Journal of Radiation Research, Vol. 58, No. 1, 2017, pp. 33–40
doi: 10.1093/jrr/rrw083
Advance Access Publication: 10 September 2016

Analysis of liver damage from radon, X-ray,or alcohol treatments in mice using aself-organizing map
Norie Kanzaki1, Takahiro Kataoka1, Reo Etani1, Kaori Sasaoka1,
Akihiro Kanagawa2 and Kiyonori Yamaoka1*
1Graduate School of Health Sciences, Okayama University, 5-1 Shikatacho, 2-chome, Kita-ku, Okayama-shi, Okayama 700-8558, Japan
Faculty of Computer Science and System Engineering, Okayama Prefectural University, 111 Kuboki, Soja-shi, Okayama 719-1197, Japan*Corresponding author. Graduate School of Health Sciences, Okayama University, 5-1 Shikata-cho, 2-chome, Kita-ku, Okayama-shi, Okayama 700-8558,
Japan. Tel/Fax: +81-86-235-6852; Email: yamaoka@md.okayama-u.ac.jp
Received April 27, 2016; Revised June 24, 2016; Accepted June 30, 2016
2

ABSTRACTIn our previous studies, we found that low-dose radiation inhibits oxidative stress–induced diseases due toincreased antioxidants. Although these effects of low-dose radiation were demonstrated, further research wasneeded to clarify the effects. However, the analysis of oxidative stress is challenging, especially that of low levelsof oxidative stress, because antioxidative substances are intricately involved. Thus, we proposed an approach foranalysing oxidative liver damage via use of a self-organizing map (SOM)—a novel and comprehensive techniquefor evaluating hepatic and antioxidative function. Mice were treated with radon inhalation, irradiated with
X-rays, or subjected to intraperitoneal injection of alcohol. We evaluated the oxidative damage levels in the liverfrom the SOM results for hepatic function and antioxidative substances. The results showed that the effects of
low-dose irradiation (radon inhalation at a concentration of up to 2000 Bq/m3, or X-irradiation at a dose of up
to 2.0 Gy) were comparable with the effect of alcohol administration at 0.5 g/kg bodyweight. Analysis using the
SOM to discriminate small changes was made possible by its ability to ‘learn’ to adapt to unexpected changes.
Moreover, when using a spherical SOM, the method comprehensively examined liver damage by radon, X-ray,and alcohol. We found that the types of liver damage caused by radon, X-rays, and alcohol have different characteristics. Therefore, our approaches would be useful as a method for evaluating oxidative liver damage caused byradon, X-rays and alcohol.
KEYWORDS: liver damage, oxidative stress, self-organizing map, radon, X-ray, alcohol

IN TR ODU CT IO N
Animals, including humans, have a defence system against oxidative
stress, and antioxidants play an important role in inhibiting theaccumulation of reactive oxygen species (ROS) generated from oxidative stress [1]. It is well known that oxidative stress is induced byvarious lifestyle-related factors, such as exposure to radiation [2], heavy drinking [3–5], smoking [6, 7], a poor diet [8], and aging [9, 10].
The metabolism of alcohol in the liver follows three pathways: thealcohol dehydrogenase, microsomal ethanol oxidation, and catalase
pathways. Each of these pathways produce ROS, including superoxide anion radicals (O2−), hydroxyl radicals (−OH), and hydrogen

peroxide (H2O2) [3]. Moreover, irradiation also generates ROS,
either directly or indirectly [2]. It has been recognized that the
ROS produced by these factors are involved with cancer, inﬂammatory immune injury, and so on [1]. On the other hand, we havereported that low-dose irradiation inhibits oxidative stress–induced
diseases in the mouse liver due to the consequent increases in antioxidative substances, including superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx), glutathione (GSH) andglutathione reductase (GR) [11–13].
A self-organizing map (SOM) is an unsupervised neural network
algorithm that models the ability of a neuron to handle information© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
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• N. Kanzaki et al.

in the primary visual area [14]. Multidimensional data are summarized in a map by data compression during the learning process.
A SOM can be used to ﬁnd clusters in the input data in order to
identify unknown data and extract new knowledge. It has been proven useful in complex analyses of biochemical data, such as in determining gene expression patterns [15–17] and for diagnostic
assistance [18, 19]. However, there are no reports comparing thedifferent effects of oxidative stress caused by the various factors.
Since the 2011 nuclear accident in Fukushima, the health effectsof low-dose irradiation have been at the forefront of public attention. A high priority for medical research is to provide accurate
information on radiation health effects to the public. We haveattempted to quantify oxidative damage after low-dose irradiation.
We have previously demonstrated that radon inhalation has antioxidative effects in the mouse liver, and that the inhibitory effects ofradon inhalation at a concentration of 2000 Bq/m3 on carbon tetrachloride–induced hepatopathy are almost equivalent to those oftreatment with ascorbic acid at a dose of 500 mg/kg or α-tocopherol at 300 mg/kg [20]. We have quantitatively shown the positiveeffects of radon inhalation; however, further analysis of the effectsof irradiation is needed.
The inhibitaion of oxidative stress following low-dose radiation
has been controversial. The objective of this study was to compareradiation-induced liver damage with alcohol-induced liver damage.
Here, we used a SOM for the analysis because it is a useful tool forthe visualization of oxidative damage induced by different sources.
To determine the antioxidative function, we focused on the changes
in antioxidants (such as SOD, GSH and Cat) in a mouse liver, andthe hepatic function parameters in serums [such as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase(GPT)] after irradiation or alcohol administration. We also analyzed
oxidative liver damage caused by radon, X-rays or alcohol by using a
SOM.

M A T E R I A L S A ND M ET H O D S

Animals
C57BL/6J mice (age, 8 weeks; sex, male; bodyweight, 21–28 g)
were obtained from Charles River (Yokohama, Japan). All experimental protocols in this study were approved by the Animal
Experimentation Committee of Okayama University. The mice werehoused under a 12:12 h artiﬁcial light cycle (8:00 a.m. to 8:00 p.m.)
and were divided into 12 groups: radon-inhaling groups (sham,
500 Bq/m3, 1000 Bq/m3, 2000 Bq/m3), X-irradiated groups (sham,
0.5 Gy, 1.0 Gy, 2.0 Gy) and 50% alcohol-administered groups(sham, 0.5 g/kg, 2.0 g/kg, 5.0 g/kg).

Radon inhalation
In the radon-inhaling group, we used a radon exposure system as
described in a previous report [21]. To generate conditions for theinhalation of speciﬁc radon concentrations, the ‘Doll Stone’ radonsource (Ningyotoge Genshiryoku Sangyo, Co., Ltd, Okayama,
Japan) was placed in a radon tank. The radon inhalation systemblows air containing radon from the tank into a mouse cage. The
radon concentration in the mouse cage was controlled using the
Doll Stone and measured using a radon monitor (CRM-510;

femto-TECH, Inc., OH, USA). Mice inhaled radon at backgroundlevel concentrations of 20 (sham), 500, 1000 or 2000 Bq/m3 for
24 h. Mice were euthanized by deep ether anesthesia immediately
after radon inhalation. Blood was collected from the heart for GOT
and GPT analyses, and intact livers were quickly removed for SOD,
GSH and Cat analyses.

X-ray exposure
In the X-irradiated group, the mice received whole-body irradiation
at doses of 0.5 Gy, 1.0 Gy or 2.0 Gy (tube voltage, 150 kV; tube
current, 20 mA; ﬁlter, 0.5 mm Al and 0.2 mm Cu; distance between
focus and target, 43.5 cm) using an X-ray generator (MBR-1520R-3;
Hitachi Power Solutions Co., Ltd, Ibaraki, Japan). Similar to thetreatment mice, the control mice were sham-exposed. The mice
were euthanized by deep ether anesthesia 4 h after X-ray exposure.
Blood was collected from the heart for GOT and GPT analyses,
and intact livers were quickly removed for SOD, GSH and Cat
analyses.

Alcohol administration
In the alcohol-administered group, mice were treated with 50% alcohol at concentrations of 0.5, 2.0 or 5.0 g/kg bodyweight intraperitoneally. The control mice (‘the sham group’) were administeredsaline. Mice were euthanized by deep ether anesthesia 24 h after
alcohol administration. Blood was collected from the heart for GOT
and GPT analyses, and intact livers were quickly removed for SOD,
GSH and Cat analyses.

Biochemical assays
Livers were homogenized on ice with 10 mM phosphate buffer(PBS; pH 7.4) for assays of SOD, t-GSH, Cat and protein. The
homogenates were centrifuged at 12 000 × g for 45 min at 4°C andthe supernatants were collected. Serum was collected from thesupernatants by centrifuging at 3000 × g for 5 min for assays of
GOT and GPT.
SOD activities in the livers were assayed based on the nitroblue
tetrazolium (NBT) reduction method using the Wako–SOD test
(Wako Pure Chemical Industry, Co., Ltd, Osaka, Japan) [22]. NBTreacts with O2− to form diformazan, and SOD reacts with O2− toform H2O2 and O2. In this assay, the rate of inhibition of diformazan production by SOD was measured at 560 nm using a spectrophotometer (Viento XS; DS Pharma Biomedical Co., Ltd, Osaka,
Japan). One unit of enzyme activity is deﬁned as 50% of the inhibition rate.
The t-GSH content in the liver was assayed using the Bioxytech
GSH-420 assay kit (OXIS Health Products, Inc., Portland, OR,
USA). Tissue samples from the liver were mixed with trichloroacetic
acid solution. The solutions were centrifuged at 3000 × g for
10 min at 4°C and the upper aqueous layers were collected. Thismethod is based on the formation of a chromophoric thione. The
absorbance measured at 420 nm using a spectrophotometer is directly proportional to the GSH concentration.
Cat activity in the livers was assayed from the H2O2 reduction
rate at 37°C [23]. Brieﬂy, 20 μl of the supernatant of the liver homogenate were mixed with 50 μl of 1 M Tris-HCl buffer containingAnalysis of liver damage from radiation or alcohol
5 mM ethylenediaminetetraacetic acid (pH 7.4), 900 μl of 10 mM
H2O2 and 30 μl of deionized water. The changes in absorbancewere monitored for 1 min. Cat activity was calculated using a molar
extinction coefﬁcient of 7.1 × 10−3 M−1cm−1.
GOT and GPT activities in the serum were assayed based on
the pyruvate oxidase-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (POP-TOOS) method using the Transaminase CII-Test Wako
(Wako Pure Chemical Industry, Co., Ltd, Osaka, Japan).
The protein content in each sample was measured by the
Bradford method [24] using the Protein Quantiﬁcation Kit-Rapid(Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

Statistical analyses

The results are presented as the mean ± standard error of the mean
(SEM). Signiﬁcant differences between the sham group and treatedgroups were analyzed by ANOVA and Dunnett’s test.

SOM
A SOM [14] has an input layer and an output layer with associatedweights (Fig. 1). It constructs an output map from multidimensional
input data by learning. We optimized the initial randomized map via
machine learning of the input data. Brieﬂy, the SOM found the best
matching unit (BMU) closest to the input data by searching theminimum value of the distance between the input data and each of
the units in the output map. The characteristics of the input data
were reﬂected in the neighborhood of the BMU. We repeatedly
updated the output map. We were able to receive information fromthe output map because the SOM places data with similar patterns
into similar locations.
We analyzed oxidative liver damage using SOM PAK (SOM
Programming Team of the Helsinki University of Technology
Laboratory of Computer and Information Science, Espoo, Finland)
and Blossom (SOM Japan, Co. Ltd, Tottori, Japan). The results ofthe biochemical assay were normalized using the following formula:x = x′/AVESham, where, x′ is the value of SOD, GSH, Cat, GOT
and GPT, and AVESham is the average of the sham groups (radon,
X-ray or alcohol). Therefore, the average of x in each sham group
was normalized (SOD, GSH, Cat, GOT, GPT) = (1, 1, 1, 1, 1) as
the control standard.
SOM PAK is a 2D map and Blossom is a spherical map. In
Experiment 1, SOM PAK was used to present liver damage by radiation, such as by radon and X-ray, on a map that summarized theFig. 1. Structure of a self-organizing map (SOM).
mi is ith unit in output map. xi is jth input data.

•

35

data for the alcohol-induced liver damage, as 2D maps pointing out
valuable information. Brieﬂy, the SOM learned the data in thealcohol-administered group and constructed the output maps.
Subsequently, the BMUs of the radon-inhaled groups (sham,
500 Bq/m3, 1000 Bq/m3 or 2000 Bq/m3) and X-irradiated groups(sham, 0.5 Gy, 1.0 Gy or 2.0 Gy) were calculated. Finally, we
mapped the relationship between the radiation-induced liver damage and the alcohol-induced liver damage. In this experiment, thewidth of the output map, the height of the output map, the numberof learnings, the learning rate, and the radius of the neighborhood
are 60 units, 40 units, 100 000 steps, 0.02, and 15 units, respectively.
In Experiment 2, Blossom was used for establishing the liver damage caused by the various factors, such as radon, X-rays and alcohol,
because spherical maps make it easier to consider the relationship ofmany clusters. Some conﬁgurations are automatically and optimallyset. Clustering was applied according to the results from Blossom.

RESULTS

Evaluation of liver damage by radon, X-rays or alcoholby using statistical analysis

We ﬁrst statistically analyzed the changes in the parameters of hepatic function (Fig. 2). GOT activity in the 2000 Bq/m3 radon–inhaling
group was signiﬁcantly lower than that in sham-inhaling group.
GPT activity in the 1.0 Gy X-irradiated group was signiﬁcantly higher than that in sham-irradiated group. GOT activity in the 5.0 g/kg
alcohol–administered group was signiﬁcantly higher than that in the
sham-administered group. GPT activity in the 5.0 g/kg alcohol–
administered group was signiﬁcantly higher than that in the shamirradiated group.
Next, we statistically analyzed the changes in the parameters ofantioxidative function due to evaluation of oxidative damage in liver(Fig. 3). Radon inhalation did not change any antioxidative substances. In the X-irradiated group, although no signiﬁcant changes
were observed in Cat activity, 0.5, 1.0 and 2.0 Gy irradiation signiﬁcantly increased SOD activity. Similarly, 2.0 Gy irradiation signiﬁcantly increased GSH content. In the alcohol-administered group,
although no signiﬁcant changes were observed in Cat activity, SOD
activity in the 5.0 g/kg bodyweight alcohol–administered group was
signiﬁcantly lower than that in the sham-administered group. GSH
content in the 5.0 g/kg bodyweight alcohol–administered groupwas signiﬁcantly lower than that in the sham-administered group.

Evaluation of liver damage by radon, X-rays or alcoholby using a SOM
Experiment 1
We constructed an output map that learned the effect of alcohol
administration on the liver (Fig. 4). The 5.0 g/kg bodyweight alcohol–administered group was put on the left half of the output map,and the other groups and control standard (SOD, GSH, Cat, GOT,
GPT) = (1, 1, 1, 1, 1), were discretely put on the right half of theoutput map. The 5.0 g/kg bodyweight alcohol–administered groupwas sharply distinguished from the other groups. However, the2.0 g/kg bodyweight and 0.5 g/kg bodyweight alcohol–administeredgroups were less sharply distinguished. Speciﬁcally, the 2.0 g/kg
bodyweight alcohol–administered group was put on the upper right36• N. Kanzaki et al.

Fig. 2. Changes in GOT and GPT activity in liver following exposure to radon, X-rays or alcohol. Number of mice perexperimental point was six. *P < 0.05, **P < 0.01, ***P < 0.001 vs sham in each group.
of the output map and the 0.5 g/kg bodyweight alcohol–administeredgroup was put on the lower right of the output map. Thus,
the output map was able to show a summarizing assessment of liver
damage.
Finally, we calculated liver damage by radiation, such as fromradon and X-rays (Fig. 4). All of the radon-inhaling and X-irradiatedgroups were put on the lower right of the above-mentioned output
map. The data indicated that the radon-inhaling and X-irradiatedgroups were of similar levels to the 0.5 g/kg bodyweight alcohol–
administered group.

Experiment 2
We analyzed liver damage by various factors (such as radon, X-raysor alcohol) using Blossom (Fig. 5). Similar to Experiment 1, the5.0 g/kg bodyweight alcohol–administered group was sharply distinguished from the other groups. The other groups were less clearly
distinguished based on the results of Experimental 2. Although not
all of the data was classiﬁed properly, we found interesting characteristics from a dendrogram (Fig. 6). The alcohol-administeredgroup and X-irradiated group were large classiﬁcations based on the
results of Experimental 2. Moreover, the data in the alcoholadministered group was classiﬁed into four subgroups based on thelevel of liver damage. These ﬁndings showed that a spherical SOM
could express the effects of alcohol-induced liver damage. The datain the X-irradiated group had no conspicuous subclassiﬁcation. This

result indicated that there were no changes between the 0.5 and
2.0 Gy administrations.
Hence, alcohol-induced liver damage had characteristics thatwere different from liver damage by X-rays. We concluded that liver
damage caused by radon at concentrations of up to 2000 Bq/m3
would be similar to that caused by 0.5 g/kg bodyweight alcohol
administration, because the 2000 Bq/m3 radon–inhaling group andthe 0.5 g/kg bodyweight alcohol–administered group were classiﬁed
together based on the results of Experiment 2. Likewise, we concluded that the liver damage caused by radon at a concentration of
500 Bq/m3 was similar to that caused by an X-ray dose of up to
2.0 Gy, because the 500 Bq/m3 radon–inhaling group and the Xirradiated group were grouped into the same class.

D IS C U S S IO N
Alcohol-induced liver damage is primarily related to oxidative stress.
It is well known that alcohol-induced liver damage sharply increases
GOT and GPT in the serum, and that acute alcohol administration
depletes GSH and reduces antioxidants in 24 h due to the detoxiﬁcation of alcohol [13, 25]. Thus, it is expected that oxidative stressis induced 24 h after alcohol administration. On the other hand,
exposure to low-dose X-rays and radon leads to the early activationof SOD. For radon exposure, it has been reported that SOD activity
in the mouse liver tended to reach a peak within 24 h [26]. For
X-ray exposure, although there were no changes with high-doseAnalysis of liver damage from radiation or alcohol•

37

Fig. 3. Changes in SOD activity, t-GSH content, and Cat activity in liver following exposure to radon, X-rays or alcohol.
Number of mice per experimental point was six. *P < 0.05, **P < 0.01, ***P < 0.001 vs sham.
X-rays (10 Gy), a low-dose increased SOD activities at 4 h after
irradiation [27]. In this study, we analyzed the liver damage afterradon inhalation for 24 h, at 4 h after X-irradiation, and at 24 h after
alcohol administration. GOT and GPT activities at 5.0 g/kg bodyweight in the alcohol-administered group were signiﬁcantly higher
than in the sham-administered group. In addition, antioxidants, such
as SOD and catalase, were signiﬁcantly reduced by 5.0 g/kg bodyweight with alcohol administration. Therefore, these results suggestthat the mouse livers were oxidatively damaged by alcohol administration at 5.0 g/kg bodyweight. In the other treated groups, GOT
and GPT activities were not sharply increased. Although there werehardly any changes to antioxidant levels, SOD activities in the
X-irradiated group and the radon-inhaling group tended to increasedepending on the X-ray dose. Therefore, our results show thatenhancement of antioxidative function by low-dose X-irradiation
and radon inhalation was extremely small and highly individual.
As mentioned previously, the effects of low levels of oxidative
stress due to irradiation and alcohol administration are uncertain
because the mechanisms are complicated by the fact that oxidative
stress–related substances are involved in quick chain reactions.
However, it is important to analyze not only hepatic function, butalso antioxidative function to verify liver damage. We have validated

38• N. Kanzaki et al.

Fig. 4. Output map for analysis of liver damage byradon, X-rays or alcohol by using a self-organizing
map (SOM). Calculated oxidative levels in the liverof radon-inhaled X-irradiated mice are expressed as
an output map for liver damage by alcohol groups.
Sources of similar oxidative levels are located in thesame area (mild oxidative stress, white area; severeoxidative stress, gray area).
diagnoses of hepatitis using a SOM [19]. In the present study, we
attempted to analyze the more difﬁcult fuzzy clustering caused byliver damage from low oxidative stress. Thus, we proposed that oxidative liver damage is best visualized by an overall assessment ofchanges in hepatic function and antioxidant levels using a SOM.
Speciﬁcally, we obtained new knowledge from the output map by
the SOM. As SOMs place data with similar patterns onto similar
locations, liver damage by radon inhalation (at a concentration of
up to 2000 Bq/m3) and X-ray irradiation (at a dose of up to
2.0 Gy) were considered similar to that caused by alcohol at
0.5 g/kg bodyweight administration because of their placement on
the map. We found that it was easy to compare liver damage due todifferent factors. Moreover, we applied a spherical SOM to analyze
oxidative liver damage. Ritter proposed that the spherical form
would be more suitable as a structure of SOMs [28]. Some problems of boundary effect information conveying and computing
speed have been solved by spherical SOMs [29, 30]. Thus, we
showed a comparison of liver damage due to radon, X-rays andalcohol using a spherical SOM. The spherical SOM adapted characteristics and made seamless changes through a comprehensive
evaluation of liver damage. Although we obtained no evidence ofspeciﬁc characteristic oxidative stress caused by radon inhalation,
the X-irradiated and alcohol-administered groups were sharply
divided in the dendrogram based on the output map of theFig. 5. Output map for analysis of liver damage byradon, X-rays or alcohol by using a spherical selforganizing map (SOM). Sources of similar oxidative
levels are located in the same area (mild oxidative
stress, white area; severe oxidative stress, gray area).
The spherical SOM shows two clusters divided
between 5.0 g/kg bodyweight alcohol-administeredgroup and the other groups.
spherical SOM. We discovered the different patterns of liver damage caused by different factors. The radon-inhaling group and Xirradiated groups were categorized into the same group as the control group or the 0.5 g/kg bodyweight alcohol-administered groupbased on the results of Experimental 2, respectively. This result
was consistent with the results of the 2D SOM. Further work isneeded to explore the differences in liver damage caused by thedifferent factors.
In our previous study, we found there were differences between
liver damage that was due to radon, X-ray or alcohol treatment.
However, it was still unclear what exactly the differences were. We
reported a radon concentration of ~3 mBq/kg in the liver of mice
after continuous radon inhalation at a concentration of 1 Bq/m3[31]. According to our report, the absorbed dose in the liver was~140 nGy under the experimental conditions. Although we reported
that radon at a concentration of 2000 Bq/m3 for 24 h or X-ray
exposure at a dose of 0.5 Gy enhances antioxidative function, theabsorbed dose of radon was much lower than that of X-ray irradiation. The results of the study also suggested that radon inhalation
at a concentration of 2000 Bq/m3 and X-irradiation at a dose of
0.5 Gy led to the same level of liver damage. Moreover, we suggested that radon inhalation at a concentration of 2000 Bq/m3, or
X-irradiation at a dose of 0.5 Gy, were comparable with the effect of
alcohol administration at 0.5 g/kg bodyweight. However, we could
not conclude why antioxidative functions were activated by radon
inhalation in spite of the low absorbed dose. Further study wasneeded at this point.
Thus, in the present study, we evaluated the oxidative damagelevels in the liver from the results of hepatic function and antioxidative substances by using a SOM to compare radiation-induced liverAnalysis of liver damage from radiation or alcohol•

39

Fig. 6. Findings based on a dendrogram by a spherical self-organizing map (SOM) for classiﬁcation of liver damage by radon,
X-rays or alcohol.
damage with alcohol-induced liver damage. We concluded thatradon inhalation at a concentration of up to 2000 Bq/m3, or
X-irradiation at a dose of up to 2.0 Gy, were comparable with theeffect of alcohol administration at 0.5 g/kg bodyweight. This novel
method using a SOM was successful in comprehensively examining
oxidative liver damage, and our approach was useful for evaluating
the oxidative stress caused by various factors. The results presented
in this study provide a substantial basis for future studies aimed at
elucidating the mechanisms involved in oxidative stress.

A C KN O W L ED G EM E NT S
The authors thank the staff of the Departments of Animal
Resources and Radiation Research at Shikata Laboratory, Advanced
Science Research Center, Okayama University, for their technicalsupport. Results from this study were presented at the International
Congress of Radiation Research 2015, and Annual Meeting ofAtomic Energy Scoiety of Japan 2016.

FUNDING
This work was supported by JSPS KAKENHI (Grant Number
26420872).

CONFLICT OF INTEREST
The authors state that there are no conﬂicts of interest.

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