AKIO AWA ABSTRACT. Radiation Effects Research Foundation, Hiroshima, Japan. Key Words. Chromosome Radiation Dicentric. Translocation.

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1 Analysis of Chromosome Aberrations in Atomic Bomb Survivors for Dose Assessment: Studies at the Radiation Effects Research Foundation from 1968 to 1993 AKIO AWA Radiation Effects Research Foundation, Hiroshima, Japan Key Words. Chromosome Radiation Dicentric. Translocation. Atomic bomb ABSTRACT Exposure to ionizing radiation causes damage to living cells, especially to DNA in the cell nucleus. The degree of this cellular damage depends on the amount of radiation administered. This review discusses current findings concerning radiation-induced chromosome aberrations that were produced in 1945 and that can still be observed in the somatic cells of atomic bomb survivors in Hiroshima and Nagasaki. The scoring methods of G-banding and fluorescence in situ hybridization are compared. In addition, some findings concerning chromosomal aberrations in citizens of the former Soviet Union a fected by the Chernobyl accident are presented. Stem Cells 1997;IS(suppl2): INTRODUCTION Chromosomes, which are composed mostly of DNA, can be observed microscopically as shown in karyotypes from a male and female (Fig. 1). After fixing and staining, the metaphase spreads appear as in the upper part of the figure, and then photographs of the individual chromosomes are cut out and arranged in order of size as shown in the lower part of the figure. Both male and female cells contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. In males, the pair of sex chromosomes consists of one X and one Y chromosome; females have two X chromosomes. In the late 1960s, new techniques to stain chromosomes were developed. One of these techniques is known as G-banding, as shown in Figure 2. The left panel illustrates a normal metaphase from a male, and the right panel shows a reciprocal translocation between chromosomes 1 and 16. This method showed that each chromosome has characteristic banding patterns, allowing subtle changes in any chromosomal region to be identified. Chromosome aberrations, which are among the most thoroughly studied effects of radiation exposure, include changes in the number and structure of chromosomes [ 11. Structural abnormalities result from the breakage of chromosomes and incorrect joining of the resulting fragments by repair enzymes (Fig. 3). Such abnormalities often cause interference with the normal segregation of chromosomes during cell division. Radiation Injury and the Chernobyl Catastrophe. STEM CELLS 1997;15(suppl2): AlphaMed Press. All rights reserved.

2 164 Chromosome Aberrations in Atomic Bomb Survivors Figure 1. Male (left) and female (right) normal human kuryotypes. The upper portion of each panel shows the metaphase chromosome spread as seen under the microscope. A photograph of the chromosomes is then cut to,form the ordered array seen below. Figure 2. G-banding patterns of human chromosomes. The upper portion of each panel shows the metaphase chromosome spread as seen under the microscope. A photograph of the chromosomes is then cut to form the ordered array seen below. The left panel shows a normal male karyotype, and the right panel a reciprocal translocation between chromosomes 1 and 16.

3 Awa 165 Induction of chromosome aberrations by ionizing radiation Normal chromosomes Meiaphase Break 1 Rejolnlng Type of aberratlons figure ring chromosome +fragment intrachromosomal exchange pericentric inversion dicentric chromosome +fragment interchromosomal exchange reciprocal translocation Figure 3. Induction of chromosome aberrations by ionizing radiation. lntrachromosomal exchanges produce ring chromosomes or inversions. Interchromosomal exchanges, involving two chromosomes, produce dicentrics or translocations. Figure 4 shows some typical examples of radiation-induced chromosomal rearrangements. The left panel shows a cell that has both ring and dicentric chromosomes, and the right panel illustrates a translocation and a pericentric inversion. Dicentric chromosomes are easily detected because of their unusual shape, and the scoring efficiency in detecting these abnormalities is therefore almost 100%. However, these forms are very unstable because the abnormalities lead to mitotic disturbances that cause cell death, as illustrated in Figure 5. During mitosis the dicentric must divide into two poles. If segregation occurs normally, each of the two daughter cells also contains a dicentric chromosome. However, if a bridge forms during mitosis (Fig. 5), cell division may result in a polyploid cell with twice the normal chromosomal complement, which may subsequently lead to cell death. In contrast to the instability of dicentrics, there are no disadvantages to undergoing normal mitosis in cells with translocated and inverted chromosomes. However, these forms can be difficult to detect, particularly if the exchanges involve very small chromosomal pieces. Figure 6 shows further evidence that cells carrying dicentrics are unstable [2,3]. These data are based on analyses of chromosomes from patients with ankylosing spondylitis who had received substantial amounts of local x radiation and from gynecological patients receiving radiotherapy. This figure shows a drastic decrease in the frequency of cells carrying dicentrics, illustrating their instability. In contrast, translocations remain very consistent with time (Fig. 7). When the surveys of chromosome aberrations were performed in 1968, which was 23 years after atomic bomb exposures, we were surprised to find virtually no dicentrics. In a search for alternative markers of radiation damage, we began to score translocations, despite the technical difficulties. Radiation-induced chromosomal damage differs according to the type of radiation to which cells are exposed. Atomic bomb radiation consisted of y rays and neutrons in both Hiroshima and Nagasaki, but the amount of neutrons was substantially larger in Hiroshima (Fig. 8). This may account for some of the different biological end points between the two cities.

4 166 Chromosome Aberrations in Atomic Bomb Survivors Figure 4. Examples of radiation-induced chromosomal aberrations. The left panel shows a cell with a ring chromsonae derivedfiom chromosome 2 (+) and a dicentric derivedfiom chromosomes 4 and 16 (v). The right panel shows a cell with a reciprocal translocation between chromosomes 11 and 13 (v), and a pericentric inversion involving chromosome 20(+). Prophase Metaphase Anaphase Telophase Daughter cells - Normal division Abnormal division (Cell death?) \ Cell with a dicentric chromosome / icronucleus Fusion of daughter cells Figure 5. Disruption of mitotic cell diviswn by dicentric chromosomes. The upper part of the figure illustrates noml mitotic cell division with the chromosomes dividing successfully into the daughter cells. The lower part of the figure illustrates the abnoml division that can occur in the presence of dicentrics. Vthe chromosomes divide successfully, i.e., with both centromeres of the dicentric traveling to the same pole, the daughter cells will each contain a dicentric themselves. However, ifa dicentric is pulled to two opposite poles, the daughter cells m y fuse instead of separating.

5 Awa 167 Hiroshima "-112 Figure 6. Chromosome analyses of (1) patients with ankylosing spondylitis, a chronic injlammatory disease of the spine and sacroiliac joints, who had received substantial amounts of fractionated local X- irradiation (square symbols, cited from Buckton, [2], and (2) patient with gynecological tumors exposed therapeutically to radium and x-irradiation (circular symbols, cited from Bauchinger, [3]). Open symbols indicate unstable chromosome aberrations (dicentrics and rings); closed symbols indicated stable chromosomal aberrations (translocations and inversions). 40 3o Nagasaki n= 81 r=o.71 I I DS86 dose (Sv) MATERIALS AND METHODS Conventional staining methods were used to study chromosomes in blood samples from atomic bomb survivors from Hiroshima and Nagasaki [4-61. The objectives of our study were: (a) to determine the frequency of stable chromosome aberrations in the lymphocytes of atomic bomb survivors and (b) to establish better dose-response curves for biological dosimetry. In 1958, a cohort of 20,000 atomic bomb survivors began to undergo biannual clinical examinations for long-term health monitoring [7]. The purposes of this Adult Health Study Cohort were: (a) to investigate late health effects of atomic bomb radiation on survivors and (b) to verify the characteristics of diseases in the survivors. The cohort of survivors was divided into three equally sized groups. The proximally exposed group was exposed within 2 km from the hypocenter, were Figure 7. Frequencies of cells with stable chromosome aberrations plotted against the DS86 radiation dose estimates for individual survivors in Hiroshima (upper panel) and Nagasaki (lower panel). - >. 0 e a 0 03 C g 0.02 m z 0.01 Nagasaki Gamma dose (Gy) Figure 8. Distribution of neutron and gamma components of the total radiation dose for cytogenetic study subjects. Hiroshima (closed squares) and Nagasaki (open squares). Reprinted with permission from RERF. with or without acute radiation symptoms, and received an estimated dose of 0.01 Gy. The distally exposed group was more than 3 km from the hypocenter. The third group, designated NIC (not in the city) consisted of subjects who were not exposed at the time of the bombing, i.e., located more than ten kilometers from the hypocenter. For the majority of the survivors in the Adult Health Study Cohort, radiation doses estimated from the physical dosimetry systems known as DS86 are available. Blood was drawn from each individual and lymphocytes were cultured for 48 h to obtain the maximum number of mitotic cells. After the cells were harvested to prepare microscopic slides, metaphases were analyzed directly under the microscope as described above. One hundred metaphases per sample were scored as

6 168 Chromosome Aberrations in Atomic Bomb Survivors a routine procedure. We then determined the proportion of cells with any kind of stable chromosome aberrations relative to the total number of cells examined. It was these stable aberrations that contributed to the dose-response relationship in atomic bomb survivors. In contrast, cells with unstable aberrations were far less frequent, suggesting that cells with such aberrations would have been selectively eliminated with time. The exclusion criteria for Adult Health Study Cohort participants were survivors whose estimated radiation dose was over 4 Gy, who had cancer diagnosed prior to cytogenetic examination (since they are likely to have received radiation therapy), and who carried cytogenetic clonal abnormalities. We also excluded cases for which we were able to analyze fewer than 100 metaphases after blood cell culture for 48 h. RESULTS Comparison of Radiation Effects in Hiroshima and Nagasaki An important feature noted in the study is the wide scatter of individual aberration frequencies for given dose ranges (Fig. 7). This is a consistent phenomenon observed in atomic bomb survivors. Such a discrepancy between aberration frequency and the radiation dose assigned to each survivor would be due to either random errors in dosimetry or differences in radiation sensitivity among survivors. As shown in Figure 8, the neutron contribution is consistently higher in Hiroshima than in Nagasaki. This observed intercity difference is partly attributable to the difference between the two cities in the amount of neutron component in the radiation dose, shown in Figure 8. The proportion of neutrons compared to they-ray dose is consistently higher in Hiroshima than in Nagasaki survivors [8]. Sposto et al. [9] reported that the shapes of dose-effect curves differ significantly by city in the strength of the quadratic component of the dose-response equation (Fig. 9). Data from Nagasaki show much more curvature upward than those seen in the aberration data from Hiroshima, which are rather linear. In other words, the dose-squared term is greater in Nagasaki than in Hiroshima. The difference in the shape of the dose response for the two cites is again due to the amount of the neutron component relative to the total dose. The results in Figure 10 also suggest a complex nonlinear interaction between radiation and age at the time of exposure. There is evidence that radiation exposure is more effective in producing these kinds of chromosome aberrations in some persons exposed at younger ages. However, the interpretation of this biological interaction is very difficult. Some of the differences are ascribed to improvement gained through experience in the techniques for culturing cells and for scoring chromosome aberrations (Fig. 11). In the early 1970s, a decision was made to reduce the volume of the material being processed, which has resulted in improvement in the quality of medical slides. Recent samples therefore show more consistent results. Himshima + Nsgasakl 1 3. o,,,,,,,,,,,, OS86 bane marrow dose (Gy) 0 " ' I ' " ' ' Mean age at expo5ure within age category Figure 9. Bone marrow dose response of chromosome aberrations for Hiroshima and Nagasaki (cited from r81). Figure 10. Effect of age at exposure on estimated chromosome aberration dose response. Dose-response slope estimates, relative to the dose response estimated for ages 25-29, are plotted against mean age at exposure (cited from [S]).

7 Awa 169 Figure 12 shows the relationship between the frequency of chromosome aberrations and radiation dose in Hiroshima using the DS86 bone marrow dose with a neutron RBE of 10. The relationship between radiation dose and chromosome aberrations appears to be linear. Another feature to notice is the fairly large scatter in aberration frequencies, namely, high aberration frequencies in the low-dose range and low aberration frequencies in the high-dose range. Sposto et al. [9] found that the dose-response relationships for frequency of aberrations using the DS86 assigned dose is significantly steeper in the subset of survivors manifesting severe epilation than in those with no epilations (Fig. 13). There is substantially more variation in the proportion of cells carrying stable aberrations among individuals at higher doses than would be expected if there were no heterogeneity in the dose response. It has been suggested that random dosimetry errors in the range of 45%-50% may explain the difference in dose responses. Such a degree of dosimetry error also accounts for the dispersion in the chromosome aberration frequencies that was shown in Figure 12. Figure 14 shows the relationship between DS86 bone marrow dose and the frequency of cells with stable chromosome aberrations as revealed by G-banding on 52 Hiroshima survivors. The G- banding technique of Seabright [ 101 was used throughout this study. The results indicate that there is a good agreement between frequency of aberrant cells and the DS86 dose estimates for individual survivors (Fig. 15) [ T $ I,,,,,,,,, / I p o S1+ Year Of assay Figure 11. Background rates of chromosome aberrations by year and by city (cited from [a]). Figure 12. Frequency of cells with chromosome aberrations in the Adult Health Study Cohort participants from Hiroshima, studies in A wide range of scatter in aberration frequencies is seen. Figure 13. Plot of observed and predicted proportion of cells with chromosome aberrations within epilation groups without adjustment for random dosimetry errors. (*) epilation, (.) no epilation. (Cited from [9/). Figure 14. Relationship between DS86 bone marrow dose and translocation frequencies (using FISH and G-banding methods) in Hiroshima A-bomb survivors. Gray circles for FISH, and black circles for G-banding, respectively.

8 170 Chromosome Aberrations in Atomic Bomb Survivors Comparison of Two Scoring Methods A major advance in molecular biology is the fluorescence in situ hybridization (FISH) technique, which makes it possible to detect chromosome translocations rapidly and efficiently. A collaborative study has been developed between the Radiation Effects Research Foundation (RERF) and the Lawrence Livermore National Laboratory (LLNL). The objectives of the study are 1) to determine if the FISH technique can detect translocations in atomic bomb survivors and 2) to compare independently the frequency of translocations detected by FISH at LLNL and by G-banding at RERF [12]. Table 1 shows the number of samples examined in this collaborative study. We have thus far analyzed blood samples from 36 atomic bomb survivors, of whom seven were controls with an estimated dose of less than Sv. For FISH, we generally score metaphases, depending on the number of chromosome aberrations detected. We scored 100 metaphases for G-banding and conventional analysis. Chromosomes 1, 2, and 4 are targeted for FISH studies, whereas all the chromosomes are analyzed with the other techniques. Figure 16 illustrates metaphases stained with chromosome painting methods. The other G-uandlng data. Hfroshima DS86 bone marrow dore (SV) Figure 15. Relationship between DS86 bone marrow dose and frequency of stable chromosome aberrations as revealed by G-banding analysis (cited from 1111.) Table 1. Number of A-bomb exposed subjects in the collaborative study Dose (Svy Male Female Total Distal Proximal OO-I t Subtotal Total ads86 bone marrow dose with an RBE of 10 for neutrons I Figure 16. Photomicrographs showing FISH with whole-chromosome probes for chromosomes 1,2, and to human metaphase spreads. Painted chromosomes are stained yellow, and unpainted as red. The left panel shows a normal set of chromosomes; the right panel shows a translocation involving a chromosome 4 (indicated by arrows).

9 Awa 171 chromosomes are stained red with propidium iodide, so that target chromosomes (which are stained yellow) can be distinguished easily. Translocated chromosomes contain a mixture of red and yellow parts. The relationship between chromosome aberration frequency and radiation dose in Hiroshima survivors is shown in Figure 16. The gray circles indicate aberration frequency as determined by the FISH method, while black circles indicate the aberration frequency as determined by G-banding for the same subjects. With a few exceptions, the two determinations agree with each other. Figure 17 illustrates the plots of translocation frequencies obtained from those survivors exposed in the open, either shielded or unshielded by a nearby Japanese house or tenement (upper panel), and inside a Japanese house or tenement (lower panel). The range of variation in translocation frequencies as determined by G-banding analysis seems to be wider for those exposed in the open. In contrast, the range of dispersion becomes narrower for survivors exposed inside the Japanese house [ 131. The size of the sample for the study is still too small to derive any conclusive evidence. However, the results suggest that the dose estimates for survivors inside Japanese houses are more accurate than the dose estimates for survivors exposed either in the open or inside the factories at the time of the bombings. Further detailed study is needed to determine the accuracy of available shielding information for estimating doses to individual survivors. The results in Figure 18 show that there is a remarkable agreement for translocation frequencies between the FISH and G-banding methods. However, the scoring efficiencies, which are important in obtaining reliable data, differ significantly with these two methods. The estimated scoring frequency is about 50 cells per day per examiner for the conventional method and cells per day per examiner for the G-banding method. In contrast, 500 metaphases per day per examiner can be scored by FISH. Since chromosomes 1, 2, and 4 together make up about 20% of the total genome, scoring 500 cells by FISH is equivalent to 100 cells by either the conventional or G-banding methods. The FISH method is therefore about six times as efficient as G-banding and thus a very feasible approach. % e J U YI F Inside a Japanese house - "=I9 I - G-banding data - I Hiroshima 1.o i I DS86 bone marrow dose (Sv) I Findings in Persons Exposed to Radiation in Chernobyl In collaboration with the International Atomic Energy Agency (IAEA) we have examined blood samples from hundreds of individuals exposed to radiation in Chernobyl, but the majority of the blood samples failed to culture because of the long duration of transportation. Nevertheless, we were able to examine samples from 24 persons from the contaminated area, and an equal number of samples from persons in the uncontaminated control t Figure 17. Translocation frequencies measured using I Ghnd (dl chrommome(~) G-banding analysis for A-bomb survivors exposed in the open (upper panel) and inside a Japanese house (lower panel). (Cited from [13]). Figure 18. Comparison of Gbanding and FISH methods in detecting translocation.

10 172 Chromosome Aberrations in Atomic Bomb Survivors area (Table 2). Interestingly, the frequency of dicentrics and rings in the control samples is about five in 1,000, which is very high relative to other unirradiated human populations [14]. The aberration frequency for samples from individuals living in the contaminated area is almost the same, with no statistical difference between the two cities. Therefore, further study of residents of the uncontaminated area seems worthwhile. Figure 19 shows two metaphases having severe chromosome damage in a sample from a fireman who worked in the area immediately after the nuclear plant disaster and thus appears to have been exposed to a very high radiation dose. After his duty, he was hospitalized for a number of days with very severe depression of his white blood count. We examined samples of his blood 31 years after the accident and found complex chromosomal aberrations, with many dicentrics and reciprocal translocations. Table 2. Results of evaluation of chromosome aberration data on Chernobyl awident cases RERF Village Number of Persons Mean Age Dicentrics and Rings: Mean per cell (SD) Contaminated 24 (MII, F13) (0.0050) Controls 24 (M20, F4) 46.25a (0.0044) IAEA Contaminatedh 43 (M27, F16) 51.O@ (0.0044) aexcludes eight with unknown age. hexcludes 13 with outlying cytogenetic values (scored at one laboratory) cexcludes three with unknown age. SD, standard deviation; M, male; F, female. Figure 19. Photomicrographs showing complex chromosome aberrations in lymphocytes of a fireman exposed to high radiation levels after Chernobyl accident.

11 Awa 173 CONCLUSIONS A major conclusion of our study of Japanese atomic bomb survivors is that translocation frequency is useful for the evaluation of dosimetry. Data on chromosome aberrations can therefore be considered as a possible surrogate for physical dosimetry in irradiated individuals for whom physical dose assessment is not possible. However, there are discrepancies between biological and physical dose estimates which may be due either to random dosimetry errors or to errors in biological measurement. Possible factors modifying the dose-response relationship in survivors include differences in shielding conditions, since a smaller variance is found for those exposed inside Japanese houses and a greater variance for those with direct (unshielded) exposure. Because of these factors, it is important to verify the uncertainties and reliability of shielding information for individual survivors. The effect of age at the time of the bombing is also an important issue. We should further investigate the possibility of age-dependent differences in radiation-dependent sensitivities. In future research, I would strongly encourage the use of the FISH technique for monitoring chromosomal translocations. ACKNOWLEDGMENTS The Radiation Effects Research Foundation (formerly the Atomic Bomb Casualty Commission, ABCC) was established in 1975 as a private nonprofit Japanese foundation, supported equally by the Government of Japan through the Ministry of Health and Welfare, and the Government of the United States through the National Academy of Sciences under contract with the Department of Energy. REFERENCES 1 Bender MA, Awa AA, Brooks AL et al. Current status of cytogenetic procedures to detect and quantify previous exposures to radiation. Mutat Res 1988;196: KE Buckton. Chromosome aberrations in patients treated with X-irradiation for ankylosing spondylitis. In: Ishihara T and Sasaki MS, eds. Radiation- Induced Chromosome Damage in Man. New York: A. R. Liss, 1983: Bauchinger M. Chromosomenaberrationen undihre zeitliche Veranderung nach Radium- Roentgentherapie gynaekologischer Tumoren. Strahlentherapie 1979;135: Awa AA, Sofuni T, Honda T et al. Relationship between the radiation dose and chromosome aberrations in atomic bomb survivors of Hiroshima and Nagasaki. J Radiat Res 1978;19: Awa AA. Radiation-induced chromosome aberrations in A-bomb survivors - a key to biological dosimetry. In: Prentice RL. Thompson DJ, eds. Atomic Bomb Survivor Data: Utilization and Analysis. Philadelphia: SIAM 1984: Awa AA, Persistent chromosome aberrations in the somatic cells of A-bomb survivors, Hiroshima and Nagasaki. J Radiat Res 1991;(suppl A): Beebe GW, Usagawa M. The Major ABCC Samples. TR 12-68, Atomic Bomb Casualty Commission, Hiroshima, Stram DO, Sposto R, Preston D et al. Stable chromosome aberrations among A-bomb survivors: an update. Radiat Res 1993;136: Sposto R, Stram DO, Awa AA. An estimate of the magnitude of random error in the DS86 dosimetry from data on chromosome aberrations and severe epilation. Radiat Res 1991;128: Seabright M. A rapid banding technique for human chromosomes. Lancet 1971;2: Ohtaki K. G-banding analysis of radiationinduced chromosome damage in lymphocytes of Hiroshima A-bomb survivors. Jpn J Hum Genet 1992;37: Lucas JN, Awa A, Straume T et al. Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. Int J Radiat Biol 1992;62: Nakano M, Kodama Y, Ohtaki K et al. How much does accuracy of information about shielding for individual survivors influence dosimetry error? RERF Update 1994;6:8. 14 International Atomic Energy Agency, The International Chernobyl Project. Technical Report. Assessment of Radiological Consequences and Evaluation of Protective Measures. Report by an International Advisory Committee, IAEA, Vienna, 1991.