Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

Radiation-induced bystander effect in large Japanese field mouse (Apodemus speciosus) embryonic cells.

Although evidence suggests that ionizing radiation can induce the bystander effect (radiation-induced bystander effect: RIBE) in cultured cells or mouse models, it is unclear whether the effect occurs in cells of wild animals. We investigated medium-mediated bystander micronucleus (MN) formation and DNA damage in un-irradiated cells from a large Japanese field mouse (Apodemus speciosus). We isolated four clones of A. speciosus embryonic fibroblasts (A603-1, A603-2, A603-3, and A603-4) derived from the same mother, and examined their radiation sensitivity using the colony-forming assay. A603-3 and A603-4 were similar, and A603-1 and A603-2 were highly sensitive compared with A603-3 and A603-4. We examined RIBE in the four clones in autologous medium from cell cultures exposed to 2 Gy X-ray radiation (irradiated cell conditioned medium: ICCM). We only observed increased MN prevalence and induction of DNA damage foci in A603-1 and A603-3 cells after ICCM transfer. The ICCM of A603-3 (RIBE-induced) was able to induce MN in A603-4 (not RIBE-induced). To assess the possible contribution of reactive oxygen species (ROS) or nitric oxide (NO) in medium-mediated RIBE, dimethyl sulfoxide (DMSO; a ROS scavenger) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; an NO scavenger) were added to the medium. A suppressive effect was observed after adding DMSO, but there was no effect after treatment with c-PTIO. These results suggest that an enhanced radiosensitivity may not be directly related to the induction of medium-mediated RIBE. Moreover, ROS are involved in the transduction of the RIBE signal in A. speciosus cells, but NO is not. In conclusion, our results suggest that RIBE may be conserved in wild animals. The results contribute to better knowledge of radiation effects on wild, non-human species.

Optimizing chemical-induced premature chromosome condensation assay for rapid estimation of high-radiation doses.

In the event of exposure to high doses of radiation, prompt dose estimation is crucial for selecting appropriate treatment modalities, such as cytokine therapy or stem cell transplantation. The chemical-induced premature chromosome condensation (PCC) method offers a simple approach for such dose estimation with significant radiation exposure, but its 48-h incubation time poses challenges for early dose assessment. In this study, we optimized the chemical-induced PCC assay for more rapid dose assessment. A sufficient number of PCC and G2/M-PCC cells were obtained after 40 h of culture for irradiated human peripheral blood up to 20 Gy. By adding caffeine (final concentration of 1 mM) at 34 h from the start of culture, G2/M-PCC index increased by 1.4-fold in 10 Gy cultures. There was also no significant difference in the G2/M-PCC ring frequency induced for doses 0 to 15 Gy between our 40-h caffeine-supplemented chemical-induced PCC method and the conventional 48-h PCC assay.

Manual Scoring with Shortened 48 h Cytokinesis-Block Micronucleus Assay Feasible for Triage in the Event of a Mass-Casualty Radiation Accident.

The cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNCs) to estimate ionizing radiation dose exposed. Despite the faster and simpler MN scoring, CBMN assay is not commonly recommended in radiation mass-casualty triage as human peripheral blood is typically cultured for 72 h. Furthermore, CBMN assay evaluation in triage often uses high-throughput scoring with expensive and specialized equipment. In this study, we evaluated the feasibility of a low-cost method of manual MN scoring on Giemsa-stained slides in shortened 48 h cultures for triage. Both whole blood and human peripheral blood mononuclear cell cultures were compared for different culture periods and Cyt-B treatment [48 h (24 h at Cyt-B); 72 h (24 h at Cyt-B); 72 h (44 h at Cyt-B)]. Three donors (26-year-old female, 25-year-old male, 29-year-old male) were used for dose-response curve construction with radiation-induced MN/BNC. Another 3 donors (23-year-old female, 34-year-old male, 51-year-old male) were used for triage and conventional dose estimation comparison after 0, 2 and 4 Gy X-ray exposure. Our results showed that despite lower percentage of BNC in 48 h than 72 h cultures, sufficient BNCs were obtained for MN scoring. Triage dose estimates of 48 h cultures were obtained in 8 min in non-exposed donors, and 20 min in 2 or 4 Gy exposed donors with manual MN scoring. One hundred BNCs could be scored for high doses instead of 200 BNCs for triage. Furthermore, observed triage MN distribution could be preliminarily used to differentiate 2 and 4 Gy samples. The number of BNCs scored (triage or conventional) also did not affect dose estimation. Dose estimates in 48 h cultures were also mostly within ±0.5 Gy of actual doses, thus showing the feasibility of manual MN scoring in the shortened CBMN assay for radiological triage applications.

Exosome-like vesicles released from ob/ob mouse adipose tissue enhance cell survival of cells with radiation-induced genomic instability.

Multiple epidemiological studies have shown that obesity is a serious risk factor for cancer development. While the underlying mechanisms between obesity and cancer are still unknown, obesity disrupts the role of adipocytes in energy homeostasis, and the alteration of adipokine, insulin and sex steroid signaling. Recently, it has been identified that adipose tissue-derived exosome-like vesicles (ELVs) regulate metabolic homeostasis. In this study, we collected ELVs from adipose tissue of an obese mouse (ob/ob) strain and control mouse (C57BL/6) strain, and checked whether adipose ELVs influence radiation-induced cell death on mouse fibroblast cells (m5S). Furthermore, we analyzed the micronucleus (MN) frequency in survived cells after radiation exposure to investigate the effect of ELVs on radiation-induced genomic instability. We first observed that ELVs from control and obese mice showed enhanced colony forming ability in un-irradiated m5S cells. However, enhanced survival was observed only in 3 Gy-irradiated m5S cells with obese ELV treatment. Despite no ELV effect on colony size, interestingly, the frequency of MN in survived m5S cells after 3 Gy irradiation was elevated when treated obese ELVs compared to control ELVs. These results suggested that obese mouse adipose ELVs could enhance the survival of irradiated cells harboring increased radiation-induced genomic instability.

Crucial role of c-Myc in the generation of induced pluripotent stem cells.

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.

Activation of recombinational repair in Ewing sarcoma cells carrying EWS-FLI1 fusion gene by chromosome translocation.

Chromosome translocation (TL) is an important mode of genomic changes underlying human tumorigenesis, the detailed mechanisms of which are, however, still not well understood. The two major modalities of DNA double strand break repair, i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ), have been hypothesized. In a typical TL+ human neoplasm, Ewing sarcoma, which is frequently associated with t(11;22) TL encoding the EWS-FLI1 fusion gene, NHEJ has been regarded as a model to explain the disease-specific TL. Using comprehensive microarray approaches, we observed that expression of the HR genes, particularly of RAD51, is upregulated in TL+ Ewing sarcoma cell lines, WE-68 and SK-N-MC, as in the other TL+ tumor cell lines and one defective in DNA mismatch repair (MMR). The upregulated RAD51 expression indeed lead to frequent focus formation, which may suggest an activation of the HR pathway in these cells. Furthermore, sister chromatid exchange was frequently observed in the TL+ and MMR-defective cells. Intriguingly, ionizing irradiation revealed that the decrease of 53BP1 foci was significantly retarded in the Ewing sarcoma cell lines, suggesting that the NHEJ pathway may be less active in the cells. These observations may support an HR involvement, at least in part, to explain TL in Ewing sarcoma.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability.

PURPOSE: To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (Apodemus speciosus). MATERIALS AND METHODS: A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 µGy/d (Hirosaki), 14.4 ± 2.4 µGy/d (Tanashio), 208.8 ± 31.2 µGy/d (Ide), 470.4 ± 93.6 µGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones. RESULTS: Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas. CONCLUSIONS: Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Assessment of chromosome aberrations in large Japanese field mice (Apodemus speciosus) in Namie Town, Fukushima.

PURPOSE: After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in Japan on March 11 2011, the surroundings became contaminated with radionuclides. To understand the possible biological effects after chronic low dose-rate radiation in contaminated areas of Fukushima, we assessed the effects in large Japanese field mice (Apodemus speciosus) by means of chromosome aberration analysis. MATERIALS AND METHODS: We collected A. speciosus in five sites around Namie Town, Fukushima (contaminated areas) and in two sites in Hirosaki City, Aomori (control areas, 350 km north of FDNPP) from autumn 2011 to 2013. The number of mice captured and ambient dose-rates were as follows: high (n = 11, 10.1-30.0 µGy h-1), moderate (n = 10, 5.7-15.6 µGy h-1), low (n = 12, 0.23-1.14 µGy h-1) and control (n = 20, 0.04-0.07 µGy h-1). After spleen extraction from rodents, spleen cell culture was performed to obtain metaphase spreads. Chromosome aberrations were assessed on Giemsa-stained metaphase spreads. RESULTS: Although the mice in the contaminated areas were chronically exposed, there was no radiation-specific chromosome aberrations observed, such as dicentric chromosomes and rings. Some structural aberrations such as gaps and breaks were observed, and these frequencies decreased annually in mice from Namie Town. CONCLUSION: These findings suggest that chromosome aberration analysis is useful to evaluate and monitor radiation effects in wild animals.

Biological Dosimetry by the Triage Dicentric Chromosome Assay - Further validation of International Networking.

Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific dicentric chromosomes in exposed individuals' peripheral blood lymphocytes. However, this method is labour-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the dicentric chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labour-demanding and time-consuming metaphase-spread analysis to 20 to 50 metaphase spreads instead of routine 500 to 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.

Cytokinesis-block micronucleus assay performed in 0 and 2 Gy irradiated whole blood and isolated PBMCs in a six-well transwell co-culture system.

PURPOSE: Cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNC) for dose estimation. Cell-cycle progression parameters of nuclear division index (NDI) and percentage of BNC (% BNC) are also evaluated. Whole blood (WB) or peripheral mononuclear cells (PBMCs) isolated from WB can be used for lymphocyte culture. Previously, 2 Gy PBMCs showed higher NDI and lower MN frequency than WB in 15 ml polypropylene tube single cultures. In this follow-up study, we wanted to assess if soluble factors present in WB but absent in PBMCs could increase MN frequency or decrease NDI in PBMCs co-cultured with WB. MATERIALS AND METHODS: Peripheral blood from four healthy donors (two males: 25, 51; two females: 23, 26 years old) was irradiated with X-ray at 1 Gy/min. CBMN assay was performed with different combinations of 0 and 2 Gy WB and PBMC (WB, WB-IR, PBMC, PBMC-IR) mono- and co-cultures in a polystyrene six-well plate. Co-cultures were separated by 0.4 µm transwell inserts. Log2 fold changes and values of NDI, % BNC and MN frequency analyzed by three scorers were obtained. RESULTS: As upper and lower wells of the same culture condition showed some significant differences, wells of the same level were compared. NDI of PBMCs increased when PBMC or PBMC-IR was co-cultured with WB or WB-IR, respectively, as compared to mono-cultures. There was no increase in PBMC-IR's MN frequency when co-cultured with WB or WB-IR. MN frequency was consistently higher in WB-IR than PBMC-IR in both mono- and co-cultures. NDI, % BNC and MN frequency were similar when WB or PBMC were co-cultured with PBMC-IR or WB-IR, respectively. Significantly lower NDI and % BNC, and higher MN frequency were also seen in some conditions of 15 ml cultures than six-well mono-cultures. CONCLUSIONS: Instead of the hypothesized decrease in NDI and increase in MN frequency, our co-culture set-up showed that in the absence of direct cell-cell interaction, soluble factors in WB increased NDI but not MN frequency in PBMCs. Moreover, radiation-induced bystander effects could not be observed. As the type of cell culture (WB, PBMC) and culture vessels could influence NDI and MN frequency, CBMN culture protocols should be kept consistent for dose-response calibration curve construction and dose estimation.

Extracellular vesicles released from irradiated neonatal mouse cheek tissue increased cell survival after radiation.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

Detailed chromosome analysis of wild-type, immortalized fibroblasts with SV40T, E6E7, combinational introduction of cyclin dependent kinase 4, cyclin D1, telomerase reverse transcriptase.

Cell immortalization enables us to expand the cultured cell infinitely. However, the process of immortalization sometimes changes the nature of the original cell. In this study, we established immortalized embryonic fibroblasts with oncogenic SV40T and human papilla virus-derived E6E7, combinational expression of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomerase reverse transcriptase (TERT) from identical primary wild-type human embryonic fibroblasts (HE16). After the establishment of immortalized cells, we compared the details of chromosome condition with the G-banding and Q-banding methods. There is no example of detailed analysis so far about chromosome abnormalities, such as trisomy, ring chromosome, reciprocal translocation, and dicentric chromosomes. The detailed chromosome analysis revealed that immortalized cells with SV40T and E6E7 showed intensive chromosome abnormalities, such as gain or loss of the chromosomes all through the genome. Furthermore, we detected that the incidence of chromosome abnormities in the immortalized cell with the combinational introduction of R24C mutant of CDK4, cyclin D1, and TERT is almost identical to that of wild-type cell. Furthermore, short tandem repeat analysis demonstrated that the origin of K4DT cell is primary HE16. These results showed that cellular immortalization with CDK4, cyclin D1, and TERT is more advantageous in keeping the chromosome's original condition than oncogenic immortalization methods.

Transition of Radioactive Cesium Deposition in Reproductive Organs of Free-Roaming Cats in Namie Town, Fukushima.

We investigated the internal contamination by radioactive cesium associated with the FDNPP accident, in the testes or uterus and ovaries of free-roaming cats (Felis silvestris catus), which were protected by volunteers in the Namie Town, Fukushima. A total of 253 samples (145 testes and 108 uterus and ovaries) obtained from adult cats and 15 fetuses from 3 pregnant female cats were measured. Free-roaming cats in Namie Town had a higher level of radioactive contamination in comparison to the control group in Tokyo, as the 134Cs + 137Cs activity concentration ranged from not detectable to 37,882 Bq kg-1 in adult cats. Furthermore, the radioactivity in the fetuses was almost comparable to those in their mother's uterus and ovaries. The radioactivity was also different between several cats protected in the same location, and there was no significant correlation with ambient dose-rates and activity concentrations in soil. Moreover, radioactive cesium levels in cats decreased with each year. Therefore, it is likely that decontamination work in Namie Town and its surroundings could affect radioactive cesium accumulation, and thus possibly reduce the internal radiation exposure of wildlife living in contaminated areas. It is hence necessary to continue radioactivity monitoring efforts for the residents living in Namie Town.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay.

PURPOSE: In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. MATERIALS AND METHODS: Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy - background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. RESULTS: >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. CONCLUSION: This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

WHO 1st consultation on the development of a global biodosimetry laboratories network for radiation emergencies (BioDoseNet).

The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17-18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agencies that provide international cooperation in biological dosimetry after radiological emergencies were reviewed. In addition, extensive reliance on the use of the relevant International Standards Organization (ISO) standards was emphasized. The results of a WHO survey of global cytogenetic biological dosimetry capability were reported, and while the survey indicates robust global capability, there was also a clear lack of global leadership and coordination. The expert group, which had a concentrated focus on cytogenetic biodosimetry, formulated the general scope and concept of operations for the development of a WHO global biodosimetry laboratory network for radiation emergencies (BioDoseNet). Follow-on meetings are planned to further develop technical details for this network.

Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT examinations in eight patients.

In our previous study, we found that chromosomes were damaged by the radiation exposure from a single computed tomography (CT) examination, based on an increased number of dicentric chromosomes (Dics) formed in peripheral blood lymphocytes after a CT examination. We then investigated whether a cumulative increase in the frequency of Dics and chromosome translocations (Trs) formation could be observed during three consecutive CT examinations performed over the course of 3-4 years, using lymphocytes in peripheral bloods of eight patients (five males and three females; age range 27-77 years; mean age, 64 years). The effective radiation dose per CT examination estimated from the computational dosimetry system was 22.0-73.5 mSv, and the average dose per case was 40.5 mSv. The frequency of Dics formation significantly increased after a CT examination and tended to decrease before the next examination. Unlike Dics analysis, we found no significant increase in the frequency of Trs formation before and after the CT examination, and we observed no tendency for the frequency to decrease before the next CT examination. The frequency of Trs formation was higher than that of Dics formation regardless of CT examination. Furthermore, neither analysis of Dics nor Trs showed a cumulative increase in the frequency of formation following three consecutive CT examinations.

Construction of fluorescence in situ hybridization (FISH) translocation dose-response calibration curve with multiple donor data sets using R, based on ISO 20046:2019 recommendations.

Purpose: Dose-response curve (DRC) generation is an important aspect in cytogenetic biodosimetry for accurate dose estimation for individuals suspected of prior irradiation. DRC construction with dicentric chromosomes after acute radiation is well-established following the publication of the IAEA EPR-Biodosimetry 2011 and ISO 19238:2014. However, the short half-life of dicentrics might not be suitable for retrospective dose estimation in radiation medical workers, radiation accident clean-up workers and the general public living in areas with higher than average amount of radiation. There is an urgent need for a chromosome translocation-based DRC, which is constructed based on translocation identification with fluorescence in situ hybridization (FISH). Despite several attempts to generate such a DRC in the past 40 years, no internationally standardized protocol has been developed until 2019, resulting in possible statistical uncertainties between DRCs previously generated.Materials and methods: Using the recently published ISO 20049:2019, a DRC from five healthy donors (four males: 23, 35, 44, 55 years old, one female: 33 years old) was generated with age-adjusted translocations scored per cell equivalent (age-adjusted Tr/CE), using a modified R-script previously published in EPR-Biodosimetry, for 60Co gamma-ray doses of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 Gy. The translocation data set used, based on probes used for chromosomes number 1, 2, and 4, was previously published by Abe et al. in 2018.Results: The results output from R include the DRC coefficients (C, α, ß), their p-values, the goodness-of-fit Pearson's chi square value and its corresponding p-value, and the DRC with its 95% confidence interval (CI). The equation of the DRC obtained was 0.0005 (±0.0001) +0.0178 (±0.0037) D + 0.0901 (±0.0054) D2. DRC generated with averaged Tr/CE had a wider 95% CI than DRC generated with pooled Tr/CE, resulting in a 1.3-1.5 times increase in estimated dose range. No outliers between α coefficients from previously published modified DRCs and our DRC were detected with robust Z-score.Conclusions: ISO 20046:2019 should be referenced for future FISH translocation-based DRC generation to ensure statistical reliability of dose estimation. Important considerations for FISH translocation-based DRC up to 1 Gy include scoring more than 2000 CE per dose, the use of multiple donors, age-adjustment of observed translocations, the use of a minimum of 5 dose points including 0 Gy, scoring of total simple translocations in only stable cells and the decision of using pooled or averaged age-adjusted Tr/CE.

Influence of anticoagulants and storage temperatures on blood counts and mitotic index of blood samples collected for cytogenetic biodosimetry.

PURPOSE: In order to establish suitable protocols of blood culture to obtain sufficient numbers of metaphases for dicentric chromosome assay (DCA), we have examined the effect of storage temperature, storage time, and anticoagulant type. MATERIALS AND METHODS: Peripheral blood was collected from five healthy donors with lithium heparin and ethylenediaminetetraacetic acid dipotassium salt (EDTA-2K). These samples were irradiated with X-rays at 3 Gy or sham; the samples were further divided into groups that were either stored at room temperature (RT) or 5.2 ± 1.0 °C. After 6, 24, 48, 72, and 168 h of storage, both blood counts and the mitotic index (MI) were analyzed. RESULTS: Heparinized blood samples stored under cold conditions exhibited low white blood cell, lymphocyte, and platelet counts. EDTA-treated blood samples did not show such obvious changes in cell counts. After 6 h of storage, heparinized blood samples stored at RT had MI of 21.5-29.3%. Similar MI was obtained in the EDTA-washed group stored for 6, 24, 48, and 72 h. CONCLUSIONS: Our study confirms that heparinized blood samples should be stored at RT to get sufficient metaphases for DCA, and that EDTA blood samples also can be used for blood culture after washing and storage under 5.2 ± 1.0 °C.

Precision of scoring radiation-induced chromosomal aberrations and micronuclei by unexperienced scorers.

Purpose: Dose assessment plays an important role in case of radiological accidents and can be performed by scoring structural changes of chromosome morphology induced in cells by ionizing radiation. The results of such a test are biased by scorer experience, therefore, simple to learn assays are recommended to be used when fast analysis of a large amount of data is needed. The aim of this study was to compare the performance of two radiobiological assays - chromosomal aberrations and micronuclei - by unexperienced scorers with the reference values generated by an expert. Materials and methods: Each participant of an EU-funded two-week radiobiology course was asked to score Chinese hamster ovary cells exposed to gamma radiation up to 4 Gy. The congruence of students' and expert's scores at each dose and the coherence of the dose-response curve parameters between the students were investigated. Results: Micronucleus test tended to be faster and easier to learn than scoring chromosomal aberrations. However, both assays carried out by inexperienced students showed reasonable dose-response curves. Conclusions: In the case of a large radiological accident involving many casualties, the unexperienced scorers would support the process of biodosimetric triage by cytogenetic biological dosimetry.