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.

Vibratome technique revealed initial carcinogenic changes that induce GST-P+ single hepatocytes and minifoci in rat liver.

The drastic initial carcinogenic changes that induce single hepatocytes and minifoci positive for GST-P (a specific biomarker of foci and nodules) identified previously in rat livers (K. Satoh, Life Sci. 2018) require elucidation. Notably, after animals were administered benzyl isothiocyanate (BITC, anti-cancer phytochemical, 0.5% by wt) in their basal diet, immunocytochemical staining of vibratome-prepared liver specimens for GST-P revealed that the canalicular networks and bile ducts of the animal livers were heavily and finely stained for GST-P even though the biomarker is a cytosolic enzyme. In addition, the mean diameter of the canaliculi was greatly enlarged. The results thus indicate that GST-P was rapidly synthesized in all hepatocytes but rapidly excreted into bile. Similar results were obtained with animals administered dietary AAF carcinogen (0.04%). The biliary excretion of GST-P was detectable not only in all hepatocytes but also within minifoci, foci and nodules. A new initiation model was therefore proposed assuming that GST-P+ single hepatocytes are formed after injury to canaliculi by carcinogens to decrease the excretion of GST-P from hepatocytes. The key findings from this study and the biomarker analysis using a vibratome technique might help elucidate the 'unknowable' mechanism of cancer initiation in rat chemical carcinogenesis.

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.

Chondroitin sulfate is not digested at all in the mouse small intestine but may suppress interleukin 6 expression induced by tumor necrosis factor-α.

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract.

Mitotic index maximization with no effect on radiation-induced dicentric chromosome frequency.

PURPOSE: The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency. MATERIALS AND METHODS: Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 µg/ml) and demecolcine　(0.05 µg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures. RESULTS: In WB-culture, MI showed the highest value (∼22%) in 5-15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose of ex vivo X-ray-irradiated blood. CONCLUSION: While MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.

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.

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.

Antimicrobial properties of L-amino acid oxidase: biochemical features and biomedical applications.

Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. KEY POINTS: •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

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.

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.

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.

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.

Metal coordination by L-amino acid oxidase derived from flounder Platichthys stellatus is structurally essential and regulates antibacterial activity.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis.

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked ß-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.

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.

Radiation-Induced Bystander Effect is Mediated by Mitochondrial DNA in Exosome-Like Vesicles.

Exosome-like vesicles (ELV) are involved in mediating radiation-induced bystander effect (RIBE). Here, we used ELV from control cell conditioned medium (CCCM) and from 4 Gy of X-ray irradiated cell conditioned medium (ICCM), which has been used to culture normal human fibroblast cells to examine the possibility of ELV mediating RIBE signals. We investigated whether ELV from 4 Gy irradiated mouse serum mediate RIBE signals. Induction of DNA damage was observed in cells that were treated with ICCM ELV and ELV from 4 Gy irradiated mouse serum. In addition, we treated CCCM ELV and ICCM ELV with RNases, DNases, and proteinases to determine which component of ELV is responsible for RIBE. Induction of DNA damage by ICCM ELV was not observed after treatment with DNases. After treatment, DNA damages were not induced in CCCM ELV or ICCM ELV from mitochondria depleted (ρ0) normal human fibroblast cells. Further, we found significant increase in mitochondrial DNA (mtDNA) in ICCM ELV and ELV from 4 Gy irradiated mouse serum. ELV carrying amplified mtDNA (ND1, ND5) induced DNA damage in treated cells. These data suggest that the secretion of mtDNA through exosomes is involved in mediating RIBE signals.

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.

Age Dependence of Radiation-Induced Genomic Instability in Mouse Hematopoietic Stem Cells.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia. Accumulating evidence suggests that ionizing radiation can induce genomic instability and promote leukemogenesis in hematopoietic stem cells (HSCs); however, the influence of age on this phenomenon has not been elucidated. In this study, infant (1-week-old) or adult (14-week-old) C3H/He mice received sham or 4 Gy whole-body irradiation, and bone marrow cells were transplanted to recipients at day 1 or 60 postirradiation. Twelve days after bone marrow transplant, we analyzed the radiation-induced genomic instability by scoring the frequency of DNA damage and micronucleus formation in colony-forming units-spleen (CFU-Ss). We observed significant increases in DNA damage and micronucleus formation in CFU-Ss of the 4 Gy irradiated adult cells transplanted at day 1 or 60 postirradiation. However, the frequency of DNA damage focus and micronucleus formation in CFU-Ss of 4 Gy irradiated infant cells transplanted at day 1 or 60 postirradiation was relatively decreased. Quantitative differences in the reactive oxygen species and cells expressing inducible nitric oxide synthase in CFU-Ss suggested that age-dependent radiation-induced genomic instability may result from chronic oxidative stress by pro-inflammatory states in HSC descendants after radiation exposure.