Hindawi

BioMed Research International

Volume 2020, Article ID 4703286, 25 pages
https://doi.org/10.1155/2020/4703286

Review Article
Space Radiation Biology for “Living in Space”

Satoshi Furukawa ,1 Aiko Nagamatsu ,1 Mitsuru Nenoi,2 Akira Fujimori,2

Shizuko Kakinuma ,2 Takanori Katsube ,2 Bing Wang ,2 Chizuru Tsuruoka ,2
Toshiyuki Shirai,2 Asako J. Nakamura ,3 Asako Sakaue-Sawano,4 Atsushi Miyawaki,4
Hiroshi Harada ,5 Minoru Kobayashi ,5 Junya Kobayashi,5 Takekazu Kunieda ,6
Tomoo Funayama ,7 Michiyo Suzuki,7 Tatsuo Miyamoto ,8 Jun Hidema ,9,10
Yukari Yoshida ,11 and Akihisa Takahashi 11
1
Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan
2
National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-
1 Anagawa, Inage-ku, Chiba 263-8555, Japan
3
Department of Biological Sciences, College of Science, Ibaraki University, 2-1-1, Bunkyo, Mito, Ibaraki 310-8512, Japan
4
Lab for Cell Function and Dynamics, CBS, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
5
Radiation Biology Center, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
6
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
7
Takasaki Advanced Radiation Research Institute, QST, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan
8
Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku,
Hiroshima 734-8553, Japan
9
Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan
10
Division for the Establishment of Frontier Sciences of the Organization for Advanced Studies, Tohoku University, 2-1-1 Katahira,
Aoba-ku, Sendai, Miyagi 980-8577, Japan
11
Gunma University Heavy Ion Medical Center, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan

Correspondence should be addressed to Akihisa Takahashi; [email protected]

Received 20 December 2019; Accepted 13 March 2020; Published 8 April 2020

Academic Editor: Shoichiro Ono

Copyright © 2020 Satoshi Furukawa et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.
2 BioMed Research International

1. Introduction by thin shielding (a few g/cm2), secondary particles produced

by nuclear reactions increase in number as shielding mass
Yuri Gagarin was the first human to journey into outer space. increases and become dominant in fluxes under thick shield-
He completed one orbit of Earth on 12 April 1961. Almost ing conditions [10, 11]. Thus, TPs play a role in increasing or
60 years have passed since this event, and space mission decreasing the exposure of astronauts to radiation in LEO. The
durations have remarkably extended. Currently, it is possible energies of TPs are generally lower than those of GCRs, and
for humans to spend more than 6 months in outer space on their maximum energy is approximately several hundred
the International Space Station (ISS). The ISS circles the MeV.
Earth at an altitude of approximately 400 km. The living Since the construction of ISS began in 1998, there have
environment on the ISS is challenging to astronauts because been more than 120 SPEs (counted by NOAA, Space Weather
microgravity (μG) induces musculoskeletal atrophy, isolated Prediction) that have affected the Earth environment over
and limited habitability causes psychological stress, and solar cycles 23 to 24. The emergency return of astronauts
exposure to space radiation potentially endangers the health following flight rules [12] due to severe SPEs has never
of the astronauts [1]. occurred before because of Earth’s protective magnetic field.
The next challenging steps for humankind include new Japan Aerospace Exploration Agency (JAXA) has con-
missions to the Moon followed by human exploration of ducted a series of monitoring experiments to evaluate the
Mars. In a Mars mission, the long distance between Earth radiation environment inside and outside the Japanese
and Mars will make the total mission duration 800–1,100 Experiment Module Kibo, which is part of the ISS with Pas-
days, of which approximately 500 days will be spent on the sive Dosimeter for Life-Science and Experiments in Space
surface of the planet, depending on the final mission design (PADLES) [9, 13, 14]: area radiation monitoring in the Japa-
[2]. As a result, radiation exposure is expected to be greater nese Experiment Module “Kibo” of the ISS (Area PADLES)
when compared with that of a 6-month mission on the ISS. [15]; dose measurements of biological samples exposed to
One major health concern in such prolonged missions is space radiation (Bio PADLES) [16–19]; radiation dosimetry
the amount of radiation exposure that accumulates over the of Asian astronauts (Crew PADLES); various kinds of interna-
duration of the lives of the astronauts. Therefore, health risks tional cooperative experiments with ISS partners (Dosimetric-
associated with exposure to space radiation are an important PADLES); measurement of the directional dependence of the
topic in a human Mars mission. The focus of this review is radiation dose inside the Kibo module (Exp PADLES) [11,
space radiation. We will initially discuss the environment of 20]; and measurement of outside doses and evaluation of
space radiation. This will be followed by a description of the shielding effect of the ISS Kibo hull (Free-space PAD-
the various kinds of research endeavors undertaken to evalu- LES). Those experiments were initiated in 2008 just after
ate and minimize adverse biological outcomes caused by attachment of the Japanese Pressurized Module (JPM) to
space radiation exposure. the ISS.
We concluded that the characteristics of the space radia-
2. Environment of Space Radiation tion environment in LEO contain the following: (i) a high
contribution from high-linear energy transfer (LET) radia-
2.1. Radiation Environment in Low-Earth Orbits (LEO). As tion that have a high-quality factor (QF) up to 30; (ii) dose
mentioned in previous reviews [3–6], important ionizing rates have values that are a few hundred times greater than
radiation (IR) sources in the ISS orbits (altitude: 300 to 400 those on the ground; (iii) the directional distribution of space
km; orbital inclination: 51.6°) include the three primary radi- radiation is nearly isotropic; and (iv) radiation effects occur
ation sources (galactic cosmic rays (GCRs), which range under μG [13, 14, 19]. Space radiation for LET greater than
widely from protons to Fe-ions, solar particle events several keV/μm causes more serious damage to living things
(SPEs), and electrons and protons trapped in the Van Allen than low-LET radiation. Measurements only of absorbed
Belts (TPs)) outside the spacecraft. These combine to pro- doses are insufficient for investigating biological effects or
duce a complex radiation environment in and around the assessing radiation risk to astronauts. Dose equivalents tak-
ISS, and the complexity of this radiation is dependent on ing into account the LET distributions of high-LET particles,
the solar cycle, altitude, and shielding of each module of the their high radiation QFs, and relative biological effectiveness
ISS. (RBE) must be measured considering space radiation
Primary GCRs comprise protons and high-energy heavy- environment.
ion (HZE) charged particles with energy spectra forming a Space radiation environments include fast neutrons
broad peak around 1 GeV/n [3]. Fluxes of less than about with a wide energy range beyond several tens of MeV.
10 GeV/n are inversely related to solar activity [7]. The The neutron dose contribution has been roughly estimated
GCR fluxes depend heavily on the ISS altitude and are diffi- through the STS-89 space shuttle mission/Mir experiment
cult to shield against using a realistic shielding mass for the with RRMD-III for charged particles and BBND for neutrons
ISS structure because of their high energy. Primary GCRs with energies less than 15 MeV, both loaded simultaneously
produce many secondary particles through projectile and tar- [21]. Neutron doses contributing to total doses in LEO and
get fragmentation in the ISS shielding materials and in the around the Moon and Mars are still being estimated with var-
bodies of astronauts. The fluxes of primary TPs increase sub- ious simulation codes. However, no practical measurement
stantially as the altitude of the ISS increases [3, 7–9]. has been established so far with a neutron personal dosimeter
Although the fluxes of primary TPs can be effectively reduced applicable to energy exceeding ~20 MeV. The most physical
BioMed Research International 3

and practical approach for estimating the high-energy neu- Cyanobacteria have created a foundation of the environ-
tron dose is to theoretically and experimentally determine ment in which most organisms live today. The ozone layer
LET values of energetic charged particles released by interac- completely absorbs harmful UV-C radiation (<285 nm),
tions with the neutrons and an anthropomorphic phantom. and through evolution, organisms were able to expand their
The dose-equivalent part of the practical dose can be habitat from water to land. The first land organisms, which
obtained using the relation between QFs and LET values resembled the liverworts, have evolved into the diverse range
via the Q–L relation ICRP 60 [22]. Therefore, dose equiva- of plant species that exist. Sunlight-driven photosynthesis
lents taking into account the LET distributions are also maintained the composition of the atmosphere, and plants
important for evaluating neutron doses. serve as a food source for animals. Although sunlight is
highly beneficial for life on Earth, it contains harmful UV-B
radiation (280–315 nm) despite its efficient absorption by
2.2. Radiation Environment beyond LEO (Deep Space, the
the ozone layer [38]. Although UV-B radiation accounts for
Moon, and Mars). The space radiation environment differs
<0.5% of the total solar energy on the surface of the Earth,
in and beyond LEO, including the surface of the Moon
its high energy causes damage to important cellular compo-
[23–28], Mars [23], deep space [29, 30], and their compari-
nents, such as DNA, RNA, protein, and lipids, as it is readily
sons [23, 31]. In past explorations, space radiation measure-
absorbed by such macromolecules [39]. Among them, DNA,
ments have been conducted by three interplanetary missions
which stores genetic information, is a major target of UV-
in the orbital environment of both the Moon and Mars to
induced damage, and UV radiation can directly alter its
generate global dosage maps and to measure energy spectra
structure. The main UV-induced photoproducts are cyclobu-
below 100 MeV [32–36]. In deep space outside Earth’s pro-
tane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone
tective magnetic field, HZE charged particles of GCRs and
(6-4) photoproducts, which are also termed (6-4) photo-
solar energetic particles (SEPs) strongly affect the dosimetry
products, and form between adjacent pyrimidines on the
of astronauts. Space radiation doses change drastically
same strand [39, 40]. CPDs account for approximately 75%
because of the varying intensity and peak amplitude of SEP
of DNA damage and the (6-4) photoproducts for the major-
events in and near the Moon and Mars environments, where
ity of the remaining 25%. DNA damage impedes replication
a protective magnetic field is almost completely absent.
and transcription, induces mutations, and may be lethal [39,
Therefore, for radiation dose management of astronauts
40]. Therefore, UV radiation causes damage to all organisms
exposed to both SEPs and GCRs, it is essential to establish
including plants. Most skin cancers are caused by UV radia-
methods for estimating organ doses and effective doses that
tion damaging the DNA in skin cells. Plants, which are ses-
are both relative to career dose limits. These are obtained
sile organisms, are at a higher risk of UV-B damage in
from the energy spectra of space radiation and doses from
comparison with motile organisms, which reduces growth
personnel dosimeters and environmental radiation monitor-
and productivity.
ing systems.
The environment of space is characterized by low gravity,
Currently, as part of the NASA Artemis program, astro-
temperature oscillation, short-wavelength solar UV radia-
nauts will land on the Moon by 2024. Under the umbrella
tion, and complex cosmic IR. In particular, space is showered
of Artemis, the Lunar Orbital Platform-Gateway, which is a
by a variety of different types of radiation, and thus, astro-
station orbiting the Moon, provides an international cooper-
nauts are exposed to a considerably large amount of space
ation platform for scientific experiments and exploration of
radiation [41, 42]. Moreover, in space, UV-C with shorter
the lunar surface. The career dose limits for gateway are still
wavelengths than UV-B are much more prevalent, and its
under coordination between international partners. Cur-
intensity is much higher than on Earth. On the surface of
rently, there is no interplanetary mission to measure the
Mars, the UV-B radiation is remarkably higher than that
space environment in Japan. Thus, we must conduct actual
on Earth and exceeds the safety limit for terrestrial life
measurements beyond LEO to determine effective materials,
[43, 44]. Therefore, to establish sustainable life support sys-
effective locations, and appropriate thicknesses or combina-
tems for securing long-term human life in space, the effects
tions on the basis of benchmark evaluations. This informa-
of the complicated space environment not only for humans
tion will be useful for interplanetary space flight and travel
but also for plants must be understood. The growth and sur-
expected in the near future.
vival of plants will be required to supply nutrients and oxy-
gen to humans under a resource-recycling system in space.
2.3. Solar Ultraviolet (UV) Radiation. UV is part of the natu-
ral energy produced by the sun. UV radiation has electro- 3. Irradiation Tests with Ground Facilities
magnetic radiation wavelengths from 10 nm to 400 nm, Similar to the Environment in Space
which are shorter than visible light (400–700 nm) but longer
than X-ray. UV radiation reaches the Earth surface. UV radi- 3.1. Low Dose Rate Irradiation Facilities. Humans are contin-
ation is classified into three regions based on their effects on uously exposed to low doses of background radiation and
biological processes: UV-C (<280 nm), UV-B (280–315 nm), may also be exposed to low doses of IR from X-ray or CT
and UV-A (315–400 nm). UV-C, which is a highly energetic scans and occupational usage of radiation as medical doctors,
wavelength, is eliminated by the stratospheric ozone layer radiologists, or nuclear power plant workers. Residents in
and is not encountered by plants. Both UV-B and UV-A high background radiation areas or space station astronauts
radiations reach the surface of Earth [37]. are exposed to low dose rates of IR for long periods. Residents
4 BioMed Research International

in the vicinity of the evacuated areas of Chernobyl and Sometimes this increases to tens of mGy/day during SPE
Fukushima Daiichi nuclear power plant disasters may also events lasting up to several days. However, this will become
have been exposed to low dose rates of IR and have health a serious health issue over long stay periods in future mis-
risk concerns because of exposure to above-average levels of sions to Mars and other planets. SEP and GCR contain vari-
IR. Biological responses toward acute irradiation from high ous radiations, including γ-ray, electron, neutron, proton,
doses of IR have been well characterized, and the molecular and heavier ions. In particular, GCR includes heavier ions
mechanisms of cell cycle checkpoints and DNA repair have up to Fe (Z = 26), and these heavier ions can significantly
been studied extensively. However, the biological effects affect crew and electric devices in a manned spacecraft
and the health risks with low dose or low-dose-rate radiation because of their high ionization density. The interesting
exposure remain poorly understood. Understanding the energy range of GCR is from 100 MeV/u to several GeV/u.
health risks (mainly cancer) due to low doses of IR has been This energy spectrum is around the peak flux of GCR
provided by epidemiological studies of atomic bomb survi- and difficult to shield against in a spacecraft. Experiments
vors [45]; however, the risk or biological effects from chronic involving cosmic radiation in space are expensive and rather
exposure to low dose rates of IR has only recently been more time-limited. Although it is difficult to reproduce the cosmic
examined. To understand the molecular mechanisms follow- radiation environment on the ground, experiments using
ing exposure to low dose rates of IR, irradiation instruments HZE accelerators are important.
for chronic exposure to low dose rates of IR have been estab- The pioneer of radiation research of HZEs was Bevalac at
lished in Japan and other countries [46]. The instruments in the Lawrence Berkeley National Laboratory (LBL), USA.
Japan use 137Cs as a radiation source and can irradiate bio- After the shutdown of Bevalac in 1993, the QST-NIRS in
logical specimens with γ-rays. Among them, the irradiation Japan started the operation of Heavy-Ion Medical Accelera-
instrument for the Institute for Environmental Sciences tor in Chiba (HIMAC) in 1994. Although NIRS is a dedicated
(IES) can expose small animals such as mice with extremely facility for heavy-ion radiotherapy, it can provide various ion
low dose rates (about 0.05 mGy/day), and other instruments beams for other experiments outside treatment hours. The
at IES can expose mice to different low dose rates (about 1 or available ions are He, C, Ar, Fe, and Xe with an energy max-
20 mGy/day). These instruments can perform chronic irradi- imum of 800 MeV/u (ion species dependent). There are five
ation for a few years and have been supplying important experimental beam lines at HIMAC, and a 3D field can be
information on the biological effects of chronic low dose rate formed by the wobbler beam delivery system. In the past 25
irradiation to mice [46]. The instruments at the Research years, many studies have been carried out, including those
Institute for Radiation Biology and Medicine (RIRBM), examining biological effects, radiation shielding, develop-
Hiroshima University, Central Research Institute of Electric ment and comparison of cosmic radiation detectors, and
Power Industry (CRIEPI), University of Occupational and radiation tests of electric devices [48].
Environmental Health (UOEH), and National Institute of At the almost same time, the GSI Helmholtz Center for
Radiological Sciences, National Institutes for Quantum and Heavy Ion Research (GSI) started the operation of SIS-18.
Radiological Science and Technology (QST-NIRS), can also GSI covers the wide research fields of nuclear physics, atomic
perform chronic irradiation with dose rates that are higher physics, material science, plasma physics, biophysics, and
than the instruments at IES. The instruments at CRIEPI clinical research, based on the heavy-ion accelerator com-
and UOEH can irradiate cultured cells, and IES and RIRBM plex. This facility can provide various ions from proton to
possess irradiation instruments for exclusive use on cultured uranium. The energy range is up to 2 GeV/u (ion species
cells. The chronic irradiation instrument at the Radiation dependent) and is suitable to study the radiation effects of
Biology Center (RBC), Graduate School of Biostudies, Kyoto GCRs. Furthermore, the new FAIR accelerator complex is
University, can be used to irradiate cultured cells and small under construction at GSI. Heavy ions with energies up to
fish [47]. The instruments have three different 137Cs radia- 10 GeV/u will be available for radiation research in the near
tion sources and stands (where the CO2 incubator for culture future [49].
cells or aquarium for small fish is placed on) that can be The Brookhaven National Laboratory (BNL) in the USA
placed at distances between 1.3 and 12 m from the radiation has operated a very large heavy-ion accelerator complex,
source. The machine exposes cultured cells or small fish to RHIC/AGS, for the study of nuclear and particle physics. In
γ-rays over the range of 0.3–1,500 mGy/day by combining 2003, the NASA Space Radiation Laboratory (NSRL) was
the radiation source choice and distance. The dose rate of founded to study the health risks of cosmic radiation to
space radiation in the ISS is 0.5 mGy/day [13–19]. Thus, the crews. NSRL uses the BNL Booster synchrotron, which can
instrument at the RBC may provide important information provide ions from protons to gold, ranging in energy from
about the health risks to astronauts at the ISS. The use of such 50 MeV/u to 2,500 MeV/u (ion species dependent) [50].
chronic irradiation instruments in Japan is expected to pro- Although one of the difficulties of ground-based experiments
vide important information to clarify the biological effects is that cosmic radiation consists of a wide variety of ion spe-
and health risks to humans under various chronic low dose cies and energy ranges, NSRL produces a GCR simulated
rates of irradiation. beam using rapid switching technology of ion species and
ion energies.
3.2. High-Energy Particle Irradiation Facilities. Dose rates In addition to these three research facilities, some insti-
from cosmic radiation such as SEP and GCR are low at tutes provide HZE beams for cosmic radiation research. In
around 0.5 mGy/day as measured inside ISS Kibo [13–19]. particular, 12 C-ion radiotherapy facilities are now in
BioMed Research International 5

operation, including the Gunma University Heavy Ion Med- diate HZEs as a microbeam. There are many international
ical Center (GHMC) in Japan and the National Centre for facilities where biological targets can be irradiated with a
Oncological Hadrontherapy (CNAO) in Italy. These facilities microbeam [55–57]; however, most of them are limited to
provide a C-ion beam from 100 MeV/u to 400 MeV/u, which irradiating only protons and alpha particles. The sites that
is close to the GCR energy range. Researchers of cosmic radi- are able to irradiate microbeams of HZE are GSI [58],
ation and the C-ion radiotherapy have common scientific Munich University [59], Institute of Modern Physics in
interests in characterizing the biological effects of HZEs and China [60], and QST-Takasaki [52, 61]. Of these four sites,
contribute to this research field. three sites, except QST-Takasaki, are only capable of irradiat-
ing cultured cells. However, to evaluate the effects of HZE on
3.3. Microbeam Irradiation Facilities. Space radiation human health, experiments using model animals are neces-
includes the HZE of GCRs, which is unlike radiation at sary. The heavy-ion microbeam at QST-Takasaki is able to
ground level. Therefore, analysis of the hit effect of HZE is irradiate cultured cells and small model animals with a
an important subject when evaluating space radiation risks HZE. Therefore, this facility has contributed to the analysis
for long-term manned missions. of radiation effects of HZE to cultured cells from the view-
HZE deposits concentrated energy along with its trajec- point of single-ion-hit effects [62] and bystander effects
tory, and this manner of microdosimetric energy deposition [63–65], as well as analyzed the effects of local HZE radiation
of HZE is called the “ion track structure” [51]. The ion track on the whole body using the nematode Caenorhabditis ele-
structure is a characteristic of HZE and explains the differ- gans [66, 67] and Medaka fish [68]. Moreover, at QST-Taka-
ence between the biological effects of HZE and that of low- saki, the development of a new microbeam beamline that
LET radiation. Because of this concentrated energy deposi- generates a finer beam spot than the current system is taking
tion of the ion track structure, a dose is deposited in close place [69]. In summary, heavy-ion microbeams will contrib-
vicinity to the ion-hit position and does not extend further ute more in future research examining the effect of HZE radi-
than a few micrometers away from this hit position. This ation, which is a significant health risk for astronauts
results in a microscopic uneven dose distribution on radia- undertaking long-term projects in space.
tion targets [52]. For example, when 1 Gy of HZEs with a
LET higher than approximately 625 keV/μm was used to 4. Adverse Events Caused by Space Radiation
irradiate a population of cells with nuclei with areas of
100 μm2 using broad field beam irradiation, less than one 4.1. DNA Damage and Detection. Radiation exposure induces
ion hits the nucleus of a cell on average. Thus, a mixture of various biological effects with the main effect being damage
cells hit and not hit by an ion occurs. Even with low-LET to DNA. There are various types of radiation-induced DNA
radiation, when the number of hit events becomes small, a damage, including base damage, single-strand breaks (SSBs),
similar uneven spatial distribution of hit events will occur. and double-strand breaks (DSBs) [70–73]. Among them,
However, the dose given to the cells with a single hit event DNA DSBs are the most severe DNA lesion. Therefore,
is too small and not sufficient to induce cellular responses. organisms have various DNA damage repair pathways to
In contrast, a single hit of HZE with high LET deposits a suf- ensure genome stability [70, 71, 73, 74]. However, if a large
ficient dose to cells that is biologically effective. Therefore, amount of damage occurs or the damage is not repaired cor-
investigating the effects of HZE on a cell population with rectly, cell death, cellular senescence, and tumorigenesis may
broad field beam irradiation faces two problems that arise be induced [71, 72, 75, 76].
from not uniformly irradiating the cell population. The first The energy of radiation is important when considering
problem is that each cell in a cell population will not be hit radiation exposure in space. Radiation exposure on the
with the same count of ions, making it difficult to evaluate ground is at low-LET radiation levels and includes X-rays
the exact effect of a single-ion hit. The second problem is and γ-rays, while GCR contains high-LET radiation such as
the radiation-induced bystander effect [53, 54], which is a energetic protons and heavy particle beams, i.e., HZE parti-
phenomenon that ion-hit cells induce radiation responses cles [77–79]. High-LET radiation exposure leads to dense
on nonhit nearby cells by transferring the hit signal via bio- ionization along their radiation tracks and induces complex
logical pathways. This second issue contributes much more DNA damage. These localized dense DNA regions of dam-
to the overall radiation effect than the situation of low-LET age, within a few helical turns of DNA, are called “complex
radiation. DNA damage (lesion)” or “clustered DNA damage (lesion)”
A microbeam is an experimental method that targets and and are difficult to repair when compared with that of normal
irradiates biological samples with a radiation spot on the DNA damage [80–83]. Therefore, even if the radiation dose
micrometer scale under microscopic observation. Because a is the same on the ground as that in space, the quality and
microbeam is able to irradiate each cell with a defined dose amount of DNA damage that occurs will be different, and
accurately, analysis of an evenly irradiated cell population is evaluating the quality and quantity of DNA damage induced
possible. Moreover, by irradiating only a part of the cell pop- by GCRs for precise assessment of the biological effects in
ulation, we are able to induce and analyze the radiation- space is required.
induced bystander effect. Therefore, a microbeam is a useful Although clustered DNA damage induced by high-LET
approach to analyze biological effects caused by radiation radiation exposure is detected using agarose gel electrophore-
having a microscopically nonuniform dose distribution like sis or the comet assay, the results are sometimes controversial
HZE. To analyze the hit effect of HZE, it is necessary to irra- because their sensitivity is limited [82, 84–88]. In recent
6 BioMed Research International

years, several papers have reported visualization of DNA mechanism needs an intact DNA template after replication,
damage induced by high-LET radiation exposure using γ- it is activated during the late S and G2 phase, whereas the
H2AX, which is a marker of DNA DSB [17, 89–91]. These NHEJ mechanism is activated at any point during the cell
data have shown the different nature of DNA damage cycle [96]. NHEJ is used preferentially in higher eukaryotes
between low-LET radiation exposure and high-LET radiation such as humans. However, high-LET radiation such as heavy
exposure. Since γ-H2AX occurs in a DSB site-specific man- particle or α-rays, which is present in space radiation, gener-
ner, it is used as a sensitive tool to detect DSB [92–94]. The ates various types of DNA damage (e.g., DSB, SSB, and oxi-
Ohnishi group was the first to report that clear tracks of the dative damage) at the point of the irradiated areas. As NHEJ
γ-H2AX signal are detected in lymphoblastoid nuclei after cannot repair such complicated DNA damage, HR is often
spaceflight [17]. Additionally, a similar track of γ-H2AX activated for repair of this DNA damage in a cell cycle-
was detected in fibroblast nuclei that had been cultured for independent manner [97]. However, cell cycle-independent
14 d at the ISS; however, these tracks were not observed for use of HR, particularly in G1 may cause misrepair and sub-
control samples on ground control samples [95]. Recently, sequent genomic instability. Hence, RIF1 and 53BP1 can
we investigated DSB formation after exposure to different repress the unexpected activation of HR in G1 and function
energy ion beams. Interestingly, our study indicated that to select the correct repair pathway (i.e., NHEJ or HR) [96].
the C-ion beam, which causes more complex DNA damage Acute exposure to 1 Gy of low-LET radiation such as a
than the He-ion, induced larger γ-H2AX foci sizes than γ-ray could generate approximately 40 DSBs in a nucleus,
exposure to the He-ion beam. Both large and small sizes of ~1,000 SSB, and more than 1,000 base damages, as well as
foci formation were observed in C-ion and He-ion mixed oxidation causing ~100,000 ionizations of various mole-
beam irradiated cells (unpublished data). These results indi- cules in a nucleus simultaneously [98]. In the case of
cate that the radiation-induced γ-H2AX foci size depends chronic irradiation by low-LET radiation, which is
on the energy of the radiation, suggesting that to correctly assumed to occur on space stations, the amount of DSB
understand biological effects, not only the spatial formation damage decreases to a negligible level. However, SSB and
of damage but also the size of damage needs to be considered. base damages remain and may represent a health risk.
Finally, radiation exposure in outer space occurs in a μG SSB damage and most types of base damage are repaired
environment. Most studies conducted only analyze DNA by base excision repair (BER), and cross-linked damage
damage caused by high-LET radiation exposure in a static between adjacent bases such as a thymine dimer is
environment, and it is unclear whether clustered DNA dam- repaired by nucleotide excision repair (NER) [99]. Some
age occurs and is repaired in a μG environment. Thus, to cor- kinds of oxidative bases cause misinsertion of a base
rectly understand the biological effects in outer space, it will against the template DNA during DNA replication. Such
be important to evaluate accurately the combined effects of misinserted bases are exchanged to correct bases by mis-
μG and high-LET radiation exposure. match repair (MMR). Mistakes or incompletion of DNA
repairs containing NHEJ and HR can lead to gene muta-
4.2. DNA Repair. As mentioned above, IR, including space tions and genomic instability, but the relationship with
radiation, generates various types of DNA damage. Among radiation carcinogenesis remains unclear.
them, DNA DSB is the most serious damage, which can lead
to tumorigenesis or cell death. Thus, organisms have devel- 4.3. Chromosomal Aberrations (CAs) and Micronuclei (MN).
oped DNA repair mechanisms to repair DSB damage. DSBs CAs are cytogenetic biomarkers for exposure to IR and other
are mainly repaired by nonhomologous end-joining (NHEJ) DNA-damaging agents [100]. CAs can be measured using
and homologous recombination (HR) in eukaryotes [74]. many types of cells including peripheral blood cells and are
Once DSB damages are generated following exposure to IR, used frequently in epidemiological studies of humans, labo-
the KU70/KU80 complex or MRE11/RAD50/NBS1 (MRN) ratory animals, and in vitro cell and tissue systems. The fre-
complex is recruited to DSB damage sites. KU70/KU80 com- quency of CAs in peripheral blood lymphocytes may be
plex activates the NHEJ pathway with DNA-PKcs and the associated with the risk of human cancer [101].
XRCC4/Lig4 complex, and these factors rejoin DSB ends. CAs are classified into unstable and stable types [102].
Since exposure of DNA to IR generates various forms of Unstable types are unrepaired broken chromosomes and
DSB ends, the resection of DSB ends by Artemis is essential rearranged acentric, multicentric, or ring chromosomes.
for NHEJ progression. Such resection can lead to the loss Unstable CAs are frequently lost with cell division because
of nucleotides and subsequent genomic instability. Hence, they are associated with impaired DNA replication of broken
NHEJ is an error-prone repair system. Recruitment of the termini without telomeres or in chromosome segregation.
MRN complex activates the HR pathway, and this complex Dicentric chromosomes are the most popular cytogenetic
initiates the resection of the DSB ends with CtIP, followed biomarker of unstable CAs. They can be identified easily with
by a longer resection with Exo1 or Dna2. As a result, more the conventional Giemsa staining because of their typical
than 30 single-stranded DNA (ssDNA) tails are formed at structure with two centromeres. Dicentric chromosomes
both DSB ends. The replication protein A (RPA) complex are the biomarker of choice for investigating recent exposure
then binds to ssDNA and is subsequently replaced with to IR.
RAD51. Such ssDNA/RAD51 ends invade intact homolo- Stable CAs are rearranged monocentric chromosomes,
gous DNA, and then error-free repair is completed using which can be transmitted stably to daughter cells after cell
the intact homologous DNA as a template. Thus, as the HR division, and hence used as biomarkers of past exposures to
BioMed Research International 7

IR. The conventional Giemsa staining cannot provide much Fe-ion beams
information about stable CAs. The fluorescence in situ hybrid- 0.1 ~ 2 Gy
ization (FISH) technique with chromosome-specific DNA
probes greatly improves the detection of stable CAs [103]. 1 month
Chromosomes can be observed only in metaphase cells in
their native forms. Premature chromosome condensation
(PCC) techniques, which can induce condensation of chromo-
somes in cells at the interphase by fusion with mitotic cells or b a
by chemical treatment, have improved CA analysis to detect
DNA damage that has occurred in interphase cells [104, 105].
The MN assay is an alternative approach to detect DNA
damage and used commonly because of its sensitivity, sim-
plicity, and the speed by which cells can be scored [100]. a b
MN are small pieces of DNA resulting from unrepaired DSBs
or mitotic spindle damage that appears near the nucleus fol- Figure 1: Wild-type EGFP (ab) fluorescence occurs as a result of
lowing cell division [106]. HR between two EGFP genes (a and b) that are both inactive
CAs in spacecraft crews have been analyzed since the because of deletions (shadowed boxes).
1960s to investigate genotoxic effects of space radiation and
to estimate the received doses [107]. The frequency of total Genomic instability (delayed effect) caused by IR was first
CAs seemed to be higher at postflight than at preflight, nota- demonstrated by Kadhim et al. after alpha particle irradiation
bly after flights longer than 180 days [107, 108]. However, the and indicated that many of the clonally derived cells that
diversity of radiation history and personal susceptibility exhibited the unstable phenotype were not likely to have been
makes it difficult for epidemiological studies to estimate the traversed by an alpha particle [114]. IR is capable of inducing
risk of space radiation exposure. In addition, our knowledge genome instability in mammalian cells, manifesting as
of the effects of HZE particles involved in space radiation on delayed HR in vitro and in vivo [115, 116], which is detected
induction of CAs is limited when compared with our under- in the progeny of an irradiated cell multiple generations after
standing of low-LET IR. Studies using FISH painting initial exposure. Genome instability is the driving force
revealed that HZE particles frequently induce highly complex responsible for radio carcinogenesis, which can initiate can-
chromosomal rearrangements when compared with the cer and augment progression [117–119].
effect of low-LET IR [109]. Induction of mitotic CAs by Cosmic radiation contains proton, various HZE particle
HZE particles is complicated by their serious effects on cell beams, and electron beams. As the heavy ion has a higher
cycle progression [110]. We recently compared induction of biological effect than proton or γ-rays, it is very important
CAs and MN in C57BL/6J Jms mice at 1 and 2 months after to study the effects in vivo and in vitro. For astronauts on
exposure to several doses of X-rays (low-LET IR) or Fe-ions space missions or people traveling in space, it is important
(HZE). FISH analysis of CAs in splenocytes showed that Fe to evaluate the risk of exposure to cosmic radiation, such as
particles are less effective at inducing translocations than X- carcinogenesis.
rays when compared at the same physical dose. DNA DSBs DNA DSBs are repaired by the NHEJ and HR pathways.
induced by Fe-ions are probably not rejoined and mostly The correct balance of NHEJ and HR is essential for prevent-
cause cell cycle arrest or cell death rather than result in induc- ing genomic instability [120, 121]. HR is essential for repair
tion of stable CAs [111]. Conversely, Fe-ions are more effec- of DSBs; however, too much HR activity can be detrimental
tive at inducing MN in bone marrow erythrocytes than X- and increase “genomic instability because HR carries the risk
rays, whereas the relative effectiveness of Fe-ions to X-rays of misalignments that cause insertions, deletions, and a loss
was higher at a low dose (0.5 Gy) than that at a high dose of heterozygosity (LOH) [122, 123]. However, there has been
(3.0 Gy) [112]. no observation of such genomic instability in animal tissues.
In recent years, a research group at Massachusetts Institute of
Technology established a model mouse system (RaDR mice)
4.4. Genome Instability. Genomic instability refers to the that enables evaluation of genomic instability using the green
accumulation of multiple changes within the genome of a fluorescence of the green fluorescence protein (GFP) as an
cellular lineage to convert a stable genome to an unstable indicator [124]. In the mouse genome, a direct repeat HR
genome. Genomic instability is characterized by varied substrate is targeted to the ubiquitously expressed Rosa26
end points, for example, CAs, amplification of genetic locus and HR between two truncated enhanced GFP (EGFP)
material, micronucleus formation, and gene mutation. expression cassettes can yield a fluorescent signal (Figure 1).
Genomic instability can be induced by a high frequency Before using the mouse model, we used an in vitro system
of DNA damage [113] as DNA damages can cause inaccurate (RKO cells), namely a GFP direct repeat homologous recom-
translesion synthesis past the damages or errors in repair, bination system. We demonstrated that DHR increases
leading to mutation. IR can cause immediate effects such as several-fold in response to low-LET X-rays and high-LET
mutation or cell death, observed within hours or a few days C-ion radiation [116, 125].
after irradiation. IR also induces delayed effects many cell Using the RaDR model mouse, we confirmed that the HR
generations after irradiation. frequency is related to thymic lymphomas. When 5 weeks
8 BioMed Research International

old, RaDR mice were irradiated with 1.8 Gy γ-rays per week GCR is also important. Chronic exposure to γ-rays or X-rays
for 4 weeks (total dose 7.2 Gy), and about half of the individ- has been reported to reduce dramatically the risk of carcino-
uals developed thymic lymphoma by 150 days. Our results genesis when compared with that of acute exposure [135,
indicated that a significant increase in GFP-positive cells 136]. Therefore, cancer risks after exposure to low-dose-
was observed in infiltrated lymphoma. Two months after rate HZE require further clarification. In future experiments,
the irradiation, the frequency of GFP-positive nucleated cells more animal data are required to determine the RBE of can-
(HR frequency) increased in the thymus, bone marrow, and cer risk after exposure to HZE particles or neutrons.
spleen. In contrast, when model mice were irradiated with
0.5 Gy Fe-ion beam, the HR frequency in bone marrow or 4.6. Central Nervous System (CNS) Response. In the last 10–
spleen cells was observed to increase significantly. Addition- 20 years, risk assessment of space radiation has focused on
ally, we found that the HR frequency significantly decreased the risks of cancer. In addition to the risk of cancer, NASA
under a radioadaptive response- (RAR-) inducible condition recently began focusing on the risks to the CNS. The CNS
when compared with that under a non-RAR-inducible con- consists of the brain and spinal cord. The brain is the body’s
dition [126]. most complex organ and its spatial architecture. There are
approximately 86 billion neurons and glia cells of the about
the same number in the human brain [137, 138], all of which
4.5. Carcinogenesis. Carcinogenesis is a major concern for communicate to form circuits and share information. It is
future space missions, especially space missions that will be therefore very difficult to evaluate the radiation risk to the
for long durations [127–129] because astronauts will be con- brain. Thus, it is necessary to evaluate the response of indi-
stantly exposed to IR from natural radiation sources. The vidual cells in the brain directly to radiation as a simple,
radiation field in space contains electrons, protons, alpha accessible model.
particles, and heavier ions up to HZE-charged particles. In The brain is a largely radioresistant organ [139]. How-
addition, inside spacecraft, various secondary radiations ever, ground-based animal studies indicate that space radia-
including neutrons are created by interactions between pri- tion alters neuronal tissue and neuronal functions such as
mary radiation and materials of the spacecraft. excitability, synaptic transmission, and plasticity. HZE parti-
The carcinogenic potential after radiation exposure has cles have been demonstrated to inhibit neuronal connectiv-
been revealed by epidemiological data from atomic bomb ity, neuronal proliferation, and neuronal differentiation and
survivors [130]. However, there is insufficient data delin- to change glial characterization [140]. We summarize the
eating the carcinogenic potential of HZE-charged particle current knowledge of neuronal and glial responses caused
radiation. Therefore, estimation of the cancer risk after expo- by HZE irradiation less than 2 Gy (Table 1).
sure to each HZE particle or neutron using animal experi- Thus, many researchers observed the response of the
ments is important. RBE values are given as the ratio of the brain to radiation using short-term, higher-dose-rate expo-
absorbed doses of two types of radiation producing the same sures of radiation, which does not accurately reflect the con-
specified biological effect under identical irradiation condi- ditions in space. The long-term effects of these doses of
tions. Cucinotta et al. [131] used RBE values from various radiation on the CNS are largely unknown. Acharya et al.
animal experiments for predicting the risk of cancer after exposed mice to chronic, low-dose-rate (1 mGy/day) radia-
exposure to HZE and fission neutrons, and the used RBE tion for 6 months to investigate how deep space travel could
values were 2 to 10 and 4 to 20, respectively. Imaoka et al. affect the CNS [152]. They found that the radiation exposure
[132] summarized RBE of the risk of cancer after exposure impaired cellular signaling in the hippocampus, a part tied to
to protons, C-ions, or neutrons to estimate secondary cancer learning and memory, and the prefrontal cortex, which plays
after radiation therapy. The RBE was less than 2 for protons a role in higher cognitive functions, resulting in learning and
and less than 20 for C-ions and neutrons. These animal data memory impairments. They predict that during a deep space
revealed that RBE values are variable for tissues type, radia- mission, 1 in every 5.1 astronauts would experience anxiety-
tion types, and age at the time of irradiation. like behavior, and 1 in every 2.8 astronauts would experience
The greater carcinogenesis effects of HZE particle radia- certain levels of memory impairments. These results suggest
tion have been analyzed from the viewpoint of a targeted that chronic, low-dose-rate radiation exposure from deep
effect (genetic change) and nontargeted effects. C-ion- space travel may pose considerable risks for cognitive perfor-
induced lymphomas showed a marginal increase in the fre- mance and health. For the assessment and management of
quency of large interstitial deletions at various sites across human health in space, it is necessary to obtain more basic
the genome when compared with that of photon-induced data of the effects of radiation on the brain. Additionally, it
lymphomas [133]. HZE particle irradiation promoted more is important for us to progress with the developments of
aggressive cancers, such as increased growth rate, transcrip- methods and protective materials that shield radiation effects.
tomic signatures, and metastasis when using a radiation/ge-
netic mammary chimera mouse model of breast cancer 4.7. Motility Disturbance. Adverse effects of high-LET radia-
[134]. This suggests that the nontargeting effects of HZE par- tion, an important component of cosmic rays, on the functions
ticles were more effective than the reference γ-radiation. of biological systems are a potential risk in interplanetary
Unfortunately, there is still a paucity of data on this subject. manned space missions. Therefore, analysis of the effects of
Considering radiation exposure in deep space, the health high-LET radiation on animals at an individual level and
risk of exposure at low dose and low-dose-rate radiation from focusing on the impact of such radiation on biological
BioMed Research International 9

Table 1: Summary of brain cellular response to HZE irradiation (doses of less than 2 Gy).

Cells Response Irradiation Dose rate (Gy/min) Time after IR Ref.

56
Fe: 1.5 Gy 0.88 1m [141]
Cell death 56
Fe: 1.6 Gy 1 12 m [142]
28
Si: 0.2, 1 Gy 1 24 h, 3 m [143]
Deficits to proliferation and differentiation 56
Fe: 0.3, 1 Gy 0.01–1 48 h, 1 m [144]
GCR (H+He+O): 0.5 Gy 0.0616 100+ d [145]
16
Neuron O, 48Ti: 0.05, 0.3 Gy 0.05, 0.25 15 w [146]
1
H: 1 Gy 0.55 3m [147]
Changes to dendritic, axonal, and 16
O, Si, 4He: 0.3 Gy
28
6w [148]
synaptic properties 56
Fe: 0.5 Gy 3m [149]
1
1 H: 0.18–0.19
H: 0.5 Gy + 16O: 0.1 Gy 16 3m [150]
O: 0.18–0.33
56
Astrocyte activation Fe: 1.6 Gy 1 12 m [142]
GCR (H+He+O): 0.5 Gy 0.0616 100+ d [145]
Glia 16 15 w,
Microglial activation O, 48Ti: 0.05, 0.3 Gy 0.05, 0.25 [146]
27 w
4
He: 0.05, 0.3 Gy 0.05 12 m [151]
h: hours; d: days; w: weeks; m: months.

functions are important for space missions. The effects of motion by a whole-body mechanism, potentially involving
high-LET radiation exposure on several behaviors including motor neurons and/or body wall muscle cells, rather than
muscle movements have been investigated using the nema- affecting motor control via the CNS and the stimulation
tode C. elegans [66, 67, 69, 153, 154], which is an experimen- response [67].
tal model organism and a powerful tool to study the effects of In studies of stress responses, disturbances to muscle cells
radiation. In this animal, locomotion, including forward and induced by various stresses and stimulations have been well
backward movements and turns, is carried out by 95 body investigated. Wang et al. showed that mitochondrial dysfunc-
wall muscle cells, for which the fate of each cell from its birth tion is related to muscle atrophy [156], and extracellular
to death can be easily determined. Locomotion (motility) of matrix (ECM) stability is necessary for maintaining muscle
adult C. elegans on an agar plate without food was reported health. In addition, Momma et al. investigated alterations of
to decrease in a dose-dependent manner immediately after Ca2+ homeostasis and mitochondrial morphology in vivo in
whole-body irradiation was administered using both high- body wall muscles of C. elegans exposed to an elevated tem-
LET radiation (12C, 18.3 MeV/u, LET = 113 keV/μm) [67, perature. The results showed that heat stress for 3 h at 35°C
153] and low-LET radiation (60Co γ-rays) [155]. The RBE increased the concentration of free Ca2+ and led to mito-
ratio of high-LET radiation relative to low-LET radiation chondrial fragmentation and subsequent dysfunction of the
for inhibition of locomotion was 1.4 [153]. If the radiation muscle cells [157]. Furthermore, it was reported that mito-
effects were mainly caused by DNA damage, it is generally chondrial dysfunction acts as an intramuscular signal that,
thought that the effects of high-LET radiation would be sev- via excessive Ca2+ release, activates ECM-degrading enzymes
eral times higher than those of low-LET radiation. Therefore, to reduce ECM content and, subsequently, results in the
the reduction of motility in C. elegans following exposure to structural and functional decline of muscles [158].
high-LET radiation is not caused by DNA damage and is Although reduction in motility of body wall muscles
likely induced by another factor. Recovery of motility shortly recovers within several hours after whole-body irradiation
after irradiation supports the hypothesis that DNA damage is with less than 1,000 Gy of high-LET radiation and the effects
not responsible for IR-induced reduction of motility. In par- are masked, the disturbance observed after whole-body irra-
ticular, an important factor that induces radiation effects is diation with more than 1,000 Gy of high-LET radiation
reactive oxygen species (ROS) produced by IR. Exposure to might be induced by the above mitochondrial mechanisms.
IR results in the formation of free radicals such as OH• or Further studies that focus on the effects of radiation to the
H•, and the reactions of free radicals cause the production homeostasis of muscle cells are required.
of ROS, including hydrogen peroxide (H2O2). Experimental
results showed that C. elegans motility was H2O2 dose- 4.8. Visualization of Adverse Events. The cellular response
dependent, indicating that radiation-induced reduction in to DNA damage varies according to the cell type, the stage
motility is caused by IR-produced H2O2 [155]. Moreover, of the cell cycle, and extent of damage [159]. More than
the results of region-specific irradiation showed that motility 50 years have passed since the first observation of cell cycle-
was not reduced significantly by irradiation of any of the dependent DNA damage was made by using synchronized
individual tissues in a ∅ 20 μm region, including the CNS, HeLa cell populations [160, 161]. These classical studies con-
intestines, and tail. This suggests that radiation reduces loco- cluded that mitotic cells are hypersensitive to X-ray
10 BioMed Research International

S/G2/M

CUL4Ddb1 SCFSkp2 Late M/G1

APCCdh1

S S S/G2/M S/G2/M Late M/G1

PCNADNA PCNADNA P hCdt1(30/120)

P
hGem(1/110)
hCdt1(1/100)Cy(–) hCdt1(1/100)

Cyclin-CDK
PCNADNA complexes
hCdt1(1/100)Cy(–) hCdt1(1/100) hCdt1(30/120)

Fucci(SA) Fucci(CA) Fucci(SCA)

NEB NEB NER NEB

M M M
G2 G2 G2
S G1 S G1 S G1

Figure 2: Cell cycle-phasing capabilities of the Fucci technology. Cell cycle regulations involving E3 ligase activities of CUL4Ddb1, SCFSkp2,
and APCCdh1. Molecules whose intracellular concentrations or enzymatic activities change in a cell cycle-dependent manner are shown in
color. PCNADNA: DNA-bound PCNA. Data adapted from Sakaue-Sawano et al. [164].

irradiation, which inactivates the DNA DSB repair pathway. (amino acids 68–70), which binds to the SCFSkp2 E3 ligase.
Cell survival was maximal when cells were irradiated during The combination of the RFP-labeled hCdt1(30/120) and
the early postmitotic (early G1) and premitotic (S to G2) GFP-labeled hGem(1/110) can be called Fucci(SA) because
phases and was minimal during the mitotic (M) and late they monitor the balance between SCFSkp2 and APCCdh1 E3
G1 or early DNA synthesis (early S) phases. However, the con- ligase activities.
ventional “arrest-and-release” methods using pharmacological Eukaryotic cells spend most of their life in interphase of
reagents or the mitotic shake-off method cause more or less the cell cycle. Understanding the rich diversity of genomic
adverse cellular perturbations and do not ensure complete regulation that occurs in interphase requires the demarcation
cell cycle synchronization of tumor cells. of precise phase boundaries in situ. Although Fucci(SA)
Recently, a variety of fluorescent protein- (FP-) based highlights the G1/S phase transition with yellow fluores-
methods for visualizing cell cycle progression at the single cence, it does not provide a fluorescent readout for distinct
cell level have been developed, enabling researchers to ana- interphase boundaries. Additionally, Fucci(SA) has a fluores-
lyze cell cycle progression without affecting normal cellular cence gap in very early G1 phase, making it difficult to con-
functions. Fucci (fluorescent ubiquitination-based cell cycle tinuously track cell positions in all phases of the cell cycle.
indicator) harnesses the cell cycle-dependent proteolysis of In 2017, we engineered the hCdt1-based probe to be sen-
Cdt1 and Geminin. Fucci highlights the cell cycle transition sitive to CUL4Ddb1 in addition to or instead of SCFSkp2 [164].
from G1 to S phase with high color contrast, like a traffic sig- As the PIP box (amino acids 1–10 of hCdt1) is a specific sub-
nal: red and green mean “stop” and “go,” respectively, for the strate of CUL4Ddb1, hCdt1(1/100), which retains both the PIP
transition (Figure 2) [162, 163]. SCFSkp2 and APCCdh1 E3 box and Cy motif, is targeted by both SCFSkp2 and CUL4Ddb1.
ligases are involved in the degradation of Cdt1 and Geminin, We also constructed hCdt1(1/100)Cy(–), which is a spe-
respectively. Over the course of the cell cycle, these two E3 cific substrate of CUL4Ddb1. By combining hCdt1(1/100)-
ligase activities oscillate reciprocally and the protein levels and hCdt1(1/100)Cy(–)-containing red-emitting probes
of their direct substrates oscillate accordingly. To label S– with hGem(1/110)-containing green/yellow-emitting probes,
G2–M-phase nuclei green, the Fucci probe has a green- we developed Fucci(SCA) and Fucci(CA) probes, respec-
emitting FP fused to the APCCdh1-mediated ubiquitylation tively, which have increased the versatility of the Fucci
domain (1–110) of human Geminin (hGem) (Fucci- technology for new biological studies of cell cycle inter-
S/G2/M-Green); this chimeric protein is the direct substrate phase regulation. Although Fucci(CA) monitors the balance
of APCCdh1 E3 ligase. To label G1-phase nuclei red, the probe between CUL4Ddb1 and APCCdh1 E3 ligase activities, Fuc-
has a red-emitting FP fused to residues 30–120 of human ci(CA) can distinguish clear interphase boundaries between
Cdt1 (hCdt1) (Fucci-G1-Red); it contains the Cy motif G1, S, and G2 phases.
BioMed Research International 11

Table 2: Biological effects of radiation and μG in space experiments.

Interactive effects Species Biological index Flight time Irradiationa Ref.

Human blood Chromosomal aberration 12 h + 32P, β-rays [167]
E. coli, S. cerevisiae DSB and SSB repair 14 d Pre-X-rays [168]
No S. cerevisiae DSB repair 10 d Pre-X-rays [169]
E. coli, S. cerevisiae SOS response 2–4 d Pre-X-rays [170]
Human blood Chromosomal aberration 8d Pre- and post-γ-rays [171]
D. melanogaster Larval mortality 45 h + 85Sr, γ-rays [172]
C. morosus Abnormality 7d No [173]
S. cerevisiae DSB repair 9d No [174]
Yes, ↑
D. melanogaster Mutation 8d No [175]
D. discoideum Spore formation 9d No [176]
Human blood Chromosomal aberration 10–485 d Pre- and post-X-rays [177]
N. crassa Cell killing, mutation 45 h + 85Sr, γ-rays [172]
Yes, ↓
D. radiodurans Cell killing 14 d Pre-γ-rays [178]
E. coli: Escherichia coli; S. cerevisiae: Saccharomyces cerevisiae; D. melanogaster: Drosophila melanogaster; C. morosus: Carausius morosus; D. discoideum:
Dictyostelium discoideum; N. crassa: Neurospora crassa; D. radiodurans: Deinococcus radiodurans; DSB: DNA double-strand breaks; SSB: DNA single-strand
breaks; h: hours; d: days. a+ means simultaneous irradiation with spaceflight.

We have demonstrated that Fucci(CA) can be used to press each other’s effects [172, 178]. There is the still no con-
sensus on whether radiation and μG have combined effects
(1) fully highlight the short G1 phase of rapidly prolifer- [179, 180]. JAXA developed not only the Cell Biology Equip-
ating mESCs ment Facility (CBEF) [181] but also a mouse habitat unit
cage (MHU) [182], which provides long-term artificial grav-
(2) continuously track cell positions in all phases of the
ity for control experiments in space. This experimental plat-
cell cycle
form provides the opportunity to investigate the specific
(3) detect cell cycle- (S phase) specific sensitivity (HeLa impacts of space radiation and μG for future human space
cells) to UV irradiation exploration [181].
The biological effects of radiation and simulated μG in
(4) explore cell cycle-specific intracellular signaling ground experiments are summarized in Table 3. To clarify
(5) visualize a cell cycle-specific response or homeostatic the effects of μG at ground level, researchers have used rotat-
balance to space radiation ing devices, such as a rotating wall vessel bioreactor (RWV;
Synthecone, Houston, TX, USA) and the random positioning
To investigate the impact of space radiation and μG on machine (RPM; Dutch Space, Netherlands), which are pieces
“Living in Space,” a variety of FP-based approaches had been of equipment that continuously rotate a sample. These
launched. Harada et al. introduced an EGFP-53BP1M FP devices can equalize the gravity vector and cancel the effect
probe to visualize the diversity of the radiation-induced of gravity, thereby simulating μG. However, there are two
DNA damage responses in real time [165]. Ishii’s group major limitations associated with this approach: (i) it is nec-
reported that B16BL6 cells in the early S phase were the most essary to stop rotation during irradiation as the sample was
susceptible to radiotherapy [166]. Live imaging technology exposed to radiation outside the incubator after or before
using FPs is expected to make significant contributions to rotation with a RWV [183–188] and (ii) nonuniformity of
the direct visualization and detailed understanding of radia- dose flatness in the irradiation area occurs because of chron-
tion adverse events. ical irradiation of a rotating sample with a RPM [189, 190].
To address these problems, we have developed a system of
5. Combined Biological Effects simultaneous irradiation in simulated-μG (SSS) using 3D
clinostat [191, 192]. Our SSS is based on technologies related
5.1. Radiation and μG. The biological effects of radiation and to X-ray irradiation with a high-speed shutter [191] and C-
μG in space experiments are summarized in Table 2. In a pre- ion radiotherapy such as accelerator systems and respiratory
vious short mission, there was no appreciable difference in gating systems [192].
results between space and ground samples because exposure Using this SSS, we reported that simultaneous exposure
to space radiation occurred at a low dose. Therefore, various of human fibroblasts to simulated μG and radiation results
living systems have been irradiated before spaceflight to clar- in a greater frequency of chromosomal aberration than in
ify the effect of μG on the radiation-induced DNA damage cells exposed to radiation alone [193]. The expression of cell
response, but there was no appreciable difference in results cycle-suppressing genes decreased and that of cell cycle-
[167–171]. However, synergistic effects between radiation promoting genes increased after C-ion irradiation under sim-
and μG have been reported [172–177], and they can sup- ulated μG [194]. Assessment of the cancer risk associated
12 BioMed Research International

Table 3: Biological effects of radiation and simulated μG in ground experiments.

Interactive effects Cells Biological index Devices Irradiationa Ref.

Lymphoblastoid Mutation, micronuclei RWV Pre-60Co, γ-rays [183]
Lymphocyte Mutation RWV Pre-60Co, γ-rays [184]
Lymphocyte γ-H2AX RWV Pre-137Cs, γ-rays [185]
Lymphoblast Apoptosis, ROS RWV Post-C-ion [186]
Yes, ↑
Fibroblast Gene induction RPM + 252Cf, neutron [189]
Neuron Apoptosis, gene induction RPM + 252Cf, neutron [190]
Fibroblast Chromosomal aberration SSS + X-rays, + C-ion [193]
Fibroblast Cell cycle-promoting genes SSS + C-ion [194]
Lymphocyte Apoptosis RWV Pre-137Cs, γ-rays [187]
Lymphoblastoid Apoptosis RWV Pre-60Co, γ-rays [183]
Yes, ↓
Lymphocyte Micro-RNA RWV Pre-137Cs, γ-rays [188]
Fibroblast Cell cycle-suppressing genes SSS + C-ion [194]
ROS: reactive oxygen species; RWV: rotating wall vessel bioreactor; RPM: random positioning machine; SSS: system of simultaneous irradiation in simulated-
microgravity. a+ means simultaneous irradiation with spaceflight.

with space radiation in the conventional manner based on of proteomic and metabolomics analysis of human primary
data of radiation quality and quantity from cells irradiated osteoblasts exposed to simulated μG suggest that μG sup-
under static conditions might underestimate the potential presses bone cell function, impairing mitochondrial energy
risk to astronauts. Nonetheless, examination of endpoints potential and the energy state of the cell [200].
and in vivo model systems under the combined effects of To plan cultivation of plants in space including Mars, we
radiation and μG are required. need to identify what plants to use and whether to use sun-
In the near future, there is also a need to investigate the light or an artificial light source for growth. Negative effects
biological effect of partial gravity such as 1/6G and 3/8G on of UV radiation can be avoided if plants are grown under
the response to radiation for manned missions to the Moon artificial light without sunlight. However, we need to address
and Mars. Two simulated partial gravity devices using the some issues. For example, (1) it is difficult to grow plants uni-
RPM, one by applying specific software protocols to drive formly in the same growth chamber, because the optimal
the RPM motors and the other involving integrating a centri- wavelength and light intensity differ for different vegetable
fuge into the RPM, should become useful tools [195]. The plants; and (2) growing plants in a growth chamber under
actual effects should be tested either in a proper centrifuge artificial light is very costly because of the consumption of
experiment on the ISS, such as CBEF [181] and MHU electric power. Conversely, if plants are grown using sunlight,
[182], or actually on the surfaces of the Moon and Mars. the potential negative effects from UV radiation are unavoid-
able. It is unclear whether various UV-B protection mecha-
5.2. Combined Effects of μG and UV Radiation on Plants. nisms, which have evolved under 1G, would function
Plants supply nutrients and oxygen to humans under a properly under lower gravity. It is thus important to investi-
resource-recycling system on Earth and also in space. All gate the potential ability of plants to adapt to the environ-
organisms, including plants, have evolved protection mecha- ment of space. For this purpose, utilization of facilities on
nisms against environmental stresses. However, the environ- orbiting space platforms such as the ISS is essential, although
ment in space differs dramatically from that on Earth. Can all we cannot repeatedly and frequently conduct experiments on
organisms adapt to the environment in space and live the ISS. To disturb the gravity direction or produce simulated
healthy? In addition, there is the possibility that the higher μG on the ground, a 3D clinostat is useful and convenient
intensity of UV radiation, which is a driving force of evolu- (Figure 3).
tion, and the complex cosmic IR in space could lead to an Therefore, it is necessary to understand the combined
increase in the mutation frequency. Currently, μG has been environmental effects of space on plants at the molecular,
reported to cause cellular oxidative stress that leads to pro- cellular, and whole-plant levels and understand not only
duction of ROS and endoplasmic reticulum stress in experi- the transient, short-term (one generation) effects but also
mental animals [196–198]. In addition, Sugimoto et al. long-term (next, subsequent generations) effects under space
reported that the environment during spaceflight induces environmental conditions through space experiments or
oxidative stress and ROS gene network activation in the experiments using equipment such as a 3D clinostat. Such
space-grown Mizuna plant [199]. The mechanisms by experiments clarify direct and/or indirect gravity effects
which μG elicits these cellular responses remain poorly on vegetative and reproductive growth, provide new evi-
understood, although very interesting results have been dence of antigravity reactions, and possibly find not only
reported recently. For example, simulated μG induces autoph- novel biological knowledge, such as molecular mechanisms
agy via mitochondrial dysfunction in human Hodgkin’s in gravity reactions, but also novel growth controls in crop
lymphoma cells [196] and TCam-2 cells [197]. The results production on Earth. In addition, experiments that include
BioMed Research International 13

induced psychological stress does not appear to synergize with

the clastogenicity of low-LET radiation in wild-type animals
[204, 205]. Interestingly, in the animal model for psychosocial
stress using 6- or 8-week-old male ddY mice (model mouse for
spontaneous IgA nephropathy) and SAMP10 mice (model
mouse for accelerated senescence), results of concurrent expo-
sure to both psychosocial stress and X-rays at a dose of 3–6 Gy
showed increased acute damage, namely, reduced 30-day sur-
vival, and decreased erythrocyte and leukocyte counts in the
peripheral blood and hypocellular bone marrow, indicating
psychological stress promotes radiosensitivity of bone marrow
in these particular mice [206]. Interestingly, investigation
using the mouse model for chronic restraint-induced psycho-
logical stress, Trp53 heterozygous C57BL/6N male mice aged 6
weeks, and high-LET Fe particle irradiation at 0.1 or 2 Gy
showed that concurrent exposure to psychological stress and
0.1 Gy Fe irradiation resulted in increased hematopoietic tox-
icity and genotoxicity measured as MN in the erythrocytes of
the bone marrow and splenocyte CAs [207–209]. In contrast,
in the mouse testis, concurrent exposure to 0.1 Gy Fe irradia-
Figure 3: A 3D clinostat quipped with a UV-visible light unit. The
tion did not induce any increased apoptosis and autophagy
UV-visible light unit is composed of white and UV-B- (280 nm)
light-emitting diodes (LEDs). inhibition [210]. These results indicate that psychological
stress does not exacerbate radiation effects. These findings
the space-specific radiation environment will help elucidate also suggest that studies on concurrent exposure should be
the combined influence of low gravity and high-level visible performed using different endpoints in different tissues in
UV and space radiations on plant growth and regeneration in both short- and long-term models for chronic restraint-
a whole growth stage. However, a study about such combined induced psychological stress. In summary, concurrent expo-
effects on organisms as well as plants has only recently been sure of wild-type mice to psychological stress and low-LET
initiated. Such studies, both on the ground and on orbiting radiation did not suggest an additive effect for induction of
space platforms such as the ISS, should be promoted to estab- hematopoietic toxicity and genotoxicity but promoted radio-
lish sustainable life support systems for securing long-term sensitivity of the bone marrow in some disease-prone mice.
human life in space and on the Moon and Mars. In contrast, concurrent exposure of Trp53 heterozygous mice
to psychological stress and high-LET radiation suggested an
5.3. Radiation and Stress. Stress refers to conditions where an additive effect for induction of hematopoietic toxicity and
environmental demand exceeds the natural regulatory capac- genotoxicity. To reduce health risks from exposure to radia-
ity of an organism, in particular situations that include unpre- tion by active intervention, further investigations are needed
dictability and uncontrollability, and psychological stress is to collect more data that provide insights into the mecha-
one of two basic kinds of stress [201]. Psychological stress nisms underlying the alterations in susceptibility due to psy-
and radiation are known to cause various adverse effects on chological stress modulation.
humans. Radiation is a carcinogen, and long-lasting psycho-
logical stress may affect the overall health and ability to cope 6. Radiation Exposure Management
with cancer. Whether psychological stress influences suscepti-
bility to radiation, radiocarcinogenesis in particular, is of To prevent IR-induced carcinogenesis, the exposure dose
great concern for both academia and the public [202]. in spaceflight is limited to a level that will not result in
Using a laboratory mouse model for chronic restraint- exposure-induced death (REID) from fatal cancer over a
induced psychological stress, the pioneering work on con- career of more than 3%, at the 95% upper confidence interval
current exposure of Trp53 heterozygous C57BL/6 mice to of the risk calculation [129]. Based on this concept, missions
psychological stress and total body γ-rays showed that psycho- in space are currently planned to last less than 180 days [211].
logical stress modulates susceptibility to radiation, causing However, it has been suggested that there are individual dif-
increased susceptibility to radiocarcinogenesis in Trp53-het- ferences in the IR-induced cancer risk within human popula-
erozygous mice underlying the mechanism of Trp53 function tions [212]. Three factors that underlie individual IR-induced
attenuation [203]. In recent years, studies using the same cancer risk, i.e., age, sex, and smoking, are already considered
chronic restraint system, wild-type C57BL/6J male mice aged when determining the safe number of days in spaceflight
5 weeks and total body exposure to 4 Gy X-rays, showed that [213]. Here, we review genetic variants in the DNA repair
psychological stress has minimal modifying effects on genes as an important factor underlying the individual differ-
radiation-induced hematopoietic toxicity and genotoxicity ences in IR-induced cancer risk.
measured as a peripheral blood histogram, MN in the erythro- In human cells, DNA repair systems monitor and repair
cytes of bone marrow, and splenocyte CAs (insertions, dicen- DNA DSBs to maintain genomic integrity. If IR-induced DSBs
trics, and fragments), suggesting that chronic restraint- are left unrepaired, they can alter the information stored in the
14 BioMed Research International

genome to cause carcinogenesis. It is thus useful to measure have been studied as radioprotective agents. Radiation
the capacity of cells to repair DSBs to understand how prone induces DNA damage both directly and indirectly through
individuals are to IR-induced carcinogenesis. The radicals generated in response to intracellular water mole-
cytokinesis-blocked MN assay, a procedure established to cules. Thus, there are numerous studies evaluating antioxi-
evaluate the capacity of cells to repair DSBs by counting MN dants that suppress radiation-induced radical generation
derived from unrepaired DSB-induced chromosomal frag- [220–224]. In particular, the effects of vitamin C and vitamin
ments, has revealed the existence of cases in which the capacity E have been studied for many years as antioxidants with
to repair DSBs has been slightly decreased by IR within radioprotective effects. Our group has assessed radioprotec-
healthy individuals and those with breast cancer [214]. The tive effects of ascorbic acid (AA) to patients before cardiac
FISH painting analysis, which monitors IR-induced unstable catheterization (CC) for diagnostic purposes. Although we
ring and multicentric chromosomes, also demonstrated the did not find satisfactory evidence to show that AA treatment
heterogeneity of the capacity to repair DSBs after IR within reduces γ-H2AX foci formation immediately after CC, a
human populations [215]. Interestingly, genome-wide associ- slight decrease in DNA damage in the group of AA treatment
ation studies (GWASs) have revealed that many nucleotide was detected [225]. However, the results vary depending on
variants in DNA repair genes are linked to an enhanced risk the animal model used, the radiation dose, and the method
of cancer in normal individuals [216]. These findings in the for evaluating the protective effect [226–230]. In addition to
fields of radiation biology and epidemiology have suggested vitamins C and E, radioprotective effects of nitroxide com-
that the personalized risk of cancer after IR exposure might pounds as strong radical scavengers have also been analyzed
be attributable to variants in DNA repair genes. [220, 231–233].
To clarify whether variants in DNA repair genes are Currently developed radioprotective drugs are unsuitable
involved in the risk of IR-induced cancer, it is informative to as radioprotectants in outer space because the situation of
compare chromosomal instability after IR exposure of radiation exposure differs to that of previous ideas. In outer
primary cells with or without the variant of interest, such as space, suitable radioprotective drugs should protect against
skin fibroblasts and peripheral blood lymphocytes. However, chronic exposure to low dose and a low dose rate of high-
the capacity of primary cells to repair DSBs is affected by the LET radiation, and not the acute high-dose radiation expo-
diverse genetic backgrounds within human populations [217]. sure found in radiotherapy. Drugs suitable for humans living
It is therefore essential to evaluate the effects of candidate in space must treat both unexpected high-dose radiation
variants on the capacity of cells to repair DSBs in a uniform exposure due to solar flares and the suppression of DNA
genetic background. Genome-editing technology is beneficial damage by space radiation that occurs constantly. Therefore,
in this regard because it enables the introduction of candi- it is necessary to validate a radioprotective drug that can be
date variants into human cultured cells with a uniform taken daily with minimal side effects. For this purpose, it
genetic background. Comparison of IR-induced chromo- may be effective to develop functional space foods with a
somal abnormalities in genome-edited cells can then reveal radioprotective effect that can be ingested continuously in
whether a candidate variant is able to repair DSBs within outer space [234, 235]. Currently, our group is examining
human populations. Previously, we used this approach to the radioprotective effect of piceatannol, which is an ingredi-
demonstrate that ataxia-telangiectasia mutated (ATM) het- ent of passion fruit and displays strong antioxidant activity.
erozygous mutations, which are present at a rate of around We have confirmed that suppression of DSB after not only
1% in human populations, are indeed associated with the indi- low-LET radiation exposure but also various high-LET radi-
vidual capacity of cells to repair DSBs [217]. Besides ATM gene ation exposures occurs when using piceatannol (unpublished
mutations, germline mutations of DNA repair genes in human data). The development of various radiation protection
populations have been reported, such as MRE11A, NBS1, agents is expected to progress in the future. We emphasize
Rad50, Artemis, and DNA Lig-IV [212]. These mutations are that there is a need for the development of protective agents
generally rare, while heterozygous BRCA1 and BRCA2 muta- against not just space radiation but also various space envi-
tions for hereditary breast and ovarian cancers are estimated ronmental risks.
to be present at a rate of 0.05–1% in human populations
[218, 219]. The extent to which these mutations contribute 7.2. Historical Overview and Perspective of Basic Research for
to the capacity to repair DSBs remains unclear but should the Development of Biological Strategies. Unfortunately, the
be resolved to achieve personalized radiation exposure man- development of a biological strategy for protection of our
agement. Further studies using an approach combining the body from space radiation has not been achieved. To accom-
fields of epidemiology and functional genomics are needed plish this, a basic knowledge about adverse effects of space
to understand the genetic basis of individual differences in radiation toward human health is required. In particular,
IR-induced cancer risk. we need to understand the radiosensitivity of each tissue. A
French oncologist, Jean Alban Bergonié, and a French der-
7. Protection from Radiation matologist, Louis Tribondeau, worked together between
1904 and 1906 and formulated a fundamental law in the field
7.1. Protective Agents. Many biological effects such as cell of radiation biology regarding the difference in radiosensitiv-
death and inflammatory responses due to radiation exposure ity of normal tissues. They observed damage in the testis of
are caused by DNA damage [71, 72, 75, 76]. Therefore, vari- male rats under a microscope after whole -body X-ray irradi-
ous drugs that aimed at decreasing induced DNA damage ation and found that biological effects of radiation were
BioMed Research International 15

Table 4: Classification of tissues based on their radiosensitivity.

Frequency of cell division Tissue Radiosensitivity

Lymphoid tissue, hematopoietic tissue (bone marrow), testicular epithelium,
++ Extremely high
follicular epithelium, and intestinal epithelium
Oropharyngeal oral epithelium, skin epidermis, hair follicle epithelium, sebaceous
+ gland epithelium, bladder epithelium, esophageal epithelium, lens epithelium, High
gastric gland epithelium, and ureteral epithelium
+/– Connective tissue, small vessel tissue, and growing cartilage/bone tissue Intermediate
Mature cartilage/bone tissue, mucous serous epithelium, sweat gland epithelium,
– nasopharyngeal epithelium, lung epithelium, renal epithelium, liver epithelium, Low
pancreatic epithelium, pituitary epithelium, thyroid epithelium, and adrenal epithelium
–– Nerve tissue and muscle tissue Extremely low

severer in the order of spermatogonia, spermatocyte, sper- radiation with complex energy spectra and diverse ionic
matid, and sperm. They generalized the result and formu- compositions. These approaches are expected to give us
lated the so-called the Law of Bergonié and Tribondeau, important information about radiosensitive tissues that
which theorized that radiation causes severer damage to a tis- should be protected from space radiation during ultralong
sue (1) when reproductive activity of cells in the tissue is spaceflights. Additionally, these approaches may lead to the
greater, (2) when the karyokinetic fate of cells is longer (in development of radioprotective agents and also a system to
other words, when the length of time that cells proliferate select an astronaut who is potentially radioresistant.
actively is longer), and (3) when morphology and function
of cells are less differentiated. Based on this, radiosensitivity 8. Radioresistant Organisms
of representative tissues is classified as a summary in Table 4.
Accumulating evidence has demonstrated that the law Organisms on Earth are protected from harmful space radia-
certainly applies to many tissues; however, there are some tion by the electromagnetic field of our planet, most organ-
exceptions. For example, Regaud claimed that spermatogo- isms including us are vulnerable to radiation, and radiation
nia in young rats are less radiosensitive than those in damage is one of the most severe risks to human health in
adults, though their proliferation rates are similar [236]. long-term space flights. Some species on our planet, however,
Using tobacco leaves, whose cell division rate significantly exhibit extraordinary resistance against high doses of radia-
decreases as they grow, Haber and Rothstein demonstrated tion. Elucidating the molecular machinery responsible for
that radiosensitivity was almost the same between dividing these extraordinary radioresistance may aid the development
and nondividing tissues [237]. Meyn and Jenkins measured of novel technologies that alleviate biological damage caused
the efficiency of DNA strand break formation in normal tis- by radiation.
sues of mice after whole-body irradiation and found that the Most of the well-known radioresistant organisms are
least breaks were produced in the gut when compared with single-cellular prokaryotic organisms, such as archaea and
those of other tissues such as the bone marrow, spleen, brain, bacteria. Deinococcus radiodurans, one of the most famous
kidney, testis, and liver [238]. Ueno et al. recently found radioresistant bacteria, is reported to survive without loss of
that quiescent melanocyte stem cells (McSCs) were more viability even after irradiation with 5,000 Gy of γ-rays [241,
radiosensitive than coexisting nonquiescent McSCs and 242]. Although the genome DNA of D. radiodurans is heavily
suggested that tissue radiosensitivity depends on the state fragmented by high-dose irradiation, the DNA fragments are
of somatic stem cells under their microenvironment [239]. rapidly repaired to a complete circular genome by extensive
The law of Bergonié and Tribondeau needs to be revisited DNA repair processes likely using their polyploid genome
to integrate current knowledge about differences in radio- [243]. Mutation in the DNA repair pathways drastically com-
sensitivity between various tissues. promises the radioresistance of D. radiodurans, suggesting
Adverse effects of space radiation have been investigated that DNA is the most vulnerable target to radiation, and
under the various limitations of experimental settings; there the powerful DNA repair system plays important roles in
is no way to separately evaluate radiosensitivity of each tissue the high radioresistant capacity of this bacterium [243]. In
using acute and monoenergetic beams [240]. To conduct addition to unicellular organisms, some animals such as tar-
more integrated analyses, our efforts in establishing a plat- digrades, bdelloid rotifers, and a sleeping chironomid, also
form for in vivo animal studies are required. We can then exhibit exceptional tolerance against high doses of irradiation
analyze the effect of multiple factors (including low gravity [244–248]. Intriguingly, these radioresistant animals also
and tissue microenvironment) on the efficiency of repair of exhibit tolerance against almost complete dehydration. In a
DNA damage caused by space radiation. In particular, dehydrated state also known as “anhydrobiosis,” they can
in vivo research using imaging techniques or genetically withstand several thousand Gy of γ-irradiation. Some tardi-
modified animals should provide spatiotemporal informa- grade species and a sleeping chironomid were reported to
tion about these factors. Moreover, we should conduct this survive direct exposure to space in a desiccated state, suggest-
research under various kinds of radiation that mimic space ing that they are resistant even against space radiation [249,
16 BioMed Research International

n.s.
⁎⁎ ⁎⁎
50 1⨯106

40
1⨯105

𝛾-H2AX foci/nucleus

Total cell number

30
1⨯104
20
n.s .
n.s . n.s. 1⨯103
10

0 1⨯102
Nonirradiated Irradiated (1 Gy) 0 1 8 10 12 dps
X-ray
Control
Dsup
Dsup
Dsup+shDsup
Dsup+shDsup
Control
(a) (b)

Figure 4: Dsup reduced X-ray-induced DNA damage (a) and improved viability of irradiated human cultured cells (b). The number of DNA-
break marker, γ-H2AX foci in nonirradiated or 1 Gy-irradiated conditions (a), and growth curves after 4 Gy-irradiation (b) are compared
among nonengineered human cultured cells (HEK293, control), Dsup-expressing cells (Dsup), and Dsup-knockdown cells (Dsup
+shDsup). Reproduced from Hashimoto and Kunieda [253] under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0
International License.

250]. Because biological damage by radiation, e.g., DNA been found in another tardigrade species, Hypsibius exem-
lesions, partly overlaps with damage caused by desiccation, plaris, which belongs to the same taxonomic family of R. var-
similar resistance machinery may be used to mitigate these ieornatus [252–254]. These findings indicate that tardigrades
two stressors, and coevolution of radioresistance and desicca- have evolved their own stress-resistant machinery in their
tion tolerance has been proposed [246]. lineages and such unique machinery can also function in
Unlike other radioresistant animals, tardigrades exhibit human cells. Radiation-resistant organisms including tardi-
high radiotolerance either in a hydrated state or in a dehy- grades are a valuable resource of undiscovered resistance
drated state, suggesting the presence of specific machinery genes and machineries, which might be used to enhance radi-
that relieves the indirect effects of radiation in this animal ation resistance in other animal species including human.
group. Ramazzottius varieornatus is one of the most radioto-
lerant species in tardigrades [244]. A recent study identified a
tardigrade-unique DNA-associating protein, termed damage 9. Conclusions
suppressor (Dsup) as a DNA-protecting agent from a chro-
matin fraction of R. varieornatus [251]. Intriguingly, in a In this review, discussion started with the environment of
human cultured cell line engineered to express the Dsup pro- space radiation followed by a variety of simulated space
tein, DNA damage caused by X-ray radiation (1–10 Gy) was radiation environments. Then, various adverse events by
reduced to nearly half of those in nonengineered cells space radiation were discussed. In that chapter, state-of-
(Figure 4). In addition, Dsup can also reduce DNA fragmen- the-art visualization technology of adverse events was dis-
tation in human cells treated with H2O2 significantly. Thus, cussed. Next, combined biological effects were discussed,
Dsup is capable of protecting DNA from both X-ray irradia- and we reported that a newly developed 3D clinostat with
tion and attack by ROS. The ability of Dsup to protect DNA synchronized irradiation capability would enable us to exam-
from ROS could explain the high radiation resistance of ine combined effects of radiation and μG. Radiation exposure
tardigrades even in wet conditions in which radiation causes management and radiation protection were then discussed.
biological damage via generation of ROS, which is known as Finally, radioprotective organisms were presented because
indirect effects. After irradiation with a near lethal dose these organisms may aid the development of novel technolo-
(4 Gy) of X-ray, nonengineered human cultured cells lose gies that alleviate biological damage caused by radiation.
their proliferative ability (Figure 4), but surprisingly, Dsup- Understanding these topics in greater detail should facil-
expressing cells survive the irradiation and even retain prolif- itate better prediction of the risks and provide risk-mitigating
erative ability that is comparable with those of nonirradiated strategies for future exploration space missions. In addition,
cells (Figure 4) [251]. Considering these results, Dsup is able meticulous use of available astronaut data, in particular
to not only confer DNA protection but also improve radioto- long-duration mission crew members, should be beneficial.
lerance to human cultured cells. A recent in vitro study also Furthermore, the use of rodent models in a Gateway program
confirmed that Dsup can protect chromatin DNA from around the Moon orbit, for example, should provide impor-
hydroxyl radicals [252]. Currently, a Dsup homolog has only tant information required for a future human Mars mission.
BioMed Research International 17

Conflicts of Interest in the Russian segment of the International Space Station,”

Physics Procedia, vol. 80, pp. 25–35, 2015.
The authors declare no conflicts of interest. [12] S. McKenna-Lawlor, the SG3.19/1.10 team, and Team mem-
bers, “Feasibility study of astronaut standardized career dose
limits in LEO and the outlook for BLEO,” Acta Astronautica,
Acknowledgments vol. 104, no. 2, pp. 565–573, 2014.
This work was supported by a MEXT Grant-in-Aid for Scien- [13] A. Nagamatsu, M. Masukawa, S. Kamigaichi et al., “Develop-
tific Research on Innovative Areas, Japan “Living in Space” ment of the space radiation dosimetry system ‘PADLES’,” in
20th Workshop on Radiation Detectors and Their Users,
(JP15H05943, JP15H05944, JP18H04992, JP18H04964,
pp. 23–36, Tsukuba, Japan, 2006.
JP18H04990, JP18H04977, JP18H04978, JP18H04969,
JP18H04991, JP18H04979, JP15H05945, JP15H05935, and [14] A. Nagamatsu, K. Murakami, S. Araki, H. Kumagai, K. Kitajo,
and H. Tawara, “Space radiation dosimetry in low Earth orbit
JP15K21745), the Institute of Space and Astronautical Sci-
by a passive and integrating dosimeter ‘PADLES’,” in 22nd
ence, JAXA Front Loading Study, and NASA Space Biology Workshop on Radiation Detectors and Their Uses, KEK Pro-
Program (80NSSC19K0133). We thank Dr. Atsushi Higashi- ceedings 2008–14, pp. 167–177, Tsukuba, Japan, 2009.
tani (Tohoku University, Miyagi, Japan), for reading the draft [15] A. Nagamatsu, K. Murakami, K. Kitajo, K. Shimada,
of this article, and the Edanz Group (Fukuoka-shi, Fukuoka, H. Kumagai, and H. Tawara, “Area radiation monitoring on
Japan) for editing the draft of this manuscript and helping to ISS increments 17 to 22 using PADLES in the Japanese
draft the abstract. A part of this study was conducted through Experiment Module Kibo,” Radiation Measurements,
the Joint Usage/Research Center Program of the Radiation vol. 59, pp. 84–93, 2013.
Biology Center, Kyoto University. [16] A. Takahashi, H. Ikeda, and Y. Yoshida, “Role of high-linear
energy transfer radiobiology in space radiation exposure
risks,” International Journal of Particle Therapy, vol. 5,
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