Journal of Radiation Research, Vol. 59, No. 1, 2018, pp. 35–42 doi: 10.1093/jrr/rrx052 Advance Access Publication: 13 October 2017 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 1 2 3 1 1 Yu Abe , Mitsuaki A. Yoshida , Kurumi Fujioka , Yumiko Kurosu , Risa Ujiie , 1 1 4 3 Aki Yanagi , Naohiro Tsuyama , Tomisato Miura , Toshiya Inaba , 5 1, Kenji Kamiya and Akira Sakai Department of Radiation Life Sciences, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima, 960–1295, Japan Department of Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University, Hirosaki, 036-8564, Japan Department of Molecular Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553, Japan Department of Pathologic Analysis, Hirosaki University Graduate School of Health Sciences, Hirosaki, 036-8564, Japan Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan *Corresponding author. Department of Radiation Life Sciences, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima, 960–1295, Japan. Tel: +81-24-547-1420; Fax: +81-24-547-1940; Email: email@example.com (Received 22 May 2017; revised 22 July 2017; editorial decision 26 August 2017) ABSTRACT 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 radi- ation 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 (chromo- some 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 correl- ation 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 none- theless 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. Keywords: dicentric chromosome; chromosome translocation; dose–response curve; biological dosimetry; Giemsa staining; centromere-FISH © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re- use, please contact firstname.lastname@example.org � 35 Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 36 � Y. Abe et al. INTRODUCTION Gamma-ray irradiation and lymphocyte culture Several methods have been reported for rapid biological dosimetry PB samples were irradiated with gamma-rays (Gamma cell 40, Best immediately following exposure to low and high doses of radiation Theratronics, Ottawa, Ontario, Canada; installation date: March, [1–8], of which the most reliable for international standardized bio- 2009.) at eight doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy). logical dosimetry is the dicentric chromosome (Dic) assay (DCA) Plastic microtubes containing whole blood were irradiated at a distance . DCA is typically used following acute radiation exposure of of 16 cm at room temperature with gamma-rays from a Co radiation between 100 mGy and 5 Gy, although recent studies report that source (1.11 TBq) at a dose rate of 26.26t (time: min) + 6.42 mGy chromosomal abnormalities such as Dics can be detected following per min, where 26.26 is the dose rate and 6.42 is the dose to the sam- chronic or low-dose radiation exposure [10–13]. In vitro experi- ple entering and leaving the irradiation source. The doses were mea- ments using human peripheral blood (PB) lymphocytes revealed sured using an ionization chamber detector for gamma-rays. that the irradiation dose and chromosome aberration frequency cor- Mononuclear blood cells were isolated from heparinized PB sam- relate down to 20 mGy . However, there is no useful method ples using BD Vacutainer CPT tubes (BD Biosciences, San Jose, CA, speciﬁcally designed for detecting chromosomal abnormalities fol- USA) according to the manufacturer’s instructions. Cells were sus- lowing exposure to doses of 100 mGy or less, and the accuracy of pended in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) con- estimation methods using the dose–response curves (DRCs) follow- taining 20% fetal bovine serum (Equitech Bio, Keilor East, Australia), ing exposure to the low doses remains unclear [15–18]. 2% phytohaemagglutinin-HA15 (Remel, Lenexa, KS, USA) and Furthermore, a DRC based on a sample from a single healthy indi- 60 μg/ml of kanamycin solution (Life Technologies, Carlsbad, CA, vidual is likely to be inadequate for estimating the exposure of a USA) in a 6-well plate. Lymphocytes were cultured in a 5% humidiﬁed large number of people following a radiation exposure disaster. The CO incubator at 37°C for 48 h. Colcemid solution (Wako, Osaka, DCA for biological dosimetry recommended by the International Japan) was added (ﬁnal concentration: 0.015–0.02 μg/ml) 2 h before Atomic Energy Agency (IAEA) is affected by gender and age, as cell harvest, then chromosome preparations were made according to a veriﬁed by the construction and comparison of DRCs by several standard cytogenetic procedure . laboratories [19, 20]. Chromosome translocation (Tr) analysis is strongly affected by factors such as age and smoking , again highlighting the problem of constructing a DRC based on a sample Staining from a single healthy individual. In this study, we attempted to con- Each slide was stained using three methods. Giemsa staining was struct a DRC compatible with estimating a lower dose range than achieved by immersing the slide in 5% Giemsa (Merck Millipore, that which DRCs use conventionally. We therefore irradiated sam- Darmstadt, Germany) solution for 15 min, then washing with dis- ples from ﬁve healthy individuals with eight gamma-ray irradiations tilled water and air drying. Centromere-FISH staining was achieved doses from 0 to 1000 mSv. Here we present the three types of using Poseidon probe (KRATECH, Amsterdam, the Netherlands) standard DRCs compatible with three methods. The ﬁrst is a clas- according to the manufacturer’s protocol, with slight modiﬁcations sical method for DCA, Giemsa staining. The second is the described previously . Chromosome painting was achieved centromere-ﬂuorescence in situ hybridization (centromere-FISH) using a Customized XCP-Mix probe (Mix-#1R-#2G-#4RG; MetaSystems, method, which likely provides higher accuracy than Giemsa staining. Altlussheim, Germany) according to the manufacturer’sprotocol, The third is a painting method for chromosome translocation (Tr) as in our previous study . Brieﬂy, nuclear DNA was denatured analysis using three probes (one each for chromosome pairs 1, 2 by incubating the slides on a hot plate at 75°C for 2 min, followed and 4), which can estimate a radiation exposure dose received sev- by incubation overnight at 37°Cinahumidiﬁed chamber to allow eral years earlier. We also comment on the difﬁculty of assessing for hybridization. The glass coverslips were removed and the slides low radiation doses. were washed in 0.4 × SSC at 72°C for 2 min. After draining, the slides were then washed in 2 × SSC/0.05% Tween-20 at room temperature (RT) for 30 s. Subsequently, the slides were brieﬂy MATERIALS AND METHODS rinsed in distilled water to avoid crystal formation and then air Ethics statement dried at RT. Finally, nuclei were counterstained with Vectashield The samples and the medical records used in this study were Mounting Medium containing DAPI (Vector, Burlingame, USA), approved by the Ethics Committee of the Fukushima Medical and the slides were covered with a glass coverslip and sealed with University School of Medicine (approval number 1577). Written nail polish. informed consent was obtained from all participants for analysis of PB samples, and the methods were carried out in accordance with the approved guidelines of the Council for International Organizations of Image capture and scoring Medical Science . Giemsa images and FISH images were captured in AutoCapt mode using two sets of AXIO Imager Z2 microscopes (Carl Zeiss AG, Blood samples Oberkochen, Germany) equippedwithCCD camerasand Metafer4 PB was collected from ﬁve healthy individuals: four males (23, 35, software (MetaSystems GmbH, Altlussheim, Germany). Chromosome 44 and 55 years old) and one female (33 years old). None of the analysis was performed according to the International Atomic Energy individuals had a history of smoking and had never been subjected Agency (IAEA) manual [9, 23] by three trained, experienced observers to radiotherapy or chemotherapy for any disease. who were not informed of the irradiation dose. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dose–response curves for chromosome aberrations � 37 Chromosomal aberrations in the FISH images were conﬁrmed Table 1. Average of dicentric chromosome results from ﬁve using ﬂuorescence imaging software (Isis FISH Imaging System, ver. donors (Giemsa staining) 5.4; MetaSystems GmbH). More than 2000 metaphases were scored Dose Number of Number of Frequency of 95%CI in each Giemsa and centromere-FISH slide . All observable aberra- (mGy) cells analyzed Dics observed (Dic-frequency) tions were classiﬁed as dicentrics or multicentrometrics (chromosomes Dics with three or more centromeres). Other chromosome- or chromatid- type aberrations were also recorded, such as rings, acentrics (aces), 0 2023.4 2.8 0.001 −0.0005–0.003 breaks and gaps. Metaphases with fewer than 45 centromeres were 10 2026.4 4.2 0.002 0.0005–0.004 omitted from analysis. For translocation analysis, more than 5000 metaphases were scored on each slide . Based on a previous 20 2020.8 2.6 0.001 0.0002–0.002 report , we included apparent one-way translocations in the 50 2051.8 4 0.002 0.0007–0.003 two-way translocation counts. For complex chromosomal abnormal- ities, the number of translocations was determined based on the 100 2020.4 4 0.002 0.0006–0.003 number of color junctions (NCJs) . We also recorded other 200 2026 7.2 0.004 0.001–0.006 chromosomal aberrations, such as Dics and aces and calculated the frequency of translocations across the whole genome using formulae 500 2010.2 27.6 0.013 0.008–0.019 published by IAEA . For scoring of translocations, the formula 1000 2066.8 78 0.038 0.029–0.045 used to calculate the frequency of translocations across the whole genome (F ) was based on the formula using three colors (chromo- G Dicentric chromosomes per cell analyzed. Conﬁdence interval. some 1: red; chromosome 2: green; chromosome 4: yellow) for the detection of translocations as follows : Table 2. Average of dicentric chromosome results from ﬁve F =F /2.05[f (−1f)+f (−1 f )+f (−1f ) G P(+124 +) 1 1 2 2 4 4 donors (centromere-FISH) −(ff + f f + f f )] 12 1 4 2 4 Dose Number of Number of Frequency of 95% CI (mGy) cells analyzed Dics observed (Dic frequency) F : the full genome aberration frequency, Dics F : the translocation frequency detected by FISH, 0 2011.2 1.6 0.001 −0.00005–0.002 f : the fraction of genome hybridized, taking into account the gender of the subjects (female: f = 0.2234, male: f = 0.2271). p p 10 2015.2 2.2 0.001 0.0005–0.002 20 2018.8 3.2 0.002 0.0004–0.003 The proportion of the genome occupied by chromosomes 1, 2 and 4 is ~23%. Therefore, F is determined by the following formula: 50 2026.2 4.8 0.002 0.001–0.003 F =× F 2.567(Female) GP 100 2021 5.6 0.003 0.001–0.004 200 2026.6 12 0.006 0.004–0.008 F =× F 2.533(Male). GP 500 2023 37.6 0.019 0.013–0.024 In order to unify the cell numbers in the analysis, we determined 1000 2057 108 0.053 0.048–0.057 F in per 2000 cells equivalents, according to the above respective formula for females or males, respectively. Dicentric chromosomes per cell analyzed. Conﬁdence interval. number of Dics formed rapidly increased at irradiation doses above RESULTS Construction of dose–response curves for DCA (Giemsa 200 mGy (see Supplementary Tables 1 and 2). Based on these results, DRCs for Giemsa staining and centromere- staining and centromere-FISH) FISH were constructed using DoseEstimate ver. 4.1 software . Approximately 80 000 metaphases were analyzed for DCA: ~2000 The results from Giemsa staining were ﬁtusing theformula: Y = metaphases per dose, and ~16 000 metaphases per individual. The 0.0013 (±0.0005) + 0.0067 (±0.0071) × D + 0.0313 (±0.0091) × D average values from these analyses are shown in Tables 1 and 2. (Y: yield of chromosome aberrations, D: dose (Gy)), and the correl- Compared with Giemsa staining, the frequency of Dic formation as ation coefﬁcient (r) was 0.9985 (Fig. 1). The results from centromere- a whole was higher in DCAs using FISH, except for 0 mGy and FISH were ﬁtusing the formula: Y = 0.0010 (±0.0004) + 0.0186 10 mGy, and was higher than at 20 mGy using Giemsa staining (±0.0081) × D + 0.0329 (±0.0104) × D (Y: yield of chromosome (Table 1). Analysis of samples from each individual showed large aberrations, D:dose(Gy)),and thecorrelation coefﬁcient (r)was variation in the frequency of Dic formation at 0 mGy by both meth- 0.9998 (Fig. 2). Therefore, we constructed two types of DRCs that ods, and the increase in the number of Dics formed did not correl- strongly correlated with the irradiation dose and the number of Dics ate with the irradiation dose up to 100 mGy. In contrast, the Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 38 � Y. Abe et al. 0.07 Construction of a dose–response curve for chromosome Y = 0.0013 (± 0.0005) + 0.0067 (± 0.0071) × D + 0.0313 (± 0.0091) × D translocation analysis r = 0.9985 0.06 Approximately 200 000 metaphases were subjected to Tr analysis: ~5000 metaphases per dose, and ~40 000 metaphases per individual. 0.05 The average values from these analyses are shown in Table 3. The 0.04 average frequency of Tr at 10 mGy was higher than that at 20 mGy. Analysis of samples from each individual showed variation in the 0.03 frequency of Tr. Variation was also observed in DCA, and the increase in the frequency of Tr did not correspond to the irradiation 0.02 dose up to 50 mGy (see Supplementary Table 3). 0.01 Based on these results, the DRC for Tr analysis was constructed using DoseEstimate ver. 4.1 software . The results were ﬁt using the formula: Y = 0.0053 (±0.0009) + 0.0259 (±0.0127) × D + 0 100 200 300 400 500 600 700 800 900 1000 0.0826 (±0.0161) × D (Y: yield of chromosome aberrations, D: Dose (mGy) dose (Gy)), and the correlation coefﬁcient (r) was 0.9995 (Fig. 3a). The slopes of DRCs of the ﬁve individuals showed no difference due Fig. 1. Dose–response curve for DCA analyzed by Giemsa to age or gender (see Supplementary Fig. 3), and therefore we suc- staining. The frequencies of chromosome aberrations per ceeded in constructing a DRC that strongly correlates with the irradi- 2000 cells in PB from ﬁve individuals induced by gamma- ation dose and the frequency of Tr. However, the frequencies of Tr at ray irradiation were plotted. Regression analysis using 0 mGy were higher than those of DCA (see Supplementary Figs 1 and DoseEstimate ver. 4.1 software was calculated from the 3), and therefore the DRC did not start from zero (Fig. 3a). Since the average value of the ﬁve samples. [Y = 0.0013 (±0.0005) translocation frequency increases withage,wesubtracted thefrequency + 0.0067 (±0.0071) × D + 0.0313 (±0.0091) × D , r = of age-dependent background reported by Sigurdson et al. from 0.9985] (Y: yield of chromosome aberrations, D:dose the frequencies of Tr, thereby resulting in the DRCs starting essentially (Gy), r = correlation coefﬁcient.) at zero (Fig. 3b). The DRCs obtained for Tr analysis focusing on the low-dose range are shown in Supplementary Fig. 3c and d. 0.07 Y = 0.0010 (± 0.0004) + 0.0186 (± 0.0081) × D + 0.0329 (± 0.0104) × D DISCUSSION r = 0.9998 0.06 DCA is the most accurate biological dosimetry method for estimat- ing radiation dose in humans and is used in conjunction with phys- 0.05 ical dosimetry, such as a personal dosimeter, during radiation exposure accidents. Radiation exposure estimation from the number 0.04 of CAs, such as Dic and Tr, require the prior construction of a 0.03 DRC using the frequency of CAs formed in lymphocytes at a series of irradiation doses. Since there are some differences in sample 0.02 preparation protocols for CA analysis, it is desirable for each labora- tory to have its own DRC. We constructed three types of DRC: 0.01 one each for Giemsa staining and centromere-FISH for DCA, and one for a painting method for Tr analysis. Each curve was con- 0 100 200 300 400 500 600 700 800 900 1000 structed using lymphocytes from ﬁve healthy individuals following Dose (mGy) irradiation with eight doses of gamma-rays. The curves showed good radiation dose responsiveness. Although there were large indi- Fig. 2. Dose–response curve for DCA analyzed by vidual differences in the frequency of CAs for radiation doses of centromere-FISH. The frequencies of chromosome 100 mGy or less, dose estimation may be possible from the lower aberrations per 2000 cells in PB from ﬁve individuals limit value of the 95% conﬁdence interval (CI). induced by gamma-ray irradiation were plotted. Regression On the other hand, CAs were observed even in non-irradiated analysis using DoseEstimate ver. 4.1 software was calculated samples in both the DCA and Tr analyses, and there was no correl- from the average value of the ﬁve samples. [Y = 0.0010 ation between the frequency of CAs and the radiation dose up to (±0.0004) + 0.0186 (±0.0081) × D + 0.0329 100 mGy. Therefore, variation was observed in the frequency of (±0.0104) × D , r = 0.9998] (Y: yield of chromosome CAs, such as in the higher number of CAs formed during a low aberrations, D: dose (Gy), r = correlation coefﬁcient.) radiation dose compared with during a high radiation dose. These variances in the low-dose range of 100 mGy or less are likely due to formed using two methods. However, comparison of the DRCs individual confounding factors such as age. It is important to ana- between individuals indicated different slopes for each individual (see lyze a large number of cells to reduce the inﬂuence of confounding Supplementary Figs 1 and 2). factors, and thus we analyzed 2000 metaphases in this study. A Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Chromosome aberrations Chromosome aberrations frequency(/Cell) frequency(/Cell) Dose–response curves for chromosome aberrations � 39 Table 3. Average of chromosome translocation analysis of ﬁve donors Dose (mGy) Number of cells analyzed Number of Trs Frequency of 95%CI a observed Trs (Tr-frequency) Cell count of analysis Cell equivalent 0 5551.6 2176.2 9.6 0.004 0.001–0.008 10 5652.6 2215.8 13.4 0.006 0.004–0.008 20 5564.4 2181.2 13.0 0.006 0.002–0.01 50 5436.4 2131.1 14.6 0.007 0.003–0.011 100 5424 2126.2 19.2 0.009 0.004–0.014 200 6058.4 2374.9 34.2 0.014 0.011–0.019 500 5701.6 2235.0 81.4 0.036 0.033–0.04 1000 5197.4 2037.4 235.8 0.116 0.109–0.122 Equivalent to full genome cell count (The formula is provided in the Materials and Methods.). Chromosome translocations per 100 cell equivalents. Conﬁdence interval. previous study reported a correlation between the radiation dose speculate is attributable to the different detection methods used for and the frequency of CAs, even at a low-dose of 20 mGy, based on DCAs and Trs. All 46 chromosomes present in a cell are analyzed in the analysis of more than 5000 cells . We therefore thought it DCA, whereas only six chromosomes (pair numbers 1, 2 and 4) are advisable to analyze more than double that number of cells in this examined in Tr analysis, and the frequency of Trs formed is calcu- study. We did not believe that age adjustment was needed for DCA lated based on the proportion of DNA in these chromosomes to the because chromosomes containing Dics are instable; thus lympho- total DNA in the cell. Therefore, the former is an actual measurement cytes containing Dics will not survive for years. However, a correl- and the latter is derived from a calculation formula. ation between the number of Dics formed and the cumulative In both DCA and Tr analysis, chromosomes are analyzed by exposure dose was recognized in an analysis of residents from a high stopping the cell cycle at the mitotic phase using colcemide. background radiation area in China . In contrast, Tr is stable; Chromosome fragments without a centromere formed in cells with thus lymphocytes containing Trs are expected to survive beyond the Dics are not distributed to the poles during cell division, and thus normal human life expectancy , which may be a confounding cells with Dics cannot proceed beyond the mitotic phase. The G2/M factor. The number of Trs increases with age and is affected by life- checkpoint is believed to delay or pause the cell cycle; subsequently, style factors such as smoking . Furthermore, race, geographic the transition to the mitotic phase may be restricted . Actually, region, and individual lifestyle are thought to affect the Trs formed when Dics and incomplete chromosome fragments lacking a telomere . We suggest that those several factors induced large variation were formed in cells by irradiation, those cells were blocked at the between individuals in the frequency of Trs formed. Therefore, bio- G2/M checkpoint . On the other hand, cells with Trs face no logical dosimetry using Tr analysis should be performed using age obstacle that affects cell division and so can transfer to the mitotic adjustment to prevent an erroneously high estimation of the number phase. Therefore, the number of Dics formed may be lower than the of CAs [21, 31]. In this study, we conducted age adjustment using actual number of Dics formed when the cell cycle is stopped at the the frequency of age-dependent background reported by Sigurdson mitotic phase using colcemid; this may lead to a lower number of et al. . However, these frequencies were obtained from summar- Dics compared with the number of Trs formed. ized analytical reports from several laboratories around the world A comparison of the two DCA methods shows that the number and were not analyzed using the same method. Most analyses were of Dics observed by centromere-FISH tends to be higher than that conducted using a one-color probe, which is different from our observed by Giemsa staining, likely because the probe used to visu- three-color painting method. Therefore, it is necessary to obtain the alize the centromere region facilitates the discrimination of Dics, background frequency for the Tr analysis based on the method of compared with Giemsa staining. analysis. Furthermore, Shin et al. reported a difference in radiosensi- Recently, Suto et al. constructed a DRC based on Tr analysis tivity between healthy individuals , suggesting it might be neces- from a single healthy individual . In the present study, we con- sary to adjust the dose estimation by factors other than age. structed a DRC based on analyses of PB from ﬁve healthy individuals The frequency of Tr formation in this study was higher than and thus recognized variation in the number of Trs formed, even for that of Dics in every sample. Theoretically, these two CAs should the same radiation dose. We propose that a DRC should ideally be con- occur with the same frequency . However, there is a difference structed using samples from multiple healthy individuals of each age in in the frequency of occurrence , with the occurrence of Trs intervals of 5 or 10 years. Sample preparation methods for radiation reported to be slightly higher than that of Dics , which we dose estimation using the number of CAs differ slightly between Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 40 � Y. Abe et al. (a) Before Age-adjustment (b) Age-adjustment 0.14 0.14 2 2 Y = 0.0053 (± 0.0009) + 0.0259 (± 0.0127) × D + 0.0826 (± 0.0161) × D Y = 0.0015 (± 0.0009) + 0.0049 (± 0.0155) × D + 0.1033 (± 0.0223) × D r = 0.9995 r = 0.9993 0.12 0.12 0.1 0.1 0.08 0.08 0.06 0.06 0.04 0.04 0.02 0.02 0 200 400 600 800 1000 0 200 400 600 800 1000 Dose (mGy) Dose (mGy) (c) (d) 0.02 0.02 0.018 0.018 0.016 0.016 0.014 0.014 0.012 0.012 0.01 0.01 0.008 0.008 0.006 0.006 0.004 0.004 0.002 0.002 0 50 100 150 200 0 50 100 150 200 Dose (mGy) Dose (mGy) Fig. 3. Dose–response curves for chromosome translocation analysis. The frequencies of chromosome aberrations per 2000 cells equivalents (Ces) in PB from ﬁve individuals induced by gamma-ray irradiations were plotted. (a) The dose–response curves before age-adjustment. Regression analysis using DoseEstimate ver. 4.1 software was calculated from the average value of the ﬁve samples. [Y = 0.0053 (±0.0009) + 0.0259 (±0.0127) × D + 0.0826 (±0.0161) × D , r = 0.9995] (Y: yield of chromosome aberrations, D: dose (Gy), r = correlation coefﬁcient). (b) The dose–response curves following age-adjustment. The regression analysis was [Y = 0.0015 (±0.0009) + 0.0049 (±0.0155) × D + 0.1033 (±0.0223) × D , r = 0.9993]. (c) The dose–response curve before age-adjustment focusing on the low-dose range. (d) The dose–response curve following age- adjustment focusing on the low-dose range. laboratories, and the criteria for determining CAs may be different; con- In conclusion, our study performing three different bio- sequently, it appears that radiation dose estimation can differ between logical dosimetry assays simultaneously on PB from ﬁve donors analyses of the same sample [19, 20, 38, 39]. Therefore, it will be neces- highlighted the difﬁculty of biological dosimetry in the low- sary to construct globally uniﬁed sample preparation methods and cri- dose range. teria if we are to improve the accuracy of biological dosimetry. We conclude that both DCA and Tr analysis following gamma-ray irradiation doses of 100 mGy or less, and especially of ACKNOWLEDGEMENTS 50 mGy or less, may lack accuracy due to poor dose responsiveness This work was supported in part by a Grant-in-Aid for Young in the number of CAs formed, as assessed by DCA and Tr analysis, Scientists (B) [No. 15K19804], a Grant-in-Aid for Scientiﬁc and variation in the dose responsiveness of samples following these Research (C) [No. 16K08970] and by funds from the Japanese irradiation doses. On the other hand, as mentioned above, the accur- Ministry of Education, Culture, Sports, Science, and Technology for acy would likely increase if we analyzed 5000 or more cells in the the development of methods for monitoring exposure to low-dose low-dose range. It would be unrealistic to expend such effort on bio- radioactivity. This work was carried out at the Joint Usage/Research logical dosimetry during an actual radiation disaster. It is therefore Center (RIRBM), Hiroshima University. The results were presented important that we learn how to estimate exposure doses of 100 mGy at the 2016 Scientiﬁc Meeting of the Japanese Radiation Research and higher quickly and accurately. Society in Hiroshima. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/35/4523771 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Chromosome aberrations Chromosome aberrations frequency(/100 CE) frequency(/100 CE) Chromosome aberrations Chromosome aberrations frequency(/100 CE) frequency(/100 CE) Dose–response curves for chromosome aberrations � 41 workers with internal deposits of plutonium. Int J Radiat Biol SUPPLEMENTARY DATA 2016;92:312–20. Supplementary data are available at the Journal of Radiation Research 14. Iwasaki T, Takashima Y, Suzuki T et al. The dose response of online. chromosome aberrations in human lymphocytes induced in vitro by very low-dose γ rays. Radiat Res 2011;175:208–13. CONFLICT OF INTEREST 15. Barquinero JF, Barrios L, Caballín MR et al. Establishment and validation of a dose–effect curve for gamma-rays by cytogenetic The authors declare that they have no conﬂict of interest. analysis. Mutat Res 1995;326:65–9. 16. Yoshida MA, Hayata I, Tateno H et al. 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Journal of Radiation Research – Oxford University Press
Published: Jan 1, 2018
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