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Analysis of radiation dose and gene expression in wild boar 10 years after the Fukushima Daiichi nuclear power plant accident

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Samples

The intestine and muscle samples from 22 wild boars were collected between September 4 and March 2, 2020, in Namie City, Fukushima Prefecture. In addition, control intestine samples were collected from three wild boars from Hyogo Prefecture. Each location is shown in Figure 1. In each case, after the licensed hunters shot the wild boar to be exterminated, only the tissue was transferred for study.

Measurement of radioactivity

Radioactivity in muscle samples was determined by gamma spectrometry using high-purity germanium (HPGe) detectors (Ortec Co., Oak Ridge, TN, USA), as described in our previous report.3. Gamma rays from 137Cs were observed.

Estimation of exposure dose

In order to estimate the internal and external dose rates of wild boars according to ICRP publication 10826, we assumed the shapes of the boars as elongated spheroids whose major axis must be the length of their bodies. The short axis was given from their weight assuming that their specific gravity was the same as that of water. The dose rates were calculated from the contribution of 137Cs, excluding natural radionuclides. Energy deposition on spheroids by beta and gamma rays from radionuclides was calculated by numerical simulation using the Particle and Heavy Ion Transport Code System (PHITS)27. For the sake of simplicity, we assumed that the spheroids were made up of muscle only, which would give overestimated values ​​because muscle contains more radiocaesium than other organs. The external exposure dose was calculated from the dose rates in the air that were observed from the monitoring station near the place of capture of the boars. The average values ​​of the dose rates in the air were obtained by fitting the data observed over two years with the decay curve. The background noise due to natural radionuclides was estimated at 0.05 µGy/h, observed before the Fukushima Daiichi accident, and was suppressed before the improvements. Dose rate half-lives in air were 2000 to 3000 days depending on the environment. Assuming that the external exposure dose has been attributed to the 137Cs included in the ground surface. The amount of the 137Cs was calculated to reproduce the observed airflows. Since the maximum beta ray range of 137Cs is a few millimeters, almost all beta rays from inside the body should be absorbed by the boar’s body, but beta rays from outside the body would stop in its fur. Beta rays contribute 100% to the internal exposure dose but 0% to the external dose. Since the linear attenuation coefficient for gamma rays of 137Cs is 0.084 cm−1= (12cm)−1, some of the gamma rays cannot stop in the body depending on the size of the body. Numerical simulation suggested that 65-90% of gamma rays from 137cs inside the body would extinguish, and 40-65% of the gamma rays of 137The Cs on the outside would pass through the body.

Pathological analysis

A piece of small intestine was fixed in 10% neutral formalin at 4°C for 24–48 h. Next, paraffin blocks were prepared for pathomorphological examination using hematoxylin and eosin (HE) staining.

Gene expression analysis

Total RNA was extracted from all gut tissue using TRIzol reagent (Life Technologies, Inc., Frederic, MD, USA) according to the manufacturer’s instructions. RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and cDNA was synthesized using random primers and SuperScript II (Life Technologies, Inc). real-time PCR for IFN-γ, TLR3and CyclinG1 was performed using Brilliant SYBR Green QPCR Master Mix III (Stratagene, La Jolla, CA, USA) with an AriaMx system (Agilent Technologies, Santa Clara, CA, USA). Primer sequences were designed using Primer-BLAST with sequences obtained from GenBank as described in the previous report4. The amplification conditions were 95°C for 3 min, 40 cycles at 95°C for 5 s and 60°C for 20 s. The 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. The quantitative differences between the groups were analyzed using AriaMx software (Agilent Technologies).

statistical analyzes

All data are presented as the mean ± standard error (SE) for each treatment group. Differences in mRNA expression between groups were determined using the you-test with Welch’s correction. (Prism: GraphPad Software Inc., La Jolla, CA, USA). The differences were considered statistically significant at one P value

Ethics approval

No animals were killed for this research. The use of all animals was secondary to control wildlife pests. We conducted the survey with permission from the local government.