Anti-CENP-C Antibody-Based Immunofluorescence Dicentric Assay: Radiation Dose-Response, Validation Studies, and Radiation Dose-Dependency on Sister Centromere Fluorescence.

Dicentric chromosome assay (DCA) is the most accepted cytological technique for the purpose of biological dosimetry in radiological and nuclear accidents, however, it is not always easy to evaluate dicentric chromosomes because of the technical difficulty in identifying dicentric chromosomes on Giemsa-stained metaphase chromosome samples. Here, we applied an antibody recognizing centromere protein (CENP) C, CENP-C, whose antigenicity is resistant to the fixation with Carnoy's solution. Normal human diploid cells were irradiated with various doses of 137Cs γ rays at 1 Gy/ min, treated with hypotonic solution, fixed with Carnoy's fixative, and metaphase chromosome spreads were stained with anti-CENP-C antibody. Dose-dependent induction of dicentric chromosomes was confirmed between 1 and 10 Gy of γ rays, and the results were compatible with those obtained by the conventional Giemsa-stained chromosome samples. The CENP-C assay also uncovered the difference in the fluorescence from the sister centromeres on the same chromosome, which was more pronounced after radiation exposure. Although the underlying mechanism is still to be determined, the result suggests a novel effect of radiation on centromeres. The innovative protocol for CENP-C-based DCA, which enables ideal visualization of centromeres, is simple, effective and reliable. It does not require skilled examiners, so that it may be an alternative method, avoiding uneasiness of the current DCA using Giemsa-stained metaphase chromosome samples.

Characterization of radiation-induced micronuclei associated with premature senescence, and their selective removal by senolytic drug, ABT-263.

Radiotherapy is well-recognized as an efficient non-invasive remedy for cancer treatment. Since 10 Gy, a weekly total dose for conventional radiotherapy, was proven to create unreparable and residual DNA double-strand breaks (DSBs), they were found to give rise to mitotic failure, such as mitotic catastrophe, which resulted in multiple micronuclei associated with premature senescence. We demonstrated that pulverization of micronuclear DNA was caspase-dependent and triggered not ATM-dependent but DNA-PK-dependent DNA damage response, including phosphorylation of histone H2AX. Pulverization of micronuclear DNA and senescence-associated secretory phenotype (SASP) worsen tumor microenvironment after radiotherapy, so that senolytic drug was applied to eliminate senescent cancer cells. Prematurely senescent cancer cells with micronuclei caused by 10 Gy of γ-irradiation were subjected to 5 µM of ABT-263, a Bcl-2 family inhibitor, and selective cancer cell death by apoptosis was observed, while ABT-263 had little effect on growing cancer cells. Western blot analysis showed augmented expression of both apoptotic and anti-apoptotic proteins in senescent cells, indicating that increased apoptotic factors are essential for selective apoptotic cell death in combination with ABT-263. Our results suggested that selective elimination of senescent cells alleviates SASP and micronuclei-mediated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation, both of which lead to unfavorable adverse effects caused by radiotherapy.

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.

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.

Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals.

In terms of biological dosimetry at the time of radiation exposure, the dicentric chromosome (Dic) assay (DCA) is the gold standard for assessing for the acute phase and chromosome translocation (Tr) analysis is the gold standard for assessing the chronic phase. It is desirable to have individual dose-response curves (DRCs) for each laboratory because the analysis criteria differ between laboratories. We constructed the DRCs for radiation dose estimation (with three methods) using peripheral blood (PB) samples from five healthy individuals. Aliquots were irradiated with one of eight gamma-ray doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy), then cultured for 48 h. The number of chromosome aberrations (CAs) was analyzed by DCA, using Giemsa staining and centromere-fluorescence in situ hybridization (centromere-FISH) and by chromosome painting (chromosome pairs 1, 2 and 4) for Tr analysis. In DCA, there was large variation between individuals in the frequency of Dics formed, and the slopes of the DRCs were different. In Tr analysis, although variation was observed in the frequency of Tr, the slopes of the DRCs were similar after adjusting the background for age. Good correlation between the irradiation dose and the frequency of CAs formed was observed with these three DRCs. However, performing three different biological dosimetry assays simultaneously on PB from five donors nonetheless results in variation in the frequency of CAs formed, especially at doses of 50 mGy or less, highlighting the difficulty of biological dosimetry using these methods. We conclude that it might be difficult to construct universal DRCs.

Analysis of chromosome translocation frequency after a single CT scan in adults.

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.

Increase in dicentric chromosome formation after a single CT scan in adults.

Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed dicentric chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.