Comprehensive analysis for detecting radiation-specific molecules expressed during radiation-induced rat thyroid carcinogenesis.

Although the association between radiation exposure and thyroid carcinogenesis is epidemiologically evident, 'true' radiation-induced cancers cannot be identified from biological evidence of radiation-associated cases. To assess the individual risk for thyroid cancer due to radiation exposure, we aimed to identify biomarkers that are specifically altered during thyroid carcinogenesis after irradiation in a time-dependent manner in an animal model. Thyroid glands were obtained from rats (n = 175) at 6-16 months after local X-ray (0.1-4 Gy) irradiation of the neck at 7 weeks of age. The gene expression profile in thyroid glands was comprehensively analyzed using RNA microarray. Subsequently, the expression levels of the genes of interest were verified using droplet digital PCR (ddPCR). The expression level of candidate genes as biomarkers for irradiated thyroid was examined in a randomized, controlled, double-blind validation study (n = 19) using ddPCR. The incidence of thyroid cancer increased in a dose- and time-dependent manner and was 33% at 16 months after irradiation with 4 Gy. The Ki-67 labeling index in non-tumorous thyroid was significantly higher in the exposed group than in the control. Comprehensive analysis identified radiation-dependent alteration in 3329 genes. Among them, ddPCR revealed a stepwise increase in CDKN1A expression from early pre-cancerous phase in irradiated thyroid compared to that in the control. The irradiated thyroids were accurately distinguished (positive predictive value 100%, negative predictive value 69%) using 11.69 as the cut-off value for CDKN1A/β-actin. Thus, CDKN1A expression can be used as a biomarker for irradiated thyroid glands at the pre-cancerous phase.

INTRODUCTION

Currently, the risk to human health due to radiation hazard has increased considerably owing to medical exposure to radiation during treatment and diagnosis for cancers and accidents at nuclear power plants. Owing to the widespread use of computed tomography, the recent medical exposure dose per person has increased by approximately seven times compared to that in the 1980s, suggesting that the incidence of radiation-related cancer will increase [1, 2]. Among radiation-associated cancers, thyroid cancer is a focus of special attention in populations exposed to ionizing radiation, particularly when children are exposed. In fact, children and adolescent atomic (A)-bomb survivors and residents of the contaminated areas after the Chernobyl power plant accident have higher rates of thyroid cancer than the general population [3, 4].

Although the association between radiation exposure and thyroid cancer risk is epidemiologically evident [5-7], ’true’ cases of radiation-induced thyroid cancer cannot be individually identified from the biological evidence of cases of radiation-associated thyroid cancer [8-11]. Radiation biomarkers are being studied intensively to accurately distinguish radiation-induced cancer cases from sporadic cases [12]. Regarding the search for biomarkers of radiation-related thyroid cancer, genomic analysis of samples stored in the Chernobyl Tissue Bank revealed associations between the overexpression of CLIP2 mRNA and radiation exposure [13, 14]. However, such attempts lack a satisfactory justification because the molecular mechanisms for radiation-induced carcinogenesis are not fully understood. To assess the individual risk for thyroid cancer due to radiation exposure, biomarkers that are specifically altered during thyroid carcinogenesis after irradiation in a time-dependent manner have to be identified. Therefore, this study aimed to comprehensively analyze the expression level of characteristic molecules altered in the precancerous stage during radiation-induced thyroid carcinogenesis in a rat model.

MATERIALS AND METHODS

ANIMALS

In total, 194 seven-week-old male Wistar Kyoto rats (WKY/Izm) (Japan SLC, Inc. Hamamatsu, Japan) with body weight ranging from 173 to 211 g were used. The animals were divided into two groups: (i) test group (n = 175) and (ii) validation group (n = 19). All rats were maintained in a pathogen-free facility at the Life Science Support Center, Nagasaki University, in accordance with the rules and regulations of the Institutional Animal Care and Use Committee (approval number: 1203290976, 1610111343).

IRRADIATION AND SAMPLING OF THYROID TISSUES

The experimental design is shown in Fig. 1. Irradiation was performed between 8:00 a.m. and noon. In the test group, 137 rats were locally irradiated at their anterior necks with 0.1, 1 or 4 Gy X-rays at a dose rate of 0.5531 Gy/min under anesthesia using an ISOVOLT TITAN32 (200 kV, 15 mA device with 0.5 mm aluminum +0.5 mm copper +5 mm aluminum filter, Toshiba, Tokyo, Japan). The rest of the body was shielded by a lead plate. Control rats (n = 38) were non-irradiated but were otherwise handled identically. In the validation group, 10 rats were locally irradiated at their anterior necks using 4 Gy X-ray, and 9 non-irradiated rats were used as control. Thyroid tissues were resected after sacrificing rats via deep anesthesia at 6, 12 and 16 months after irradiation (6, 12 and 16 M, respectively) in the test group, and at 16 M after irradiation in the validation group. Both lobes of the thyroid glands were divided into two parts along the horizontal axis as shown in Fig. 1 and used for histological and molecular analyses.

Fig. 1.

Experimental design of the study. Rat was locally irradiated with 0.1, 1 or 4 Gy of X-ray, and the thyroid gland was collected at 6, 12 or 16 M after irradiation. The resected thyroid gland was divided into two parts for histological analysis with hematoxylin–eosin (H & E) staining/immunohistochemistry and molecular analysis.

IMMUNOHISTOCHEMISTRY

The resected thyroid glands were fixed in 20% buffered formalin and then embedded in paraffin blocks to cut sections for immunohistochemistry. Deparaffinized 4-μm sections were pre-treated by autoclaving for 20 min at 120°C in target retrieval solution (pH 6.0) (Dako, Glostrup, Denmark). The sections were incubated for 1 h at 37°C with 1:50 dilution of anti-Ki-67 mouse monoclonal antibody (MIB-5, Dako) or 1:200 dilution of anti-p21 mouse monoclonal antibody (CP74, Abcam, Cambridge, UK) in a humidified chamber. After incubation with EnVision+ system-HRP-labeled polymer anti-mouse (Dako) for 60 min, immunoreactivity was visualized by incubation with 3,3-diaminobenzidine (DAB) using a liquid DAB+ substrate chromogen system (Dako). The number of Ki-67-positive cells in thyroid follicular cells was counted as the Ki-67 labeling index (LI; the number of positive cells per one high-power field). The level of p21 immunoreactivity in thyroid follicular cells was assessed by scoring the highest intensity of staining in one high-power field as follows: negative, 0; faint nuclear staining, 1; moderate nuclear staining, 2; intense nuclear and cytoplasmic staining, 3.

COMPREHENSIVE ANALYSIS OF MRNA EXPRESSION IN IRRADIATED THYROID GLANDS

Total RNA was extracted from the snap-frozen thyroid glands (5–12 mg) using an QIAcube system (Qiagen, Venlo, The Netherlands) and an AllPrep DNA/RNA mini kit (Qiagen) according to the manufacturer’s instructions. The quantity and integrity of the extracted RNA were confirmed using agarose gel electrophoresis, NanoDrop quantification (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). After RNA amplification, cDNAs were synthesized and labeled using a Low Input Quick Amp Labeling kit (Agilent) and were hybridized on SurePrint G3 Rat GE microarray 8 × 60 K using a gene expression hybridization kit and RNA spike-in kit (Agilent) following the manufacturer’s instructions. This analysis was performed with samples extracted from non-tumorous thyroid tissues at 6 M (n = 4), 12 M (n = 4) and 16 M (n = 7) after 4 Gy radiation, and 6 M (n = 4), 12 M (n = 4) and 16 M (n = 6) from the control group. The data obtained were analyzed using the Gene Spring software (Agilent).

DROPLET DIGITAL POLYMERASE CHAIN REACTION ANALYSIS TO VERIFY THE LEVEL OF MRNA EXPRESSION

To verify the results of comprehensive analysis for identifying candidate genes as biomarkers in irradiated thyroid glands, droplet digital polymerase chain reaction (ddPCR) was performed using the QX200 droplet digital PCR system and EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). Takara Prime Script RT reagent kit with gDNA eraser (Perfect real Time) (Takara Bio Inc. Shiga, Japan) was used for reverse transcription (RT). cDNA was prepared from RNA diluted to 0.1 μg/μl according to the protocol of the RT kit. The expression of selected candidate biomarkers was quantitatively measured using cDNA from two follicular-cell cancers at 16 M after 4 Gy radiation, and non-tumorous thyroid tissues at 6 M (n = 9), 12 M (n = 7) and 16 M (n = 10) after 4 Gy radiation and at 6 M (n = 6), 12 M (n = 11) and 16 M (n = 15) from the control group. The primers used for ddPCR are listed in Supplementary Table 1, see online supplementary material. Gene expression was shown as the ratio to β-actin expression, which was used as an internal control. We determined a statistically appropriate cut-off value for the expression level of candidate biomarkers to distinguish the irradiated thyroid glands from non-irradiated controls and assessed the receiver operating characteristic (ROC) curve.

EVALUATION OF CANDIDATE GENES AS BIOMARKERS OF IRRADIATED THYROID GLANDS IN THE VALIDATION GROUP

The expression level of candidate genes as biomarkers in irradiated thyroid glands was blindly examined with a randomized validation set with non-tumorous thyroid tissue samples (n = 19) using ddPCR as mentioned above.

STATISTICAL ANALYSIS

The Mann–Whitney U test was used to examine the differences of Ki-67 LI and score of p21 immunoreactivity between irradiated and non-irradiated thyroid glands. The differences in the level of mRNA expression were analyzed using Student’s t-test. The logistic regression model and ROC curve were used to evaluate the significance of the expression level of candidate genes as biomarkers of irradiated thyroid glands. All statistical analyses were performed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA) with a statistical significance level of 0.05. The boxplots and ROC curves were created in R statistical language version 3.6.1 using ggplots, dplyr and pROC packages.

RESULTS

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS IN IRRADIATED RAT THYROID GLANDS

Both invasive cancers and benign nodules, such as hyperplasia or adenomatous goiter, were observed in rat thyroid glands. Invasive cancers were pathologically classified as follows: (i) follicular cell-derived cancer showing nuclear findings similar to those of human papillary carcinoma, and (ii) medullary carcinoma expressing calcitonin. The incidence of thyroid nodules in the test group is summarized in Table 1. The histological features of the thyroid nodules are shown in Fig. 2. Thyroid cancers were histologically confirmed only in the irradiated thyroid. The incidence of thyroid cancer increased in a time- and dose-dependent manner and reached the highest rate of 33% at 16 M after irradiation with 4 Gy. No significant histological changes were observed in non-tumorous thyroid tissues between irradiated and control groups or those with and without cancer. However, immunohistochemistry revealed that Ki-67 LI was significantly (P = 0.0233) higher in non-tumorous thyroid tissues surrounding the cancer at 16 M after exposure to 4 Gy (1.96 ± 0.80) than in their control thyroid tissues (0.63 ± 0.49) (Fig. 3). Ki-67 LI in non-tumorous thyroid tissues showing no cancer at 16 M after exposure to 4 Gy was lower (1.40 ± 0.90) than those in the surrounding cancer but higher than in the control thyroid tissues, although no statistical difference was observed between these two groups.

Table 1

The incidence of thyroid nodules

Dose	Pathological type	6 M		12 M		16 M
		n	%	n	%	n	%
Control (n = 38)	Carcinoma	0		0		0
	Benign lesion	0		0		2	10.5
	Normal	8	100	11	100	17	89.5
	Total	8	100	11	100	19	100
0.1 Gy (n = 47)	Carcinoma	0		1	8.3	2	8.3
	Benign lesion	0		3	36.0	2	8.3
	Normal	11	100	8	66.6	20	83.3
	Total	11	100	12	100.0	24	100
1 Gy (n = 46)	Carcinoma	1	10	2a	16.7	5a	20.8
	Benign lesion	1	10	5	41.6	4	16.7
	Normal	8	80	5	41.6	15	41.6
	Total	10	100	12	100	24	100
4 Gy (n = 44)	Carcinoma	1	9.1	2	16.7	7b	33.3
	Benign lesion	0		0		4	19.0
	Normal	10	90.9	10	83.3	10	47.6
	Total	11	100	12	100	21	100

aIncluding one case of medullary carcinoma.

bIncluding two cases of medullary carcinoma.

Fig. 2.

Representative images of rat thyroid nodules at 16 M after exposure to 4 Gy radiation. (A–C) and (D–F) show benign nodule and invasive cancer, respectively. The nuclear findings of thyroid cancer are similar to those of human papillary carcinoma (F). (A and D: macroscopic appearance; B and E: original magnification, ×20, C and F: original magnification, ×400).

Fig. 3.

Immunohistochemistry for Ki-67 expression in non-tumorous thyroid tissue at 16 M after exposure to 4 Gy radiation. Ki-67-positive cells are indicated by arrow heads. Boxplots show the maximum, third quartile, median, first quartile and minimum values. Values are the mean ± standard deviation. The Ki-67 labeling index was significantly higher in thyroid tissue surrounding cancer than in the control. *P = 0.0233 using the Mann–Whitney U test.

PROFILE OF MRNA EXPRESSION IN IRRADIATED THYROID GLANDS

The median RNA integrity number of the extracted total RNA was 8.3 (range: 7.4–9.4), assuring the quality of the RNA used for molecular analysis. Our microarray analysis demonstrated that the expression levels of 5635 genes were significantly altered (fold-change > 1.1; increase: 2644 genes, decrease: 2991 genes) (P< 0.05) in non-tumorous thyroid glands at 16 M after irradiation with 4 Gy (Fig. 4). Among them, 3329 genes were considered to be candidate biomarkers in irradiated thyroid gland (increase: 1486 genes, decrease: 1843 genes), as the remaining genes were also associated with ageing (Supplementary Tables 2 and 3, see online supplementary maerial). Furthermore, 142 pathways were extracted from 3329 genes using pathway analysis (Supplementary Table 4, see online supplementary material). Among these, the expression levels of 12 genes were verified as biomarkers of irradiated thyroid gland using ddPCR. These 12 genes included ATM, TP53 and 53BP1, which were involved in the DNA damage response pathway, XRCC4 and RAD51, involved in the DNA damage repair pathway, CDKN1A, CDKN2A, CDK1 and CDK2, involved in the cell cycle pathway, and CKDN4, CLDN9 and CTNNB1, involved in the cell adhesion pathway.

Fig. 4.

Results for comprehensive analysis of mRNA expression in non-tumorous thyroid tissues after 4 Gy irradiation using RNA microarray. (A) The volcano plot shows the alterations in gene expression post-irradiation compared to that in the control at the respective time points. Red dots indicate significantly upregulated genes, while blue dots indicate significantly downregulated genes (P < 0.05, fold change > 2). (B) The Venn diagram shows the number of genes that were significantly altered in a time-dependent manner after irradiation or due to ageing (P < 0.05, fold change > 1.1). In total, 3329 genes were identified to be radiation-dependent.

VERIFICATION OF THE EXPRESSION LEVELS OF CANDIDATE GENES USING DDPCR

The expression levels of the 12 candidate biomarkers in irradiated thyroid gland were assessed using ddPCR, as shown in Fig. 5. The levels of ATM, 53BP1, XRCC4 and CTNNB1 were significantly reduced in the order of control, irradiated non-tumorous thyroid and radiation-induced cancer at 16 M post-4 Gy irradiation; in contrast, CDKN1A expression increased significantly at 16 M. Furthermore, CDKN1A expression increased in irradiated thyroid glands in a time-dependent manner, and was higher in irradiated thyroid glands than in the controls at all time points, namely, 6, 12 and 16 M after irradiation. Among cell cycle pathway-related genes, the levels of CDKN2A and CDK1 were significantly increased in radiation-induced cancers, whereas the expression of all three genes related to the cell adhesion pathway, namely, CLDN4, CLDN9 and CTNNB1, were significantly reduced in radiation-induced cancers.

Fig. 5.

Quantitative analyses for expression levels of 12 genes as candidate biomarkers in irradiated thyroid gland using ddPCR. The y-axis represents the ratio of the expression level of each gene to β-actin expression. CDKN1A expression was significantly higher than that of the control at all time points and increased in a time-dependent manner.

VALIDATION OF THE SIGNIFICANCE OF CDKN1A EXPRESSION AS A BIOMARKER FOR IRRADIATED THYROID GLANDS

We focused on CDKN1A expression as a biomarker for distinguishing the irradiated thyroid glands from non-irradiated thyroid. Verification of the results of comprehensive analysis using ddPCR showed the area under the curve (AUC) to be 0.928, suggesting reliable detection of irradiated thyroid glands at 16 M after irradiation with 4 Gy (Supplementary Fig. 1, see online supplementary material). The sensitivity and specificity were 82 and 95%, respectively, when 11.69 was considered the cut-off value of CDKN1A/β-actin for detecting irradiated thyroid glands at 16 M post-4 Gy irradiation. Our randomized, controlled, double-blind validation study using ddPCR revealed that when 11.69 was considered the cut-off value of CDKN1A/β-actin for detecting irradiated thyroid glands, the positive and negative predictive values (PPV and NPV) were 100 and 69%, respectively (Fig. 6). Immunohistochemical expression of p21, which is the product of the CDKN1A mRNA, was also examined in irradiated non-tumorous thyroid glands and compared to that in the non-irradiated control thyroid glands. The representative images of p21 immunoreactivity are shown in Fig. 7. In cases heterogenous expression, the scoring was assessed at a high-power view showing the highest immunoreactivity. The mean scores of p21 immunoreactivity was higher in thyroid tissues (2.2 ± 0.9) at 16 M after 4 Gy irradiation than in their control thyroid tissues (1.6 ± 0.7). However, no statistically significant difference was observed between the two groups (P = 0.13, Mann–Whitney U test). The mean score (2.9 ± 0.4) of p21 immunoreactivity in tumors (both benign and malignant), tended to be higher than in surrounding irradiated non-tumorous thyroid tissues (2.2 ± 0.9), but no significant difference was evident (P = 0.10, Mann–Whitney U test).

Fig. 6.

Comparison of CDKN1A expression level in non-tumorous thyroid glands at 16 M between 4 Gy exposure and control groups in a randomized, controlled, double-blind study. Using 11.69 as the cut-off value for CDKN1A/β-actin for detecting irradiated thyroid glands, irradiated thyroid glands could be distinguished with high accuracy. The positive and negative predictive values were 100 and 69%, respectively.

Fig. 7.

Comparison of p21 immunoreactivity in non-tumorous thyroid tissues at 16 M between 4 Gy exposure and control groups. The level of p21 staining was scored as follows: negative, 0; faint nuclear staining, 1; moderate nuclear staining, 2; intense nuclear and cytoplasmic staining, 3. No statistical difference was observed between the two groups. P = 0.13 using the Mann–Whitney U test.

DISCUSSION

In our rat model, thyroid cancers were frequently (33%) induced by 4 Gy irradiation but were not observed in the non-irradiated control. Using this model, comprehensive analysis with RNA microarray demonstrated significant alteration in the expression of several genes in rat thyroid glands from the early pre-cancerous phase, such as 6 M after irradiation. After pathway analysis, we selected 12 genes as candidate biomarkers for the irradiated thyroid glands. ddPCR confirmed that among these genes, the expression of CDKN1A increased significantly in non-tumorous thyroid tissues in a time-dependent manner after exposure to 4 Gy. Furthermore, after setting a statistically appropriate cut-off value (11.69, AUC 0.928) of the CDKN1A/β-actin ratio in ddPCR, we were able to accurately distinguish the irradiated thyroid glands from the control (PPV: 100%, NPV: 69%) in a randomized, controlled, double-blind study. Previous reports have described upregulation of CDKN1A in different cell lines at the acute phase post-irradiation [15-17]. This is the first study to demonstrate a stepwise increase in CDKN1A expression from the early pre-cancerous phase during radiation-induced thyroid carcinogenesis in vivo.

Epidemiological data on A-bomb survivors have revealed that a long latency period is required for the onset of radiation-related solid cancers, and that cancer risk persists for more than half a century after the exposure [18]. We have previously reported activation of spontaneously occurring DNA damage response in non-neoplastic epidermal cells surrounding basal cell carcinoma in aged A-bomb survivors [19]. These findings suggested that irradiated cells may survive in the human body throughout life and may exhibit cancer phenotypes at the molecular pathological level with ageing, such as abrogated DNA damage response/repair, abnormal cell cycle regulation, and genomic instability, from the early phase of carcinogenesis during which histological changes are not recognized. Indeed, this comprehensive analysis identified altered expression of >3000 genes in irradiated non-tumorous thyroid glands, which were histologically comparable to the non-irradiated control but showed enhanced cell proliferation as evident from the Ki-67 LI, suggesting induction of growth stress in the pre-cancerous phase after irradiation. Upregulation of CDKN1A may play a pivotal role in suppressing this growth stress.

As shown by the quantitative analysis of selected genes as candidate biomarkers for irradiated thyroid glands using ddPCR, a stepwise decrease in ATM, 53BP1, XRCC4 and CTNNB1 expression was observed in the non-irradiated thyroid, irradiated non-tumorous thyroid, and radiation-induced thyroid cancer at 16 M after 4 Gy irradiation, suggesting failure of the DNA damage response/repair and cell–cell adhesion at the late phase of radiation-induced thyroid carcinogenesis. In addition, CTNNB1, CLDN4 and CLDN9 expression were significantly reduced in radiation-induced cancers, indicating involvement of loss of cell–cell adhesion in cancer cells. In contrast, CDKN2A and CDK1 expression was markedly upregulated in radiation-induced cancers. As CDKN2A and CDK1 are negative and positive regulators of the normal cell cycle, respectively, dysregulation of cell cycle-related molecules, including CDKN1A overexpression in irradiated thyroid, can directly accelerate radiation-induced carcinogenesis [20, 21].

CDKN1A encodes p21, a cyclin-dependent kinase inhibitor, which promotes growth arrest and cellular senescence [22]. As its functions are upregulated via the ATM–p53–p21 pathway after irradiation, aberration of these molecules can induce solid cancers due to failure of growth arrest and cellular senescence [23]. However, results of our immunohistochemical staining did not confirm increased p21 immunoreactivity in non-tumorous thyroid tissue at 16 M post-irradiation compared to that in the control thyroid tissue, indicating post-transcriptional modification of p21 expression. We have reported that although 4 Gy radiation induces p53 phosphorylation, neither p21 nor cleaved casapase-3 is upregulated in rat thyroid tissue at the acute phase (3–24 h) after irradiation [24]. Reports have shown that p21 is not detected in human embryonic stem cells after induction of DNA damage because of post-transcriptional regulation by microRNAs irrespective of upregulation of TP53 and CDKN1A expression [25]. Thus, further studies are required to clarify the regulatory mechanism underlying p21 expression following CDKN1A overexpression in irradiated thyroid glands at acute and chronic pre-cancerous phases.

Moreover, to validate the significance of our data in humans, samples must be obtained by non-traumatic procedures such as blood testing. Several studies have recently attempted to identify candidate biomarkers for radiation exposure in blood samples. A recent proteomic analysis using high-throughput techniques searched for candidate radiation-exposure protein markers [26]. A novel approach measuring mRNA and microRNA expression levels in blood exosomes has also been reported [27]. Interestingly, Nongrum et al. demonstrated an increased level of expression of the CDKN1A gene in ex vivo irradiated human whole blood samples by DNA microarray [28]. Thus, in future studies we will focus on identifying analytic targets in blood samples instead of tissue samples of our animal model.

This is the first study to demonstrate a stepwise increase in CDKN1A expression from the early pre-cancerous phase during radiation-induced thyroid carcinogenesis using an animal model. Using quantitative analysis of CDKN1A expression with ddPCR, we were able to accurately distinguish irradiated thyroid glands from the control. Thus, CDKN1A expression can be used as a biomarker to estimate irradiated thyroid glands at the pre-cancerous phase. In this study, the selected candidate biomarkers of irradiated thyroid glands were limited to molecules, the carcinogenic functions of which are well established. However, as shown in Supplementary Table 5 (see online supplementary material), >3000 radiation-associated genes were altered in irradiated thyroid glands. Although several genes exhibited greater fold-change in expression than CDKN1A, their biological significance remains unknown. Further analysis of the remaining genes is required to elucidate the molecular pathology of radiation-induced thyroid carcinogenesis.

CONFLICT OF INTEREST

None declared.

FUNDING

This work was supported partially by the Atomic Bomb Disease Institute, Nagasaki University, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture [No. 20 K07424], and the Program of the Network-Type Joint Usage/Research Center for Radiation Disaster Medical Science.

SUPPLEMENT FUNDING

This supplement has been funded by the Program of the Network-type Joint Usage/Research Center for Radiation Disaster Medical Science of Hiroshima University, Nagasaki University, and Fukushima Medical University.

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