Kazuo Ohtaki

Stable Chromosome Aberrations in Atomic Bomb Survivors: Results from 25 Years of Investigation

Abstract Kodama, Y., Pawel, D., Nakamura, N., Preston, D., Honda, T., Itoh, M., Nakano, M., Ohtaki, K., Funamoto, S. and Awa, A. A. Stable Chromosome Aberrations in Atomic Bomb Survivors: Results from 25 Years of Investigation. Radiat. Res. 156, 337–346 (2001). Frequencies of stable chromosome aberrations from more than 3,000 atomic bomb survivors were used to examine the nature of the radiation dose response. The end point was the proportion of cells with at least one translocation or inversion detected in Giemsa-stained cultures of approximately 100 lymphocytes per person. The statistical methods allow for both imprecision of individual dose estimates and extra-binomial variation. A highly significant and nonlinear dose response was seen. The shape of the dose response was concave upward for doses below 1.5 Sv but exhibited some leveling off at higher doses. This curvature was similar for the two cities, with a crossover dose (i.e. the ratio of the linear coefficient to the quadratic coefficient) of 1.7 Sv (95% CI 0.9, 4). The low-dose slopes for the two cities differed significantly: 6.6% per Sv (95% CI 5.5, 8.4) in Hiroshima and 3.7% (95% CI 2.6, 4.9) in Nagasaki. This difference was reduced considerably, but not eliminated, when the comparison was limited to people who were exposed in houses or tenements. Nagasaki survivors exposed in factories, as well as people in either city who were outside with little or no shielding, had a lower dose response than those exposed in houses. This suggests that doses for Nagasaki factory worker survivors may be overestimated by the DS86, apparently by about 60%. Even though factory workers constitute about 20% of Nagasaki survivors with dose estimates in the range of 0.5 to 2 Sv, calculations indicate that the dosimetry problems for these people have little impact on cancer risk estimates for Nagasaki.

Human Fetuses do not Register Chromosome Damage Inflicted by Radiation Exposure in Lymphoid Precursor Cells except for a Small but Significant Effect at Low Doses

Abstract Ohtaki, K., Kodama, Y., Nakano, M., Itoh, M., Awa, A. A., Cologne, J. and Nakamura, N. Human Fetuses do not Register Chromosome Damage Inflicted by Radiation Exposure in Lymphoid Precursor Cells except for a Small but Significant Effect at Low Doses. Radiat. Res. 161, 373–379 (2004). Human fetuses are thought to be highly sensitive to radiation exposure because diagnostic low-dose X rays have been suggested to increase the risk of childhood leukemia. However, animal studies generally have not demonstrated a high radiosensitivity of fetuses, and the underlying causes for the discrepancy remain unidentified. We examined atomic bomb survivors exposed in utero for translocation frequencies in blood lymphocytes at 40 years of age. Contrary to our expectation of a greater radiosensitivity in fetuses than in adults, the frequency did not increase with dose except for a small increase (less than 1%) at doses below 0.1 Sv, which was statistically significant. We interpret the results as indicating that fetal lymphoid precursor cells comprise two subpopulations. One is small in number, sensitive to the induction of both translocations and cell killing, but rapidly diminishing above 50 mSv. The other is the major fraction but is insensitive to registering damage expressed as chromosome aberrations. Our results provide a biological basis for resolving the long-standing controversy that a substantial risk of childhood leukemia is implicated in human fetuses exposed to low-dose X rays whereas animal studies involving mainly high-dose exposures generally do not confirm it.

Ratios of radiation-produced chromosome aberrations as indicators of large-scale DNA geometry during interphase

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise. Stable chromosome aberrations that exemplify inter- and intra-chromosomal exchanges are, respectively, translocations and pericentric inversions. By comparing the number of these for each chromosome one can infer results on the randomness of DSB induction or exchange formation and on large-scale chromosome geometry. We analyze frequencies of translocations and pericentric inversions in lymphocytes from 38 A-bomb survivors, using G-banding. A total of 636 translocations and 102 pericentric inversions were found. The 636/102 ratio of translocations to pericentric inversions is approximately 14 times smaller than predicted by a random model, in general agreement with earlier results and results on the ratio of dicentrics to centric rings for in vitro irradiation. Presumably the excess of intra-chromosomal exchanges is due to a spatial proximity effect, implying a localization of chromosomes within the cell nucleus during and shortly after irradiation. The distribution of the pericentric inversions among different chromosomes indicates this proximity effect is roughly the same for all chromosomes, regardless of DNA content; i.e., the ratio of pericentric inversions for two different chromosomes approximately equals the ratio given by a model which takes into account chromosome lengths and centromere locations but otherwise assumes randomness. Possible exceptions are chromosomes 7 and 12, which show some excess of pericentric inversions. The percentage of translocations involving each chromosome corresponds roughly to the percentage expected assuming randomness, except that for chromosome 1 there is a significant excess.

Detection of stable chromosome aberrations by FISH in A-bomb survivors: comparison with previous solid Giemsa staining data on the same 230 individuals

Purpose : To evaluate the relative abilities of the solid Giemsa staining (conventional) and fluorescence in situ hybridization (FISH) methods in the detection of stable chromosome aberrations in the peripheral blood lymphocytes of A-bomb survivors. Materials and methods : Lymphocytes from a total of 230 A-bomb survivors for whom prior chromosome aberration data had been obtained by the conventional method were recently examined afresh using FISH in which chromosomes 1, 2 and 4 were painted with composite probes. Results : It was found that the early use of the solid Giemsa staining method had allowed the detection of translocations with a mean frequency of 73% of the value for the genome-equivalent translocation frequency (F G) that was now obtained using FISH. The disparity may at least in part be due to the reciprocal exchange of seemingly identical amount of chromosome material; such exchanges can escape detection by the conventional method but can be readily identified using FISH. Conclusion : It has previously been established that the conventional method can detect about 20% of radiation-induced translocations as abnormal monocentric chromosomes. Present results indicate that an additional 50% can be detected if proper karyotyping is conducted and the remaining 30% are not likely to be detected unless FISH or banding methods are used. Thus, solid Giemsa staining accompanied by karyotyping may not be quite as unsuitable as is generally assumed for retrospective biodosimetry analyses, which deal mainly with stable aberrations.

G-Banding analysis of radiation-induced chromosome damage in lymphocytes of hiroshima A-bomb survivors

SummaryThe present report describes the G-band analysis of somatic chromosomes in lymphocytes from 63 A-bomb survivors in Hiroshima to determine the type and frequency of radiation-induced chromosome aberrations. (1) The cells with stable-type chromosome aberrations (Cs cells) predominated among the aberrant cells, and showed a dose-dependent increase. All stable chromosome aberrations were classified into nine categories: reciprocal translocations, translocations of complex type, insertions, complex exchanges, peri- and paracentric inversions, terminal and interstitial deletions, and unidentified rearrangements. The frequencies of aberrations were found to increase with increasing dose for all aberration categories. Reciprocal translocations predominate in all dose ranges and among the chromosome aberrations classified. (2) The linear model was fitted to test the dose-response relationship for Cs cell frequencies. Employing a constant neutron RBEs of 10, an estimated linear slope of 15.2%/Sv was obtained for DS86 bone marrow dose with an intercept of 2.9% at dose 0. (3) Statistical analysis of data on 3,370 break sites showed good correlations betweens relative DNA content and the distribution of chromosome breaks involved in translocations, although the involvement of chromosome 1 is significantly higher.

Chromosome Aberrations do not Persist in the Lymphocytes or Bone Marrow Cells of Mice Irradiated In Utero or Soon after Birth

Abstract Nakano, M., Kodama, Y., Ohtaki, K., Nakashima, E., Niwa, O., Toyoshima, M. and Nakamura, N. Chromosome Aberrations do not Persist in the Lymphocytes or Bone Marrow Cells of Mice Irradiated In Utero or Soon after Birth. Radiat. Res. 167, 693–702 (2007). Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (≤0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated when ≥6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53−/− mice and 1.4% in p53+/− mice) compared to the frequency in the p53−/− mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

Paracentric inversion of a human chromosome 7

SummaryIn the course of chromosome studies of atomic bomb survivors in Hiroshima using the trypsin-G-banding and Q-banding methods, a 40-year-old male was found to have an abnormal banding pattern in the long arm of a chromosome 7, although no such abnormality was detected by ordinary staining method. Since all other chromosomes apparently had normal banding patterns, the abnormality was determined to be a paracentric inversion of a chromosome 7, which is described as 46,XY,inv(7)(q22q31). This is the first demonstration of a possible paracentric inversion in man.

FISH examination of lymphocytes from Mayak workers for assessment of translocation induction rate under chronic radiation exposures

Purpose: To estimate the translocation-induction rate under chronic exposure conditions by measuring chromosome aberration frequencies in lymphocytes from Mayak nuclear workers using fluorescence in situ hybridization (FISH). Materials and methods: Lymphocytes were examined from 27 nuclear workers at the Mayak Production Association and two control individuals using FISH with probes for chromosomes 1, 2 and 4. Official doses derived from worker film-badge records varied from 0 to 8.50 Gy. Results: The mean (- SD) genome-equivalent translocation frequency (F G) was 2.30 (- 0.75)% in the zero-dose group (n = 7), and Poisson regression analysis provided the best-fit equation of F G (%) = 2.96 (- 0.39) + 0.69 (- 0.14) D + 0.12 (- 0.05) A, where D is the film-badge-derived dose (Gy), and A is age centred at 67 years. The induction rate would increase to nearly 1% Gy -1 if the radiation dose to bone marrow, one of the major organs for lymphocytes and where their precursor cells reside, is considered. Conclusion: The estimated induction rate in vivo appeared substantially smaller than linear coefficients estimated from various in vitro studies.

Estimating the Number of Hematopoietic or Lymphoid Stem Cells Giving Rise to Clonal Chromosome Aberrations in Blood T Lymphocytes

Abstract Nakano, M., Kodama, Y., Ohtaki, K., Itoh, M., Awa, A. A., Cologne, J., Kusunoki, Y. and Nakamura, N. Estimating the Number of Hematopoietic or Lymphoid Stem Cells Giving Rise to Clonal Chromosome Aberrations in Blood T Lymphocytes. Radiat. Res. 161, 273–281 (2004). Quantifying the proliferative capacity of long-term hematopoietic stem cells in humans is important for bone marrow transplantation and gene therapy. Obtaining appropriate data is difficult, however, because the experimental tools are limited. We hypothesized that tracking clonal descendants originating from hematopoietic stem cells would be possible if we used clonal chromosome aberrations as unique tags of individual hematopoietic stem cells in vivo.Using FISH, we screened 500 blood T lymphocytes from each of 513 atomic bomb survivors and detected 96 clones composed of at least three cells with identical aberrations. The number of clones was inversely related to their population size, which we interpreted to mean that the progenitor cells were heterogeneous in the number of progeny that they could produce. The absolute number of progenitor cells contributing to the formation of the observed clones was estimated as about two in an unexposed individual. Further, scrutiny of ten clones revealed that lymphocyte clones could originate roughly equally from hematopoietic stem cells or from mature T lymphocytes, thereby suggesting that the estimated two progenitor cells are shared as one hematopoietic stem cell and one mature T cell. Our model predicts that one out of ten people bears a non- aberrant clone comprising >10% of the total lymphocytes, which indicates that clonal expansions are common and probably are not health-threatening.

Clonally Expanded T-Cell Populations in Atomic Bomb Survivors Do Not Show Excess Levels of Chromosome Instability

Abstract Kodama, Y., Ohtaki, K., Nakano, M., Hamasaki, K., Awa, A. A., Lagarde, F. and Nakamura, N. Clonally Expanded T-Cell Populations in Atomic Bomb Survivors Do Not Show Excess Levels of Chromosome Instability. Radiat. Res. 164, 618– 626 (2005). Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.