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.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months.

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

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.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Combined Environment Simulator for Low-Dose-Rate Radiation and Partial Gravity of Moon and Mars.

Deep space exploration by humans has become more realistic, with planned returns to the Moon, travel to Mars, and beyond. Space radiation with a low dose rate would be a constant risk for space travelers. The combined effects of space radiation and partial gravity such as on the Moon and Mars are unknown. The difficulty for such research is that there are no good simulating systems on the ground to investigate these combined effects. To address this knowledge gap, we developed the Simulator of the environments on the Moon and Mars with Neutron irradiation and Gravity change (SwiNG) for in vitro experiments using disposable closed cell culture chambers. The device simulates partial gravity using a centrifuge in a three-dimensional clinostat. Six samples are exposed at once to neutrons at a low dose rate (1 mGy/day) using Californium-252 in the center of the centrifuge. The system is compact including two SwiNG devices in the incubator, one with and one without radiation source, with a cooling function. This simulator is highly convenient for ground-based biological experiments because of limited access to spaceflight experiments. SwiNG can contribute significantly to research on the combined effects of space radiation and partial gravity.

Space Radiation Biology for "Living in Space".

Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

Detection of space radiation-induced double strand breaks as a track in cell nucleus.

To identify DNA damage induced by space radiations such as the high linear energy transfer (LET) particles, phospho-H2AX (gammaH2AX) foci formation was analyzed in human cells frozen in an International Space Station freezer for 133days. After recovering the frozen sample to the earth, the cells were cultured for 30 min, and then fixed. Here we show a track of gammaH2AX positive foci in them by immuno-cytochemical methods. It is suggested that space radiations, especially high LET particles, induced DSBs as a track. From the formation of the tracks in nuclei, exposure dose rate was calculated to be 0.7 mSv per day as relatively high-energy space radiations of Fe-ions (500 MeV/u, 200 keV/microm). From the physical dosimetry with CR-39 plastic nuclear track detectors and thermo-luminescent dosimeters, dose rate was 0.5 mSv per day. These values the exposed dose rate were similar between biological and physical dosimetries.

Astronaut's organ doses inferred from measurements in a human phantom outside the international space station.

Space radiation hazards are recognized as a key concern for human space flight. For long-term interplanetary missions, they constitute a potentially limiting factor since current protection limits for low-Earth orbit missions may be approached or even exceeded. In such a situation, an accurate risk assessment requires knowledge of equivalent doses in critical radiosensitive organs rather than only skin doses or ambient doses from area monitoring. To achieve this, the MATROSHKA experiment uses a human phantom torso equipped with dedicated detector systems. We measured for the first time the doses from the diverse components of ionizing space radiation at the surface and at different locations inside the phantom positioned outside the International Space Station, thereby simulating an extravehicular activity of an astronaut. The relationships between the skin and organ absorbed doses obtained in such an exposure show a steep gradient between the doses in the uppermost layer of the skin and the deep organs with a ratio close to 20. This decrease due to the body self-shielding and a concomitant increase of the radiation quality factor by 1.7 highlight the complexities of an adequate dosimetry of space radiation. The depth-dose distributions established by MATROSHKA serve as benchmarks for space radiation models and radiation transport calculations that are needed for mission planning.

COMPARISON OF COSMIC-RAY ENVIRONMENTS ON EARTH, MOON, MARS AND IN SPACECRAFT USING PHITS.

Estimation of cosmic-ray doses is of great importance not only in aircrew and astronaut dosimetry but also in evaluation of background radiation exposure to public. We therefore calculated the cosmic-ray doses on Earth, Moon and Mars as well as inside spacecraft, using Particle and Heavy Ion Transport code System PHITS. The same cosmic-ray models and dose conversion coefficients were employed in the calculation to properly compare between the simulation results for different environments. It is quantitatively confirmed that the thickness of physical shielding including the atmosphere and soil of the planets is the most important parameter to determine the cosmic-ray doses and their dominant contributors. The comparison also suggests that higher solar activity significantly reduces the astronaut doses particularly for the interplanetary missions. The information obtained from this study is useful in the designs of the future space missions as well as accelerator-based experiments dedicated to cosmic-ray research.

Somatic mutation in larvae of the silkworm, Bombyx mori, induced by heavy ion irradiation to diapause eggs.

In order to investigate whether eggs of the black-striped strain (P(S)) of the silkworm, Bombyx mori, represent an appropriate model for estimating the biological effect of cosmic radiation, radiosensitivity of the eggs against X-rays and heavy ion particles was examined as ground-based experiments. The exposure of diapause eggs to X-rays or heavy ion particles resulted in somatic mutations appearing as a white spot on the black integument during larval stage. Irradiation of non-diapause eggs with X-rays demonstrated a significant difference in frequency of the mutation between fractionated and single administration doses, but no difference was observed in diapause eggs. Incidence of the mutation as induced by carbon ion beams for 15-day old eggs was higher for eggs that had been kept at 15 degrees C than those kept at 25 degrees C. Neon beam irradiation of diapause eggs displayed dose- and linear energy transfer (LET)-dependent effects, causing a maximal rate of the mutation at 150 keV/microm. These results confirm that B. mori eggs represent valid models for estimating the biological effects of cosmic radiation.