Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation

Mutation Research 759 (2014) 1–8

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Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation Qing-Jie Liu a,∗ , Xue Lu a , Xiao-Tao Zhao b , Jiang-Bin Feng a , Yu-Min Lü c , En-Hai Jiang d , Shu-Lan Zhang e , De-Qing Chen a , Ting-Zhen Jia e , Li Liang e,∗∗ a China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, PR China b Department of Cardiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, PR China c Henan Institute of Occupational Medicine, Zhengzhou 450052, PR China d Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, PR China e Department of Tumor Chemotherapy and Radiation Sickness, Peking University Third Hospital, Beijing 100191, PR China

a r t i c l e

i n f o

Article history: Received 9 March 2013 Received in revised form 25 May 2013 Accepted 5 July 2013 Available online 16 November 2013 Keywords: Radiation accident Retrospective dose estimation Fluorescence in situ hybridization Background translocation frequency

a b s t r a c t The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0–5 Gy 60 Co ␥-rays in vitro, and dose–effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose–effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose–effect curves established in the present study follow a linearquadratic model, regardless of the background translocation frequency. Estimated doses according to two dose–effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of dicentrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in underestimation of the biological doses compared with values obtained through analysis of di-centrics and tooth-enamel EPR. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Many endpoints for biological and physical dose estimation that could be used immediately following exposure to external ionizing radiation are available [1–3]. Among these endpoints, scoring of radiation-induced di-centric chromosomes plus centromeric

∗ Corresponding author at: National Institute for Radiological Protection, China CDC, 2 Xinkang Street, Deshengmenwai, Beijing 100088, PR China. Tel.: +86 10 62389629; fax: +86 10 62012501. ∗∗ Corresponding author. E-mail addresses: [email protected] (Q.-J. Liu), [email protected] (L. Liang). 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.07.016

rings (analysis of di-centrics) has become the “gold standard” for bio-dosimetry assessment after accidental exposure [4]. However, when retrospective dose assessment is carried out for exposures that have taken place years or decades ago or for chronic cases, results are usually inaccurate. The most advanced biophysical method for retrospective dose estimation is electron paramagnetic resonance (EPR) spectroscopy with tooth enamel. The EPR technique allows estimation of absorbed doses by detection of paramagnetic centers, such as free radicals or point defects that are specifically generated by ionizing radiation [5]. The distinct advantages of tooth-enamel EPR measurement are that the analysis is non-destructive, which allows repeated measurements of the same sample, that it detects a wide dose range (from 100 mGy

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to 100 Gy), and that the EPR signal is stable for a very long time (106 years) [6]. Many studies have shown that tooth-enamel EPR measurement is reliable for accurately estimating the retrospective radiation dose received by individuals regardless of whether exposure to radiation was chronic or acute, or recent or long before analysis [7]. Such radiation may have resulted from exposure to A-bombs in Hiroshima and Nagasaki [8,9], or from radiation accidents, such as the Chernobyl and Southern Urals accidents [10,11]. Tooth-enamel EPR measurement could effectively determine the accumulated lifetime-dose of radiation. Thus, EPR-estimated radiation doses could be used as a reference for dose reconstruction with new dosimetry methods if tooth-enamel samples are available. Tooth-enamel EPR measurement requires an exfoliated tooth so the sample is not usually available when dose estimation is performed. Translocation analysis by fluorescence in situ hybridization (FISH) is an alternative choice for retrospective dosimetry. Chromosomal translocations in cells can survive through mitosis and these defects are passed on to the daughter cells. Thus, these defects persist long enough for successful reconstruction of radiation doses received months or years before blood sampling [12]. Generally, only parts of the genome are detected during FISH analysis. For retrospective biological dosimetry, a single-color FISH for a triple cocktail of target chromosomes (usually chromosomes 1, 2 and 4), as recommended by the IAEA, appears to be sufficient for analysis [4]. A number of reports show that FISH analysis can be successfully applied to dose estimation for chronically exposed subjects, such as retired workers in Sellafield nuclear facility, residents near Techa River, or individuals who live in a radioactive environment [13–15]. However, the literature also shows different results for dose estimations of victims of acute radiation accidents. Some studies have shown that radiation doses estimated by FISH-based translocation analysis carried out 28 years after exposure match the estimated doses obtained by analysis of di-centrics immediately after the accident [16–19]. Other studies have shown that doses estimated with FISH-based translocation analysis are generally lower than values obtained by analysis of di-centrics when measurements are done only a short time after the accident [20–22]. Aside from the background translocation frequency, which is usually unknown, several other factors can influence the doses estimated by FISH, including the period between exposure and translocation analysis [23], the uniformity of the radiation exposure [24], the type of translocation recorded [23,25] and the exposure levels [26,27]. Thus, retrospective bio-dosimetry estimation by FISH for acute exposures cannot be obtained reliably from the remaining chromosomal translocations in cells, and further investigation of FISH-based systems for retrospective quantitative dose assessment is still required. The optimal method for validating the FISH method is by enrolling individuals with known biological dosimetry estimates to conventional analysis of di-centrics immediately following radiation exposure. Some studies have shown that both persisting stable translocations and EPR-spectroscopy signals are suitable with similar efficiencies for retrospective biodosimetry after acute whole-body exposure [28,29]. EPR tooth-enamel measurements may be used to validate the translocation analysis by FISH in the absence of physical dosimetry and data from analysis of di-centrics. By comparing doses obtained by FISH with doses estimated by analysis of di-centrics or tooth-enamel EPR measurement, the feasibility of retrospective dose estimation by FISH could be validated. In the present study, we compared reconstructed doses by FISH with biological doses obtained by analysis of di-centrics soon after the accident or tooth-enamel EPR doses obtained at the same time when FISH analysis was carried out. Measurements were done on five adults previously exposed to accidental radiation in the past. We also carried out FISH analysis for a child victim three times at different times after exposure to a radiation accident. Data from

physical dose estimation and from bio-dosimetry with dicentrics were not obtained for the child victim at the time of the accident. 2. Material and methods 2.1. Subjects Two types of subject were enrolled in the present study. This work was conducted at National Institute for Radiological Protection (NIRP), Chinese Center for Disease Control and Prevention. The Ethics Committee of NIRP approved all of the experiments in the present study. The scope of the study was explained to each subject and written informed consent was obtained. 2.1.1. Normal subjects to establish a calibration curve Three males were enlisted to establish the radiation-induced chromosome translocation dose–response curve. All subjects were 24–26 years old, healthy, and without any history of chronic disease, substance abuse or exposure to toxic chemicals. No radiation exposure or viral infection during the months preceding the study was documented. 2.1.2. Victims of previous accidental exposure Six subjects were collected in the present study. All had been previously exposed to accidental radiation and displayed acute radiation syndrome of varying severity after exposure (Table 1). Victim A, a 44-year-old male, was a scrap-metal dealer. Seven years ago, he purchased an unused 60 Co radiation source (the source radioactivity was 21.1 TBq) without knowing its nature and potential toxicity and kept the radiation source in his bedroom. The radiation source was reclaimed after he had been exposed to radiation for about 9.3 h. The clinical report and the dose estimation based on unstable chromosome aberrations were published elsewhere [1,30]. Victim B, a 43-year-old male, was accidentally exposed to a 60 Co source (source radioactivity, 40 TBq) for about 20 s when he manually cleaned the source rod with gauze 6 years ago. The estimated physical doses for the right and left hands were about 20–26 Gy and 12–18 Gy, respectively. The biological dose was estimated on the basis of chromosome aberrations (di-centrics and rings) 15 d after exposure [31]. The clinical diagnosis was severe acute radiation injury of bilateral hand skin and mild acute radiation sickness. Victim C, a 39-year-old female, was exposed intermittently to a 60 Co source (radioactivity, 396 TBq) for several days in a radiation accident 16 years earlier [32]. Her whole-body biological dose was estimated on the basis of analysis of chromosomes in peripheral blood lymphocytes on the 41st day following the accident. She was diagnosed with a moderate bone-marrow form of acute radiation sickness [33]. Victim D, a 45-year-old male, worked as flaw detection X-ray radiographer in a machinery factory for 8 years. He worked without radiation-protective measures for 7 years and in the operation room during his last year of work. He was accidentally exposed to X-ray radiation at a distance of 42 cm from the machine for 4 min about 3 years ago. Physical dose-estimation data were collected after the accident, and estimated doses using the two-tooth enamel EPR method were also obtained when the FISH analysis was carried out [34]. Victim E, a 62-year-old male, had been working as Cobalt-source operator and was accidentally exposed to radiation twice in a span of 4 years. During the first exposure, he exchanged the source using simple protection measures. During the second exposure, which happened 33 years ago, he manually returned the fallingout source to the right position. No physical radiation dosage record was reported. Two-tooth samples were collected for EPR analysis at the same time as the FISH analysis [34]. Victim F, a 15-year-old boy, accidentally received an electric shock and was sent to a local hospital 6 years ago. He was asked to undergo a computer tomography (CT) scan on the head, chest, and abdomen during 4 days. The victim experienced a convulsion at the second day after the last CT examination. No physical dose estimation or biological dose estimation based on unstable chromosome aberrations was reported after the accident. After the first translocation analysis with FISH, two blood samples were drawn and analyzed with FISH at intervals of 2 years. 2.2. Tooth-enamel EPR measurement Tooth-enamel samples were separated according to the alkaline treatment method described previously [35]. For victim E, the two front teeth were cut into two parts (buccal half and lingual half) to minimize the effect of exposure to sunlight [36]. Because the mass of one lingual half enamel was below the detection limit, the lingual and buccal halves for each tooth were pooled for the measurement. The tooth-enamel samples were crushed into a powder with a grain size-range of 500–1000 ␮m, followed by etching with 42% phosphoric acid under shaking. EPR measurements were carried out after thorough drying of the enamel samples at 40 ◦ C, which were placed in pure quartz tubes. EPR spectra were recorded at room temperature with an EPR spectrometer (JEOL ES-IPRITS/TE, Japan) operating in the X-band for microwave cavity. The following EPR spectrometer parameters were utilized: microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.32 mT; conversion time, 50 ms; time constant, 100 ms; magnetic field sweep, 336.6 mT; sweep time, 60 s; and number scan, 10. ESR sample measurements

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Table 1 Estimated physical doses, biological doses, and clinical diagnoses of radiation-accident victims [1,30–34]. Victim

Gender

Age at the time of the accident (year)

Physical dose (Gy)a

Biological dose (Gy)

Grade of ARSb

Pattern of radiation exposure

A B

Male Male

37 37

2.55(1.28–3.82) 1.00(0.70–1.30)

2.61(2.40–2.80)c 1.44(1.27–1.61)c

Moderate Mild

C

Female

23

2.0

2.30(2.07–2.50)c

Moderate

Uniform Non-uniform (hands and anterior body exposure) Non-uniform (cranial exposure)

D

Male

42

0.83(0.65–1.01)

0.64(0.54–0.75)d 0.45(0.36–0.53)d

Mild

Non-uniform (left lateral body exposure)

E

Male

29

N.A.

3.09(2.69–3.50)d 3.27(2.88–3.66)d

Moderate

Uniform

F

Male

9

N.A.

N.A.

Mild

N.A.

N.A., not available. a Mean value and 95% confidence interval of absorbed whole body doses estimated by experimental simulation. b Hematopoietic form of acute radiation syndrome. c Average doses and 95% confidence interval estimated by analysis of di-centrics. d Average doses and 95% confidence interval estimated by tooth-enamel EPR method (two teeth for both victims D and E).

were performed simultaneously with a Mn2+ standard sample set in the same resonator. Each sample was measured three times with removal of the sample tube and sample shaking. Measurements were repeated three times at different occasions. Two methods, the calibration-curve method and the addictive irradiation method, were used to estimate the enamel dose. For victim D, energy-correction factors were applied while considering the exposure geometry (Table 1) [37]. 2.3. Sample collection and irradiation of normal human peripheral blood samples Peripheral blood samples of normal people and accident victims were collected in heparinized vacutainer tubes (Becton Dickinson, USA) by venipuncture. Normal human peripheral blood samples were divided into 6 aliquots and irradiated with 0 Gy (sham-irradiation), 1 Gy, 2 Gy, 3 Gy, 4 Gy, and 5 Gy at room temperature by use of a [60 Co]-gamma-ray source provided by Secondary Standard Dosimetry Laboratory of NIRP, Chinese Center for Disease Control and Prevention. The source of radioactivity was 130 TBq, the homogeneous irradiation field was 10 cm × 10 cm, and the dose-rate was 1.00 Gy/min. The exposure set-up was calibrated by physical measurement using an ionizing chamber. After irradiation the blood samples were incubated at 37 ◦ C for 2 h before initiation of the cell culture. 2.4. Cell culture and chromosome preparation Lymphocytes were isolated from the whole-blood samples with Ficoll-Paque (Amersham Pharmacia Biotech). The cells were washed three times with fresh RPMI 1640 medium (Invitrogen, Carlsbad, USA) and chromosomes were prepared by adding colcemid to the start culture according to a method described previously [38] with some modifications. Briefly, lymphocytes were grown for 48 h at 37 ◦ C in fresh RPMI 1640 medium with 20% fetal bovine serum (HyClone, USA), 2 mM lglutamine, 1% penicillin/streptomycin, 1% phytohaemagglutinin (PHA, Invitrogen), and colcemid (0.04 ␮g/mL, Invitrogen). To harvest the culture, cells were treated with 37 ◦ C hypotonic solution (0.075 M KCl) for 30 min, followed by three cycles of fixation with fresh fixative (mixture of methanol and glacial acetic acid, 3:1, v/v). A few drops of the cell suspension in fixative were dropped onto dried, alcoholpre-cleaned slides. The slides were then placed above a container filled with heated water for 20–60 s, depending on the ambient humidity [39]. 2.5. Fluorescence in situ hybridization Each slide for FISH was pretreated with RNase A (100 ␮g/mL in 2× SSC, Boehringer Mannheim, USA) and proteinase K (Sigma–Aldrich, Santa Clara, USA) and fixed in 1% formaldehyde according to the method described by Pinkel et al. [40]. The slides were denatured by incubation in 70% formamide and 2× SSC at 70 ◦ C for 1–2 min, followed by dehydration through an ice-cold ethanol series (70%, 90%, and 100%). The denatured probes of chromosomes 1, 2, and 4, which were directly labeled with FITC fluorochrome (Cell Bank, Kuming Institute of Zoology, Chinese Academy of Sciences), were applied to the denatured chromosome slides and hybridization occurred at 37 ◦ C overnight. Three post-hybridization washes were performed at 42 ◦ C in 50% formamide and 2× SSC for 5 min each, followed by three washes in 0.1× SSC at 50 ◦ C for 5 min each. Chromosome counterstaining was performed with 0.15 ␮g/mL 4 ,6-diamidino-2-phenylindole (DAPI). The slides were stored in a dark refrigerator at −20 ◦ C before analysis. 2.6. Image capture and scoring criteria The FISH slides were viewed with a Zeiss Axioplan 2 Imaging fluorescence microscope (Zeiss, Oberkochen, Germany) with a cooled charge-coupled device (AxioCam HRM, Zeiss) and Isis FISH analysis software from MetaSystems, Germany. Two-color

(green and blue) images were captured, merged, and stored. About 500–2000 cells were scored for each sample. Metaphases were considered suitable for analysis only if they appeared to be complete, the fluorochrome signals were sufficiently bright, the centromeres were morphologically detectable, and all three pairs of chromosomes were visible in the cell. A conventional scoring system was used. Each translocation was checked by two observers. The cells were scored as normal when all of the signals on three pairs of chromosomes were complete and positioned accurately. Reciprocal translocation was recorded if more than six chromosomes were observed with green color and two chromosomes were observed with green-blue color.

2.7. Dose–response curve of 60 Co gamma-ray-induced genomic translocation frequency The observed translocation frequency for each dose level was calculated according to the formula Fp = x/n, where Fp is the translocation frequency detected by FISH, x is the number of observed translocations, and n is the number of analyzed cells. The observed translocation frequency was converted into the genomic translocation frequency by means of the Lucas formula FG = Fp /2.05fp (1 − fp ) [16], where Fp is the translocation frequency detected by FISH, FG is the full genomic translocation frequency, fp is the fraction of genome hybridized: for chromosomes 1, 2 and 4 this is 22.7% for males, and 22.3% for females [41]. In the present study, two dose–response curves were established regardless of whether or not the background translocation frequency was considered. The dose–response curve without considering the background translocation frequency was established with the genomic translocation frequency calculated above the absorbed dose. Previous studies unanimously agree that the background translocation frequency increases with age, although this trend is not clear for other factors. Only the effect of age on the background translocation frequency was investigated in this study. The background translocation frequency for each blood donor was calculated according to the formula proposed by Lucas et al. [42]: Y = 7 × 10−4 + 6.9 × 10−6 A + 1.35 × 10−6 A2 , where Y is the background translocation frequency, and A is the age of donor. The net radiation-induced genomic translocation frequency was calculated by subtracting the background translocation frequency from the genomic translocation frequency. The dose–response curve considering the background translocation frequency was established with the net radiation-induced genomic translocation frequency and the absorbed dose.

2.8. Dose estimation for victims Firstly, the observed translocation frequency for each victim was calculated with the formula Fp = x/n, as described above. Secondly, the genomic translocation frequency was obtained with the Lucas formula, FG = Fp /2.05fp (1 − fp ). Thirdly, the estimated dose without considering the background translocation frequency was reconstructed according to the corresponding dose–response curve. The background translocation frequency for each victim was calculated according to the formula established by Lucas et al. [42], and the net radiation-induced genomic translocation frequency was determined. Retrospective dose estimation was carried out with the dose–response standard curve and its formula was established in the present study. The mean absorbed doses and 95% confidence intervals were calculated for each victim. The 95% confidence intervals for estimated dose were calculated from both standard errors of the yields of aberrations and the error due to the dose–response calibration curve.

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Fig. 2. Dose–response curves of the irradiation dose (0–5 Gy) of [60 Co] gamma-rays and genomic translocation frequency: (A) the dose–response curve without considering the background translocation frequency and (B) the dose–response curve considering the background translocation frequency.

Fig. 1. Normal metaphase and translocations detected by FISH with wholechromosome probes of chromosomes 1, 2, and 4: (a) a normal human metaphase with FITC-painted chromosomes 1, 2, and 4 (green) and counterstained with DAPI (blue); and (b) a reciprocal translocation involving chromosome 2 (yellow arrows) and other reciprocal translocations involving chromosome 1 (white arrows).

2.9. Data analysis Statistical analysis was performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as a mean ± standard deviation or average (95% confidence interval). The 2 test was used to compare the genomic translocation levels of different dose groups and the Poisson Z-test was used to compare two different dose groups. Analysis of variance was used to analyze the significance of the regression equation of the dose–response curve. All reported P values were two-sided and a significance level of 0.05 was used for determination of significance.

3. Results 3.1. Dose–response curves Dose–response curves should be established between the absorbed doses of normal human peripheral lymphocytes and the genomic translocation frequency in order to estimate the absorbed doses of each victim. Peripheral blood samples of three healthy adults were irradiated with 1–5 Gy Co-60 gamma rays in vitro, and chromosome reciprocal translocations were analyzed by FISH with painting probes for chromosomes 1, 2, and 4 (Fig. 1). All observed translocations of three adults were pooled together. The reciprocal translocation for each absorbed dose level was fitted to the Poisson distribution (Table 2). The observed translocation frequency and the genomic translocation frequency were both enhanced with increasing absorbed dose (2 = 5478.73,  = 5, P < 0.005). The

difference between every two absorbed dose levels was significant (Poisson Z-test, P < 0.001 or P < 0.0005). Without considering the background translocation frequency, the fitted dose–response curve for absorbed dose level and genomic translocation frequency followed a linear-quadratic model (Fig. 2A). The significance of the regression equation of the dose–response curve was confirmed by ANOVA (1 = 2, 2 = 3, F = 2.633 × 104 , P < 0.001). If the background translocation frequency was considered, the net radiation-induced genomic translocation frequency was calculated by use of the above expression (genomic translocation frequency minus the background translocation frequency), which was obtained from the equation established by Lucas et al. [42]. The dose–response curve of the absorbed dose vs the net radiation-induced genomic translocation frequency also fitted the linear-quadratic model well (Fig. 2B). 3.2. Dose estimation for the victims The reciprocal translocations in all analyzed cells of six radiation accident victims were analyzed with FISH. The observed translocation frequency and the genomic translocation frequencies were calculated (Table 3). The estimated doses for victims were calculated based on the linear-quadratic equation Y = 0.0037 + 0.0062D + 0.043D2 , which does not take into account the background translocation frequency. If the background translocation frequency was considered, this frequency was calculated for each victim. The net radiation-induced genomic translocation frequencies were obtained and the absorbed dose for each victim was reconstructed by use of this frequency, according to the linear-quadratic equation Y = 0.0020 + 0.0062D + 0.043D2 (Table 3). The two estimated doses for each victim were in good agreement with each other and the 95% confidence interval estimated with the net radiation-induced genomic translocation frequency was narrow. Thus, the doses reconstructed with the dose–response

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Table 2 Cell-to-cell distribution of translocation in human lymphocytes irradiated with 0–5 Gy [60 Co] gamma-rays. Absorbed dose (Gy)

No. of cells scored

No. of translocation observed

0.00 1.00 2.00 3.00 4.00 5.00

3000 3000 2400 2400 1800 1560

3 60 159 357 462 624

a b

Cell distribution of translocation 0

1

2997 2940 2241 2043 1338 936

3 60 141 321 365 461

2

9 15 32 51

3

2 10 15

 2 /ya

ub

0.999 0.980 1.047 0.969 1.012 0.985

−0.047 −0.78 1.63 −1.07 0.36 0.42

4

4

y is the mean number of translocations observed per cell;  2 is the variance. u is the value used for deciding compliance with the Poisson distribution for each dose.

Table 3 Estimated doses by fluorescence in situ hybridization (FISH). Victim

No. of cells scored

Observed translocations

Observed translocation frequency (/cell)

A B C D E F

500 500 2000 1000 500 500

54 17 24 12 26 41

0.108 0.034 0.012 0.012 0.052 0.082

a b c

± ± ± ± ± ±

Genomic translocation frequency, GTF (/cell)a

Estimated dose with GTF (Gy)

Background genomic translocation frequency (/cell)b

Net genomic translocation frequency, NGTF (/cell)c

± ± ± ± ± ±

2.55(2.15–2.88) 1.39(0.99–1.70) 0.77(0.59–0.92) 0.76(0.50–0.95) 1.74(1.36–2.07) 2.21(1.82–2.55)

0.0037 0.0036 0.0030 0.0033 0.0063 0.0011

0.297 0.091 0.031 0.030 0.138 0.227

0.015 0.008 0.002 0.003 0.010 0.013

0.300 0.095 0.034 0.033 0.145 0.228

0.042 0.022 0.006 0.008 0.028 0.036

± ± ± ± ± ±

0.041 0.021 0.005 0.007 0.027 0.036

Estimated dose with NGTF (Gy)

2.55(2.16–2.88) 1.37(0.99–1.67) 0.75(0.60–0.88) 0.74(0.51–0.92) 1.71(1.32–2.03) 2.22(1.82–2.55)

Calculation results according to the Lucas formula [16]. Calculation results according to the formula established by Lucas et al. [42]. Obtained by genomic translocation frequency minus the background translocation frequency.

curve considering background translocation frequencies were used in subsequent analysis.

(1.82–2.55 Gy) (Table 5). Follow-ups by FISH were carried out every 2 years afterwards. Results showed that the estimated FISH doses decreased rapidly. Lower dose levels were reconstructed twice and done every 2 years.

3.3. Comparison of the estimated doses for adult victims by different methods

4. Discussion Doses estimated by means of FISH for 5 adult victims were compared with biological doses obtained by analysis of di-centrics or tooth-enamel EPR measurement (Table 4). The FISH-derived doses of three victims who were exposed to accidental irradiation 3, 6, and 7 years prior to analysis were consistent with the dicentricbased bio-dosimetry or tooth-enamel EPR doses. The FISH-derived doses of two other adult victims who were accidentally exposed to irradiation 16 or 33 years before analysis appeared to be underestimated, with calculated values about two to three times lower than the dicentric-based bio-dosimetry or tooth-enamel EPR doses.

We report a method of retrospective dose estimation of six Chinese accidental radiation victims. In the present study we used FISH with whole-chromosome probes, also called chromosome painting. The results of this study provide further evidence of the validity of the FISH method to measure translocations as a reliable retrospective biological dosimeter after accidental exposure to radiation [43]. Generally, the dose–response curve for FISH is established with normal human peripheral blood and acute exposure to ionizing radiation in vitro. In the present study, human peripheral blood samples from three normal adults were used to establish a dose–response curve. The dose–response curves fitted the linearquadratic model well, similar to the established dose–response curves for di-centrics and micronuclei [1,2]. Dose–response curves for FISH translocation from other studies were established with two kinds of irradiation: some studies employed chronic irradiation whereas others used single acute irradiation [44,45]. Comparison

3.4. Follow-up of the 15-yr old victim with FISH For victim F, no physical or biological doses estimated by analysis of dicentrics were available immediately after the accident, because no doctor in the local hospital was familiar with the signs of acute radiation sickness. The first FISH analysis was performed 6 years after the accident and the estimated dose was 2.22 Gy

Table 4 Comparison of doses estimated by FISH and those estimated by analysis of di-centrics or tooth-enamel EPR measurement for adult victims. Victim

Years after accident

Biological dose (Gy)

Dose estimation with FISH (Gy)

Pattern of radiation exposure

A B C

7 6 16

2.61(2.40–2.80) 1.44(1.27–1.61) 2.30(2.07–2.50)

2.55(2.16–2.88) 1.37(0.99–1.67) 0.75(0.60–0.88)

Uniform Extremely non-uniform Non-uniform

D

3

0.74(0.51–0.92)

Non-uniform

1.71(1.32–2.03)

Uniform

E a b c d

33

0.64(0.54–0.75)a 0.45(0.36–0.53)b c

d

3.09(2.69–3.50) 3.27(2.88–3.66)

Estimated EPR dose of mandibular right second premolar. Estimated EPR dose of mandibular right first molar. Estimated EPR dose of maxillary left central incisor. Estimated EPR dose of maxillary right central incisor.

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Table 5 Changes in genomic translocation frequency and dose estimation with FISH for victim F. Years after accident

Age

No. of cells scored

Translocation Observed translocation recorded frequency (X¯ ± S)

5 7 9

15 17 19

500 1000 2000

41 23 10

a

0.082 ± 0.013 0.023 ± 0.005 0.005 ± 0.002

Background genomic translocation frequencya

Net radiation-induced genomic translocation frequency (X¯ ± S)

Estimation dose (Gy)

0.0011 0.0012 0.0013

0.227 ± 0.036 0.063 ± 0.014 0.013 ± 0.005

2.22(1.82–2.55) 1.13(0.81–1.38) 0.48(0.11–0.63)

Calculation results according to the formula established by Lucas et al. [42].

of the alpha- and beta-coefficients of the dose–response curves between the present study and other single acute irradiation studies showed that the beta-coefficient of the dose–response curves in the present study agree with the values found in previous studies [46,16]. The alpha-coefficient in the present study was lower than those reported in the literature for acute, low-dose exposure or chronic exposure of human lymphocytes [47]. Because translocation is a persistent type of chromosome aberration, the background translocation frequencies are higher than that of di-centrics [48,49]. Taking the translocation background into account when attempting retrospective bio-dosimetry, particularly for low-dose exposures, is thus important. While studies unanimously agree that the translocation rate increases with age, a review shows that this trend is not clear for other factors [50]. According to the two dose–response curves established in the present study, corresponding retrospective doses for each victim were similar because all of the victims showed symptoms of acute radiation syndrome and all absorbed irradiation doses received were considerably higher than 0.5 Gy. The irradiationinduced translocation frequency was significantly higher than the background translocation frequency, which suggests that the latter can be ignored if the accidental radiation exposure involves a sufficient number of samples and the predicted exposed dose level is significantly higher than 0.5 Gy. There is no unequivocal information available on how long the translocation aberration could be considered stable, since the estimated radiation doses based on FISH analysis are not significantly different from the radiation-dose estimates obtained using analysis of di-centrics soon after exposure. Three victims were exposed to accidental radiation less than 10 years before analysis (3, 6, and 7 years ago) and participated in the present study. The retrospective doses obtained with FISH were in agreement with values determined by the two other methods, regardless of whether or not the pattern of exposure was uniform. These findings may have resulted from the sufficiently long average lifespan of circulating T-lymphocytes and the low number of complex chromosomal aberrations induced by irradiation at levels below 3 Gy [25]. These findings confirm that translocation analysis by FISH could be used for retrospective dose estimation in accidental exposures, especially for cases that occur less than a decade before analysis [51]. In the present study, the retrospective doses estimated by FISH for two victims who had been exposed to accidental radiation 16 and 33 years prior to analysis were significantly lower than biological dose estimates obtained by analysis of di-centrics soon after the accident or tooth-enamel EPR measurement long after the accident. FISH-based doses estimates longer than 10 years after exposure may lead to underestimation of the dose compared with the initial dose level. For retrospective biological dosimetry performed longer than a decade after exposure, the estimated dose by use of the FISH method represents average doses derived from the active bone marrow. This is because the original exposure affects the stem-cell precursors of lymphocytes that are scored [49]. In addition, a translocation can still fail to pass cell division if an unrelated and unstable structure, such as a di-centric or an excess acentric fragment is also present in the same cell [52]. Such findings

underline the need to consider stability not only of individual types of aberration but of the cell as a whole. Because FISH-based doses estimates longer than 10 years after exposure underestimate the initial exposure-dose level, incorporation of appropriate correction factors to the necessary equations is required. In the present study, one victim was aged 9 years when he was exposed to accidental radiation. The first retrospective dose estimation by FISH analysis was performed 6 years after exposure, when he was 15 years old. Two follow-up analyses were carried out every 2 years afterwards (8 and 10 years after the accident) when he was 17 and 19 years old. The results showed that doses estimated by FISH decreased considerably with time. This finding is different from the results of the dose analysis of adult victims. The variations observed may be related to changes that occur during puberty, as well as instability of the translocation aberration [53]. The physical growth rate during teenage years is very high and the amount of bone marrow increases rapidly during puberty, such that bone marrow cells containing translocations may be replaced rapidly. Values calculated for translocation frequency in peripheral blood lymphocytes may also be lowered by unaffected cells entering the circulation. A study on a 13-year-old boy exposed to protracted low dose-rate, whole-body, and short-time partial-body irradiation from a radiation accident in Estonia in 1994 showed that the calculated yields of reciprocal translocations also reveal a gradual but significant reduction [54]. All these results suggest that radiation doses estimated by FISH for children or adolescents may be underestimated. 5. Conclusion FISH-based reciprocal translocation analysis could be used for retrospective dose estimation for adults who have been exposed to accidental radiation less than a decade before. If radiation exposure occurred more than a decade ago, the dose determined by FISH may be underestimated. The proposed method of retrospective dose estimation for adolescents by FISH requires further evaluation. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements All authors thank the participants in the present study for their contributions. This work was supported by the National Natural Science Foundation of China (Nos. 30570551 and 81172593), and the Scientific Research Fund of Ministry of Health, People’s Republic of China (No. 98-2-46). References [1] Q. Liu, J. Cao, Z.Q. Wang, Y.S. Bai, Y.M. Lü, Q.L. Huang, W.Z. Zhao, J. Li, L.P. Jiang, W.S. Tang, B.H. Fu, F.Y. Fan, Dose estimation by chromosome aberration analysis and micronucleus assays in victims accidentally exposed to 60 Co radiation, Br. J. Radiol. 82 (2009) 1027–1032.

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