Gene expression analyses of the small intestine of pigs in the ex-evacuation zone of the Fukushima Daiichi Nuclear Power Plant
© The Author(s). 2017
Received: 25 June 2017
Accepted: 9 November 2017
Published: 15 November 2017
After the accident at the Fukushima Daiichi Nuclear Power Plant, radioactive contaminants were released over a widespread area. Monitoring the biological effects of radiation exposure in animals in the ex-evacuation zone should be continued to understand the health effects of radiation exposure in humans. The present study aimed to clarify the effects of radiation by investigating whether there is any alteration in the morphology and gene expressions of immune molecules in the intestine of pigs and inobuta (wild boar and domestic pig hybrid) in the ex-evacuation zone in 2012. Gene expression analysis was performed in small intestine samples from pigs, which were collected from January to February 2012, in the ex-evacuation zone. Pigs lived freely in this zone, and their small intestine was considered to be affected by the dietary intake of radioactive contaminants.
Several genes were selected by microarray analysis for further investigation using real-time polymerase chain reaction. IFN-γ, which is an important inflammatory cytokine, and TLR3, which is a pattern recognize receptor for innate immune system genes, were highly elevated in these pigs. The expressions of the genes of these proteins were associated with the radiation level in the muscles. We also examined the alteration of gene expressions in wild boars 5 years after the disaster. The expression of IFN-γ and TLR3 remained high, and that of Cyclin G1, which is important in the cell cycle, was elevated.
We demonstrated that some changes in gene expression occurred in the small intestine of animals in the ex-evacuation zone after radiation. It is difficult to conclude that these alterations are caused by only artificial radionuclides from the Fukushima Daiichi Nuclear Power Plant. However, the animals in the ex-evacuation zone might have experienced some changes owing to radioactive materials, including contaminated soil, small animals, and insects. We need to continue monitoring the effects of long-term radiation exposure in living things.
After the Great East Japan Earthquake on March 11, 2011, a huge amount of radioactive cesium was released following an accident at Fukushima Daiichi Nuclear Power Plant (FNPP) , and the Japanese government set the evacuation zone within 20-km radius from FNPP (ex-evacuation zone now). People were seriously concerned about food safety. Therefore, the Ministry of Agriculture, Forestry and Fisheries officially informed the local government around the FNPP about management guidelines for feeds. However, rice hay was contaminated over a large area owing to wind, and many cows were found to be contaminated, including those that were far from FNPP . Consumers required thorough radioactivity inspection for food products. Fukuda et al. previously reported a correlation between 137Cs radioactivity in whole peripheral blood and that in the organs of cattle and established an approach to estimate muscle contamination with a small amount of blood . Therefore, the level of contamination in the muscles of cows could be estimated using a blood test. However, many other livestock were abandoned in the evacuation zone at that time. Pigs have a different feeding habit and different physiological properties compared with those of cows. Therefore, raising concern about contaminated rice hay with regard to pigs was not necessary. Pigs are omnivorous animals, and they eat small insects or tree nuts, such as acorn, with soil. Large physiological differences exist between cows and pigs, and it is important to understand the biological effects of radioactive cesium in pigs in the ex- evacuation zone.
The immune system and physiological functions of pigs are very similar to those of humans [4–6]. Therefore, knowledge about the responses in pigs to radioactive contamination can be useful to understand radiation effects and responses in humans. The intestine can be greatly affected by radiation through internal exposure after oral intake of contaminated food. The intestinal epithelium constitutively and greatly proliferates from stem cells, which exist in the crypt . It is well known that proliferative cells are highly sensitive to radiation, and a previous report mentioned that a dose of radiation as low as 0.01–0.05 Gy can induce apoptosis in these cells . The gastrointestinal tract is not only an organ for digestion and absorption of nutrients, but also an important organ in the host defense mechanism. In the gastrointestinal tract, immune and nonimmune cells work together to expel pathogens, as part of the mucosal barrier. In addition, the immune system in the intestine is closely linked to physiological functions, including metabolism and chronic inflammation.
In this study, we focused on the intestine and aimed to clarify the effects of radiation by investigating whether there is any alteration in morphology and gene expressions of immune molecules in the intestine of pigs and inobuta (wild boar and domestic pig hybrid) in the ex-evacuation zone in 2012. Additionally, we assessed samples obtained from wild boars in the ex-evacuation zone in 2015.
We collected intestine and muscle samples from euthanized 13 pigs between 18 January and 16 February 2012 at 5 km southwest of the FNPP and three inobuta on 28 February 2012 at 8 km southwest of the FNPP. The animals were sacrificed by the following method according to the Regulation for Animal Experiments and Related Activities at Tohoku University by the veterinary doctors belonging to the Livestock Hygiene Service Center of Fukushima Prefecture . Estimates of the amounts of 134Cs and 137Cs deposited on the ground have been reported . Intestine samples were collected from five wild boars on 18 November 2015, in Namie town. Control intestine samples were obtained from three healthy pigs present in an uncontaminated pigsty in Miyagi Prefecture in 2012. Each experimental protocol was approved by the Institutional Ethics Commissions for Animal Research at Tohoku University, Fukushima University, and Miyagi University.
Measurement of radioactivity
Radioactivity in the muscle samples was determined with gamma-ray spectrometry using high-purity germanium (HPGe) detectors (Ortec Co., Oak Ridge, TN, USA), as described in our previous report . Gamma rays from 134Cs and 137Cs were observed and radioactivity ratios of 134Cs to 137Cs (decay corrected to March 11, 2011) were 0.9–1.0 which corresponded to other samples polluted by the FNPP accident.
Small pieces of the small intestine were slit longitudinally, laid flat with the mucosal surface facing down, and rolled around a wooden stick (Swiss roll). Paraffin blocks were prepared for pathomorphological examination using hematoxylin and eosin (HE) staining and Masson trichrome (MT) staining.
Gene expression analysis
List of Primers
Primer sequences (5′ to 3′)
Real-time PCR was performed using Brilliant SYBR Green QPCR Master Mix III (Stratagene, La Jolla, CA, USA) with an MX3000P system (Stratagene, La Jolla, CA, USA). Amplification conditions were as follows: 95 °C for 3 min, 40–50 cycles at 95 °C for 5 s, and 60 °C for 20 s. Fluorescence signals measured during the amplification were analyzed. Ribosomal RNA primers were used as an internal control, and all data were normalized to constitutive rRNA values. Quantitative differences between the groups were calculated according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA).
All data are presented as mean ± standard error (SE) for each treatment group. Differences in mRNA expression among the groups were determined using the t-test (Prism: GraphPad Software Inc., LaJolla, CA, USA). Differences were considered statistically significant at a P-value <0.05.
Gene expression in the small intestine of pigs in the ex-evacuation zone
The number of significantly differentially expressed genes (>2-fold) linked to immune responses by microarray data analysis
The number of ≥2-fold change genes in category
Innate immune response in mucosa
Immune effector process
Immune system process
Immune complex clearance
Immune complex clearance by monocytes and macrophages
Regulation of immune system process
Regulation of immune effector process
Positive regulation of immune effector process
Positive regulation of immune complex clearance by monocytes and macrophages
Regulation of immune complex clearance by monocytes and macrophages
T-helper 1 type immune response
Type 2 immune response
Innate immune response
Regulation of innate immune response
Positive regulation of innate immune response
Gene expression in inobuta
Relationship between gene expression and radiation levels in blood
Gene expressions in the small intestine for samples obtained in 2015
There are major concerns about several biological effects of radioactive contamination caused by nuclear power plant accidents. Previous studies on animals and plants in the Fukushima ex-evacuation zone have described biological changes after exposure to radioactive contaminants [14–18]. However, the health consequences of nuclear accidents are still unclear. Therefore, it is important to perform studies in several fields, including developmental biology, immunology, and oncology continuously over a long period. We have been working on host defenses against gastrointestinal nematode parasites in pigs for a long time [6, 19]. As mentioned above, the gastrointestinal tract is an important local immune organ and has extreme sensitivity to radiation, because of the high proliferative activity of epithelial cells. Therefore, whether the intestinal cells in pigs and wild boars are affected after radiation exposure is of interest.
We are members of the ‘Group for Comprehensive Dose Evaluation in Animals from the Area Affected by the Fukushima Nuclear Power Plant Accident’. The sampling method and schedule for sampling, which are discussed in this paper, have been described in previous reports [2, 3, 20, 21]. In this study, we focused on the alterations of gene expressions in the small intestine of pigs in the ex-evacuation zone. Previous research reported that low-dose radiation induced biological responses, such as inflammatory responses, innate immune system activation, and DNA repair . Therefore, we selected several genes associated with inflammation, DNA repair, and the cell cycle for further analysis after microarray analysis. We found that AIFM1 and IFN-γ gene expressions were elevated in pigs from the ex-evacuation zone (Fig. 1). IFN-γ is one of the most important cytokines in type 1 immune responses, and if excess IFN-γ is produced, the intestine shows an inflammatory status. In addition, interferon stimulates several genes, including mediators of apoptosis . In pigs from the ex-evacuation zone, there might be stimulation to eliminate damaged cells in the intestine caused by radiation, inducing apoptosis. Meanwhile, HE and MT staining revealed that there were no morphological changes, including fibrosis, in all tissue samples (Fig. 5). Chronic inflammation in tissue can lead to fibrosis and tissue remodeling; however, the results of this study suggested that the elevation of inflammatory genes was not severe enough to cause morphological changes. Therefore, intestinal homeostasis appeared to be maintained at that time, although inflammatory cytokine and apoptosis-related genes were activated.
The gene expression analysis demonstrated that TLR3 was also increased. TLRs are very important in the innate immune system. Additionally, they regulate tight junctions in the mucosal barrier system of the intestine. Takemura et al. reported that TLR3 deficient mice showed substantial resistance to gastrointestinal syndrome (GIS) . TLR3 bound to cellular RNA leaking from damaged cells owing to radiation and caused crypt cell death, which can result in GIS. Therefore, the elevation of TLR3 gene expression might reflect cell response against radiation.
It is important to carefully consider the findings of these gene expressions, because these genes might have been activated by infection. When we obtained samples from the animals, they had no symptoms of infection, such as fever, diarrhea, cough, and wasting. To clarify the relationship between elevated gene expression and radiation, we investigated the relation between the gene expression ratio and the concentration of 137Cs in muscles. There were positive relations between AIFM1 and IFN-γ expressions and the concentration of 137Cs, but not between Cyclin G1 and TLR3 expressions and the concentration of 137Cs (Fig. 3). The elevations of IFN-γ and TLR3 in pigs from the ex-evacuation zone might not be regulated by the same mechanism. A previous study by Shea-Donohue et al. investigated the influence of radiation on the cytokine balance in the small intestine and colon in non-human primates and demonstrated an increase in the gene expression of inflammatory cytokines, such as IFN-γ, after irradiation . These findings suggest that radiation exposure might result in the alteration of immune genes in animals in the ex-evacuation zone.
Four years after the Great East Japan Earthquake, the air radiation dose has dropped and decontamination of the land has progressed. The government has gradually reduced the ex-evacuation zone. However, there are many wild animals in the evacuation zone. The number of wild boars has increased dramatically, as they breed freely in the ex-evacuation zone. We collected tissue samples from wild boars in the ex-evacuation zone in 2015 to examine gene expressions in the small intestine. AIFM1, Cyclin G1, IFN-γ, and TLR3 gene expressions tended to be higher in wild boars than in control pigs (Fig. 4). Especially, Cyclin G1 and IFN-γ gene expressions were significantly higher in wild boars than in control pigs. These results suggested that some biological effects might be present in the small intestine after radiation; however, the mechanism is unknown.
We demonstrated that some changes in gene expression occurred in the small intestine of animals in the ex-evacuation zone after radiation. It is difficult to make a conclusion that these alterations are caused by only artificial radionuclides from the FNPP. However, the animals in the ex-evacuation zone might have experienced some changes due to radioactive materials, including contaminated soil, small animals, and insects. The effects of long-term radiation exposure in living things need to be monitored.
We thank all people associated with the livestock production field in Fukushima, including dairy farmers and stockman, for their cooperation and full understanding of this work.
This study was funded by the Emergency Budget for the Reconstruction of Northeastern Japan, MEXT, Japan; Discretionary Expense of the President of Tohoku University; the Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries, MAFF, Japan; the Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry, BRAIN, Japan and Grant-in-Aid for Scientific Research (KAKENHI): 15H01850 and 26,253,022, JSPS, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MM analyzed all data and was a major contributor in writing the manuscript. AK performed PCR and the histological examination of the small intestine. KO, YaK, YoK and TS measured radioactivity in the muscle samples. YoK, TF, EI and MF performed pathological diagnosis. JK and YA analyzed microarray data. MM, JK, KO, YaK, YoK, YA, TS, TF, EI, and MF provided sampling tissues by dissecting in the ex-evacuation zone. EI and MF directed the project. All authors have read and approved the final manuscript.
Each animal experimental protocol was approved by the Institutional Ethics Commissions for Animal Research at Tohoku University, Fukushima University, and Miyagi University. This study is one of the national projects associated with the Great East Japan Earthquake and has been entirely endorsed and supported by the Japanese government. Written consent from the owner was obtained by the Livestock Hygiene Service Center of Fukushima Prefecture.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Harada KH, Niisoe T, Imanaka M, et al. Radiation dose rates now and in the future for residents neighboring restricted areas of the Fukushima Daiichi nuclear power plant. Proc Natl Acad Sci U S A. 2014;111:E914–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Fukuda T, Kino Y, Abe Y, et al. Distribution of artificial radionuclides in abandoned cattle in the evacuation zone of the Fukushima Daiichi nuclear power plant. PLoS One. 2013;8:e54312.View ArticlePubMedPubMed CentralGoogle Scholar
- Fukuda T, Kino Y, Abe Y, et al. Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: analyses of cattle within the evacuation zone of the Fukushima Daiichi nuclear power plant. Anim Sci J. 2015;86:120–4.View ArticlePubMedGoogle Scholar
- Bode G, Clausing P, Gervais F, et al. The utility of the minipig as an animal model in regulatory toxicology. J Pharmacol Toxicol Methods. 2010;62:196–220.View ArticlePubMedGoogle Scholar
- Swindle MM, Makin A, Herron AJ, et al. Swine as models in biomedical research and toxicology testing. Vet Pathol. 2012;49:344–56.View ArticlePubMedGoogle Scholar
- Dawson HD, Beshah E, Nishi S, et al. Localized multigene expression patterns support an evolving Th1/Th2-like paradigm in response to infections with Toxoplasma gondii and Ascaris suum. Infect Immun. 2005;73:1116–28.View ArticlePubMedPubMed CentralGoogle Scholar
- Potten CS. Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond Ser B Biol Sci. 1998;353:821–30.View ArticleGoogle Scholar
- Yamashiro H, Abe Y, Hayashi G, et al. Electron probe X-ray microanalysis of boar and inobuta testes after the Fukushima accident. J Radiat Res. 2015;56:i42–7.View ArticlePubMedGoogle Scholar
- Chaudhry MA. Biomarkers for human radiation exposure. J Biomed Sci. 2008;15(5):557–63.View ArticlePubMedGoogle Scholar
- Barcellos-Hoff MH, Cucinotta FA. New tricks for an old fox: impact of TGFbeta on the DNA damage response and genomic stability. Sci Signal. 2014;7:re5.View ArticlePubMedGoogle Scholar
- Aherne SA, O'Brien NM. Protection by the flavonoids myricetin, quercetin, and rutin against hydrogen peroxide-induced DNA damage in Caco-2 and Hep G2 cells. Nutr Cancer. 1999;34:160–6.View ArticlePubMedGoogle Scholar
- Pollard KM, Cauvi DM, Toomey CB, et al. Interferon-gamma and systemic autoimmunity. Discov Med. 2013;16:123–31.PubMedPubMed CentralGoogle Scholar
- Takemura N, Kawasaki T, Kunisawa J, et al. Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome. Nat Commun. 2014;5:3492.View ArticlePubMedPubMed CentralGoogle Scholar
- Bonisoli-Alquati A, Koyama K, Tedeschi DJ, et al. Abundance and genetic damage of barn swallows from Fukushima. Sci Rep. 2015;5:9432.View ArticlePubMedPubMed CentralGoogle Scholar
- Hayashi G, Shibato J, Imanaka T, et al. Unraveling low-level gamma radiation--responsive changes in expression of early and late genes in leaves of rice seedlings at Iitate Village, Fukushima. J Hered. 2014;105:723–38.View ArticlePubMedGoogle Scholar
- Ochiai K, Hayama S, Nakiri S, et al. Low blood cell counts in wild Japanese monkeys after the Fukushima Daiichi nuclear disaster. Sci Rep. 2014;4:5793.View ArticlePubMedPubMed CentralGoogle Scholar
- Hiyama A, Nohara C, Taira W, et al. The Fukushima nuclear accident and the pale grass blue butterfly: evaluating biological effects of long-term low-dose exposures. BMC Evol Biol. 2013;13:168.View ArticlePubMedPubMed CentralGoogle Scholar
- Taira W, Hiyama A, Nohara C, et al. Ingestional and transgenerational effects of the Fukushima nuclear accident on the pale grass blue butterfly. J Radiat Res. 2015;56:i2–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Morimoto M, Zarlenga D, Beard H, et al. Ascaris suum: cDNA microarray analysis of 4th stage larvae (L4) during self-cure from the intestine. Exp Parasitol. 2003;104:113–21.View ArticlePubMedGoogle Scholar
- Takahashi S, Inoue K, Suzuki M, et al. A comprehensive dose evaluation project concerning animals affected by the Fukushima Daiichi nuclear power plant accident: its set-up and progress. J Radiat Res. 2015;56:i36–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Fukuda T, Hiji M, Kino Y, et al. Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples. Anim Sci J. 2016;87:842–7.View ArticlePubMedGoogle Scholar
- Hekim N, Cetin Z, Nikitaki Z, et al. Radiation triggering immune response and inflammation. Cancer Lett. 2015;368:156–63.View ArticlePubMedGoogle Scholar
- Chawla-Sarkar M, Lindner DJ, Liu YF, et al. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis. Apoptosis. 2003;8:237–49.View ArticlePubMedGoogle Scholar
- Shea-Donohue T, Fasano A, Zhao A, et al. Mechanisms involved in the development of the chronic gastrointestinal syndrome in nonhuman primates after total-body irradiation with bone marrow shielding. Radiat Res. 2016;185:591–603.View ArticlePubMedPubMed CentralGoogle Scholar