 30, 60 and 90 days were also observed within this dose range. The results indicate that irradiation with 0.5 to 2 Gy on day 15 of gestation caused permanent alterations in offspring that were dependent on dose. The alterations include permanent growth retardation, morphological malformations in main organs, including microcephaly, diminished reflex attainment, delayed appearance of physiological markers, and changes in adult behavior. Exposure to 1 to 2 Gy of radiation resulted in growth retardation and behavioral alterations that persisted throughout life. Accelerated carbon ions generally induced more detrimental effects than X rays.

Effects of carbon-ion radiation on the postnatal development of mice and on the yield of white spots in the mid-ventrum and tail tips.

Pregnant female C57BL/10JHir mice were irradiated whole-body at 9 days of gestation with a single acute dose of carbon-ion radiation. The average linear energy transfer (LET) of the carbon ions was 50 keV/microm within a spread-out Bragg peak (SOBP). The effects were studied by scoring changes in the postnatal development of the mice as well as in the pigmentation of the cutaneous coats and tail tips of their offspring 22 days after birth. The percentage of live births was reduced in mice exposed to carbon ions at doses greater than 0.5 Gy. The survival to day 22 was also reduced in mice exposed to carbon ions at doses greater than 0.75 Gy. Moreover, the body weight at day 22 was reduced in mice exposed to carbon ions at doses greater than 0.1 Gy. A comparison of the survival to day 22 after exposure to carbon ions with our previous results for 60Co gamma rays indicated that carbon ions were twice as effective as gamma rays. White spots were found in the mid-ventrum as well as in the tail tips of offspring exposed to carbon ions in utero. The frequency and the size of the white spots in the mid-ventrum and in the tail tips increased as the dose increased. Carbon ions appear to be slightly more effective than the gamma rays used in our previous study. In the ventral white spots, no melanocytes were observed in the epidermis, dermis and hair follicles. These results indicate that prenatal exposure to carbon ions has a greater effect on the postnatal development and survival of mice than does exposure to gamma rays, and that the relative biological effectiveness is greater than that for effects on melanocyte development.

An improved culture system of mouse peripheral blood lymphocytes for analysis of radiation-induced chromosome aberrations.

There is an incentive to develop a culture system of mouse peripheral blood lymphocytes (PBLs) to serve as models for studying genotoxic effects in humans exposed to mutagens, including ionizing radiation. However, many past approaches have been laborious, complex and only partly reproducible. In the present study, we established an improved culture system of mouse PBLs by removing blood and/or plasma, which was found to inhibit in vitro mitotic stimulation or proceeding cell cycles of lymphocytes. We compared the reactions of isolated PBLs to mitogens between the classical method and the present improved one. Then, we applied this method to the cytogenetic analysis using chemically induced premature chromosome condensation (PCC) as well as the conventional analysis, and demonstrated that the frequency of excess fragments observed in PCC cells might be useful to quantify the radiation-induced damages on chromosomes.

Comparative analysis of G2 arrest after irradiation with 75 keV carbon-ion beams and 137Cs gamma-rays in a human lymphoblastoid cell line.

Heavy-ion beams are more effective than gamma-rays in causing G2 arrest. In this study, we investigated the expression of Wee1 and Cdc2 protein levels in order to analyze the G2 arrest caused by carbon-ion beam irradiation. Human lymphoblastoid TK6 cells were exposed to a 75 keV carbon-ion beam or 137Cs gamma-rays. Although the levels of Wee1 and Cdc2 protein were increased after exposure to either beam, Wee1 protein levels were influenced more by carbon-ion beam irradiation than by gamma-rays. To the contrary, Cdc2 protein levels were increased more by gamma-rays than by carbon-ion beams. These findings suggest that the G2 arrest produced by heavy-ion beams, such as the carbon-ion irradiation used in this study, might be associated with the overexpression of the Wee1 protein and of Cdc2 phosphorylation regulated by Wee1. Together, these events may act to prolong the length of G2 arrest.