The effect of exposure on cattle thyroid after the Fukushima Daiichi nuclear power plant accident.

Nuclear plant accidents can be a risk for thyroid cancer due to iodine radioisotopes. Near the Fukushima Daiichi nuclear power plant, cattle were exposed to radiation after the accident occurred in May 2011. Here we estimated the total radiation exposure to cattle thyroid and its effects on thyroid function. Until October 2016, the estimated external exposure dose in Farm A was 1416 mGy, while internal exposure dose of 131I, 134Cs, and 137Cs were 85, 8.8, and 9.7 mGy in Farm A and 34, 0.2, and 0.3 mGy in Farm B, respectively. The exposed cattle had thyroid with relatively lower weight and lower level of stable iodine, which did not exhibit any pathological findings. Compared with the control, the plasma level of thyroid-stimulating hormone (TSH) was higher in Farm A cattle born before the accident, while the plasma thyroxine (T4) was higher in Farm A cattle born after the accident, suggesting that exposed cattle showed slight hyperactivation of the thyroid gland. In addition, Farm A cattle have higher level of cortisol, one of the anterior pituitary gland-derived hormones. However, we did not observe a causal relationship between the radiation exposure and cattle thyroid.

Assessments of DNA Damage and Radiation Exposure Dose in Cattle Living in the Contaminated Area Caused by the Fukushima Nuclear Accident.

Since the Fukushima nuclear accident in 2011, various abnormalities have been reported in animals living in the contaminated area. In the present study, we examined DNA damage in cattle living in the "difficult-to-return zone" by 8-hydroxy-2'-deoxyguanosine, comet, and micronucleus assays using their peripheral blood. The radiation exposure dose rate at the sampling time was approximately 0.25 or 0.38 mGy/day and the cumulative dose was estimated at approximately 1000 mGy. Significant increase in DNA damage was not detected by any of the three methods. As DNA damage is a stochastic effect of radiation, it might be occurring in animals living in the contaminated area. However, the present results suggest that radiation-induced DNA damage in the cattle did not increase to the level detectable by the assays we used due to the low dose rate in this area.

Pathological characteristics of thyroid glands from Japanese Black Cattle living in the restricted area of the Fukushima Daiichi Nuclear Power Plant accident.

To study the effect of ionizing radiation on thyroid glands, 66 Japanese Black cattle residing in the restricted area of the Fukushima Daiichi Nuclear Power Plant in 2013-2017 were pathologically examined. There were no clinical symptoms of thyroid disease in these cattle. Three cases of goiter and seven of atrophy were found in two among the four farms examined. Cases of goiter exhibited normal morphological structure without mass or nodule formation in thyroid glands. Cellular atypia or capsular invasion of the follicular epithelium was absent. The estimated integrated dose of external radiation in goiter cases ranged from maximum 797 mSv to minimum 24 mSv. All lobules in the seven atrophic thyroid glands were affected, but pathological findings, such as inflammatory cell infiltration or stromal fibrosis, were not observed. The estimated integrated dose of external radiation in atrophic thyroids ranged from maximum 589 mSv to minimum 8 mSv. Immunohistochemical analysis of anti-nitroguanosine and the TUNEL method in goiter and atrophic thyroid glands did not reveal any positive findings. The present study indicates that there was no significant relationship between a radiation effect and pathological findings in any thyroid glands.

Comparison of urine and blood as a convenient and practical sample for estimating the contamination level of live cattle with radioactive cesium.

Blood and urine were compared to clarify which is a better sample for estimating the concentration of radioactive cesium in muscles of live cattle. The most probable concentration of 137 Cs in muscles was 21.0 times that in blood, and the error of this estimation was 28%. The concentration of 137 Cs in blood was estimated accurately using urine samples. The estimation error did not increase so much (33%), even when the concentration of 137 Cs in muscles was estimated using urine samples. On the other hand, the maximum volume of blood that can be collected with one syringe is 50 ml, whereas it is easy to collect 1,000 ml of urine. It took at least 360 min to confirm that a cow meets the legal standard by inspecting 50 ml of blood. However, with 1,000 ml of urine, a 20 min measurement time was sufficient for this purpose. This difference in the required measurement time is critically important for practical use. In addition, urine can be collected by farmers themselves, whereas the blood collection requires a veterinarian. Therefore, urine is a more convenient and practical sample for estimating the contamination level of live cattle with radioactive cesium.

RETROSPECTIVE ESR RECONSTRUCTION OF CATTLE TOOTH ENAMEL DOSES FROM THE RADIOACTIVE NUCLEI RELEASED BY THE ACCIDENT OF FUKUSHIMA DAI-ICHI ATOMIC POWER PLANTS.

The method of electron spin resonance (ESR) tooth enamel dosimetry was successfully applied to cattle molar teeth exposed in the accident of Fukushima Dai-ichi atomic power plants. Total of 10 samples from 5 cattle were examined and the doses were retrospectively reconstructed to be up to 1.2 Gy (enamel dose). The dose values are roughly consistent with those estimated from the monitored environmental dose rate and the durations of the exposure. This first successful result on ESR reconstruction of doses in the actual radiation accident indicates that ESR tooth enamel dosimetry with cattle is practically useful in the dose range of ~1 Gy.

Decreased blood cell counts were not observed in cattle living in the "difficult-to-return zone" of the Fukushima nuclear accident.

White blood cells, especially lymphocytes, are susceptible to radiation exposure. In the present study, red blood cell, total white blood cell, and lymphocyte counts were repeatedly measured in cattle living on three farms located in the "difficult-to-return zone" of the Fukushima nuclear accident, and compared with two control groups from unaffected areas. Blood cell counts differed significantly between the two control groups, although almost all the values fell within the normal range. The blood cell counts of the cattle in the "difficult-to-return zone" varied across sampling times even on the same farms, being sometimes higher or lower than either of the two control groups. However, neither a statistically significant decrease in blood cell counts nor an increase in the rate of cattle with extremely low blood cell counts was observed overall. The estimated cumulative exposure dose for the cattle on the most contaminated farm was within a range of 500-1000 mSv, exceeding the threshold for the lymphopenia. Because of the low dose rate on these farms, potential radiation damages would have been repaired and have not accumulated enough to cause deterministic effects.

Pathological findings of Japanese Black Cattle living in the restricted area of the Fukushima Daiichi Nuclear Power Plant accident, 2013-2016.

Fifty-one Japanese black cattle from four farms in the evacuation zone of the Fukushima nuclear accident were examined pathologically during the period, 2013-2016. We found no evidence of a radiation effect on pathological findings in any of these autopsy cases, although nine cases (3.7%) of enzootic bovine leukosis (EBL) and three cases (1.2%) of goiter were diagnosed. Estimated integrating dose of external exposure in EBL cases ranged from a maximum of 1200 mSv to a minimum of 72 mSv. Clinically, five cases showed wobble, dysstasia or paralysis. Exophthalmos was observed in three cases. Macroscopically, enlarged lymph nodes, multiple irregular masses of intra-abdominal and intrapelvic adipose tissue, diffuse thickening of the abomasal submucosa with ulceration, and numerous white nodules of myocardium were observed. Histologically, neoplastic lymphoid cells were extensively proliferated and infiltrated in the lesions. Lymphoid tumor cells showed mature or large lymphoblastic appearance. Immunohistochemically, tumor cells were positive for BLA-36, CD20 and CD5, and negative for CD3. Three cases showed diffuse enlargement of the thyroid gland with no mass or nodule lesion. Histologically, diffuse proliferation of follicular epithelium was observed with preservation of normal structures. There were no malignant findings such as cellular atypia or invasion to capsule.

Whole-Body Counter Evaluation of Internal Radioactive Cesium in Dogs and Cats Exposed to the Fukushima Nuclear Disaster.

As a result of the 2011 nuclear incident that occurred at the Fukushima Nuclear Power Plant, a large number of abandoned dogs and cats were left within the disaster zone. A small number of these animals were rescued and cared for at shelters. Prior to the dispersal of these animals to their owners or fosterers, we evaluated the degree of internal radiocesium contamination using a specially designed whole-body counter. We conducted 863 non-invasive measurements of gamma rays due to internal radioactive cesium for 68 dogs and 120 cats at one shelter. After plotting graphs of 137Cs density we generated exponential functions of decay from seven dogs and six cats. From the regression formulae, we were able to determine the biological half-lives as 38.2 days for dogs and 30.8 days for cats. We found that in dogs there was a correlation between the biological half-life of radioactive cesium and age. Using our data, we estimated whole-body densities for each cat and dog at the time when they were rescued. We found that there were deviations in the data distributions among the different species, likely due to the timing of rescue, or living habits prior to rescue. A significant correlation was found when extracted feline reproductive organs were analyzed; the coefficients for the estimation of whole-body densities were approximately 7-fold higher than those based on the extracted feline reproductive organs. This may be due to the fact that majority of the radioactive cesium accumulates within muscular tissue with less distribution in other organs. It is possible to plan the appropriate management period in an animal shelter based on the use of the biological half-life of radioactive cesium calculated in this study. We believe that the correlations we uncovered in this work would be of great use for the management of companion animals in the event of a future nuclear accident.

Local variation of soil contamination with radioactive cesium at a farm in Fukushima.

Radioactive cesium concentration in soil was measured at 27 sections with 5 points per section, and surface dose of ground was measured at 10 sections with 13 points per section at a farm in Fukushima to assess local variation of soil contamination with radioactive cesium. As for the cesium in soil, averages of the coefficient of variance (CV) and the maximum/minimum ratio in each section were 49% and 4.9, respectively. As for the surface dose, average of its CV in each section was 20% and the maximum/minimum ratio reached a maximum of 3.0. These findings suggest that exact evaluation of soil contamination with cesium is difficult. Small changes or differences in soil contamination may not be detected in studies of the environmental radioactivity.

Predicting metastatic potential of gastrointestinal stromal tumors in dog by ultrasonography.

Gastrointestinal stromal tumor (GIST), a mesenchymal neoplasm affecting the gastrointestinal tract, shows a variety of clinical behaviors from inactive benign to aggressive malignant in dogs. In this study, the feasibility of using clinically significant ultrasonographic features to predict the metastatic potential of canine GIST was investigated through comparison with actual metastatic incidence and findings of malignancy obtained by postoperative pathological examination. Ultrasonographic features, including large tumor size, irregular margin and heterogeneous internal echogenicity with large hypoechoic areas, related closely with the presence of metastasis as well as a high-risk ranking by the human classification system according to pathological findings. Based on these ultrasonographic features, the potential of metastasis in canine GIST could be preoperatively predicted.

Vulnerability of feline T-lymphocytes to charged particles.

An analysis of ionizing radiation-induced damage in peripheral lymphocytes has been employed to predict the prognosis of radiotherapy in terms of toxicity in normal tissues. Therefore, understanding the sensitivity of lymphocytes to high linear energy transfer (LET)-charged particles would be indispensable for utilizing charged particle therapy in veterinary medicine. However, the availability of such information is very limited. This study aimed to compare the radiosensitivity of feline T lymphocytes to gamma-rays (0.2 keV/microm) and 4 different types of charged particles with LET values ranging from 2.8 to 114 keV/microm. It was observed that the relative biological effectiveness, inactivation cross-section, and isodose-induced apoptosis increased in an LET-dependent manner. On the other hand, no difference in apoptosis frequency was observed in the cells exposed to an isosurvival dose of all the radiation types tested. This is the first study that demonstrates the LET dependence of cell killing and apoptosis induction in feline T lymphocytes. Our results suggest that lymphocytes can be effectively used to predict the prognosis of charged-particle therapy in cat patients.

Distinct modes of cell death by ionizing radiation observed in two lines of feline T-lymphocytes.

We have examined in vitro radiosensitivities and radioresponses to (60)Co gamma-rays irradiation in feline T-lymphocyte cell lines, FeT-J and FL-4. There seemed to be no significant difference in clonogenic survival between the two lines. The mean lethal dose for both was both 1.9 Gy, and surviving fraction at 2 Gy was 0.30 and 0.48 for FeT-J and FL-4 cells, respectively. However, TUNEL assay indicated much higher degrees of apoptosis induction in FeT-J cells (>40%) than in FL-4 cells (<10%) at 4 days after 15 Gy irradiation. Microscopic examination revealed a larger population of multi-nucleate cells in FL-4 cells (60.3%) than in FeT-J cells (16.0%) at 4 days after 15 Gy irradiation, suggesting that a larger ratio of mitotic catastrophe occurred in FL-4 cells. These results suggest that FeT-J is more likely to be induced into apoptosis and FL-4 is more likely to fall into mitotic catastrophe, and eventually necrosis; both of them showed a similar surviving fraction against gamma-rays. The results also indicate that FL-4 cells follow a process other than apoptosis to cell death, suggesting the presence of a regulatory mechanism that may control the relationship between mitotic catastrophe and apoptosis in feline T-lymphocytes.

Probenecid: its chromatographic determination, plasma protein binding, and in vivo pharmacokinetics in dogs.

Pharmacokinetics (PK) of probenecid including plasma probenecid concentrations, in vitro plasma protein binding properties, and in vivo PK parameters were determined in dogs. Probenecid concentrations were best determined by HPLC, which showed good linearity and good recovery with simple plasma preparation. The quantification limit of probenecid was approximately 50 ng/ml at S/N ratio = 3, by simple procedure with HCl and methanol treatment. Probenecid showed two types of binding characteristics, i.e., high-affinity with low-capacity and low-affinity with high-capacity binding. This result indicated 80-88% of probenecid was bound to plasma protein(s) at observed concentrations (< 80 microg/ml) in vivo at an intravenous dose of 20 mg/kg. Plasma probenecid concentration-time profile following i.v. administration in dogs showed biphasic decline and well fitted a two-compartment open model. The total body clearance was 0.34 +/- 0.04 ml/min/kg, volume of distribution at steady-state was 0.46 +/- 0.07 l/kg, elimination half-life was 18 +/- 6 hr, and mean residence time (MRT) was 23 +/- 6 hr. Since probenecid has been known as a potent inhibitor of renal tubular excretion of acidic drugs and highly binds to plasma proteins, our observation in relation to plasma protein binding and PK parameters will serve as the basic information concerning drug-drug interactions in dogs and in other mammalian species.

Killing of feline T-lymphocytes by gamma-rays and energetic carbon ions.

High linear energy transfer (LET) heavy charged particles have previously been applied clinically to human cancer radiotherapy because of their excellent physical properties of selective dose distribution and higher relative biological effectiveness (RBE) for human; however, such an approach has yet to be applied to cat patients. The present study investigates the biological effectiveness of low-LET gamma-rays (0.2 keV/micro m) compared to high-LET carbon ions (114 keV/micro m) in feline T- lymphocyte FeT-J cells. Clonogenic survival analysis revealed that the RBE value of carbon ions was 2.98 relative to a 10% survival dose (D(10)) by gamma-rays, and that the inactivation cross-section in cells exposed to gamma-rays and carbon ions was 0.023 and 38.9 micro m(2), respectively. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) analysis revealed that TUNEL-positive frequency in carbon-irradiation cells is higher than for gamma-irradiated cells against exposure to the same physical doses, but that very little difference in TUNEL-positive frequency is observed between cells exposed to the respective D(10) dose of gamma-rays. Our data thus indicate that carbon ions are more effective for cell killing than gamma-rays at the same physical doses, but kill cells to an extent that is comparable to gamma-rays at the same biological doses. Carbon ion radiotherapy is therefore a promising modality for cat patients.

Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation.

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.

Detection of radiation-induced apoptosis using the comet assay.

The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.

The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay.

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by gamma-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells.

Clearance rates of biotinylated ferritins in canine: a possible physiological role of canine serum ferritin as an iron transporter.

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T 1/2) of liver ferritin (H/L subunit ratio = 0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L = 3.69) was 9.3 to 25.0 min. T 1/2 of biotinylated canine liver ferritin was independentof iron content, whereas canine heart apoferritin (T 1/2 = 31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T 1/2 = 153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T 1/2 = 26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration.

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The Km value obtained from iron uptake kinetics was 4.5 microM, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.

Detection of DNA damage in individual cells induced by heavy-ion irradiation with an non-denaturing comet assay.

Investigating the biological effects of high-LET heavy-ion irradiation at low fluence is important to evaluate the risk of radiation in space. It is especially necessary to detect radiation damage induced by a precise number of heavy ions in individual cells. We thus compared the number of ions traversing a cell and the DNA damage produced by ion hits. We applied a comet assay to measure the DNA damage in individual cells. Cells attached on ion-track detector (CR-39) were irradiated with 17.3 MeV/u 12C, 15.7 MeV/u, 10.4 MeV/u 20Ne ion and 7.2 MeV/u 40Ar beams at TIARA, JAERI-Takasaki. After irradiation, CR-39 was covered with 1% agarose. The agarose was allowed to solidify on a glass slide, and then the electrophoresis was performed. Afterward, the CR-39 was taken off the glass slide. The agarose gel on the CR-39 was stained with ethidium bromide and the opposite side of the CR-39 was etched with a KOH-ethanol solution at 37 degrees C. We observed that heavy ions with higher LET values induced heavier DNA damage, even with the same number of ion hits within the irradiated cells. The result indicated that the amount of DNA damage induced by one particle depended on the LET value of the heavy ions.