A method to differentiate between the levels of ESR signals induced by sunlight and by ionizing radiation in teeth from atomic bomb survivors.

Electron spin resonance (ESR, or electron paramagnetic resonance, EPR) analysis of tooth enamel is an effective method for the retrospective estimation of individual radiation doses. One problem with this technique is that the observed ESR signal may include a contribution from ultraviolet (UV) light exposure from sunlight, especially in front teeth. Thus there has been a need to find ways to estimate the UV-light effect in the total signal so that the net ESR dose from ionizing radiation can be determined. To examine this issue, we measured 96 teeth of various types, but with buccal and lingual parts measured separately, from a control group of atomic bomb survivors (estimated dose <5 mGy). We found that, except for molars, the mean ESR-estimated dose for the buccal halves was, on average, nearly twice that from the lingual side, which indicates that the UV-light-induced lingual dose equals the difference between the two halves. Using these corrections for UV-light exposure to front teeth that had been exposed to both ionizing radiation and UV light, it was found that the estimated radiation doses closely approximated the previously estimated ESR dose to molars from the same donors or the estimated dose arrived at with cytogenetic methods. We concluded that, when using ESR to estimate radiation dose, measuring molars is the first choice, but if only front teeth are available, separate measurements to the buccal and lingual parts can provide an estimation of the mean UV-light contribution to the ESR-determined dose.

Clonally expanded T-cell populations in atomic bomb survivors do not show excess levels of chromosome instability.

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.

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.

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.

Prediction of clonal chromosome aberration frequency in human blood lymphocytes.

We recently conducted a large-scale screening for clonal aberrations among atomic bomb survivors and proposed a model for the gross clonal composition of blood lymphocytes. Here we show an application of the model indicating that the number, m,of clones detectable by cytogenetic methods in an individual is predictable by the equation m= (1.8 + 6.4FG) x FP x n/500, where FG represents the estimated translocation frequency in the 46 chromosome set, FP is the observed translocation frequency with FISH or other methods, and nis the number of cells examined. Application of the equation to the results of seven other reports gave close agreement between the observed and calculated numbers of clones. Since the model assumes that clonal expansion is ubiquitous, and any translocation can be the constituent of a clone detectable by cytogenetic means, the vast majority of observed clonal expansions of these somatic cells are likely the result of random-hit events that are not detrimental to human health. Furthermore, since our model can predict the majority of clonal aberrations among Chernobyl workers who were examined 5-6 years after irradiation, clonal expansion seems to occur primarily within a few years after exposure to radiation, most likely being coupled with the process of recovery from radiation-induced injury in the lymphoid and hematopoietic systems.

Estimating the number of hematopoietic or lymphoid stem cells giving rise to clonal chromosome aberrations in blood T lymphocytes.

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.

Stable chromosome aberrations in atomic bomb survivors: results from 25 years of investigation.

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.

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.

Organ doses from radiation therapy in atomic bomb survivors.

Previous surveys of radiation therapy among the Life Span Study (LSS) population at the Radiation Effects Research Foundation (RERF) revealed that 1,670 (1.4%) of the LSS participants received radiation treatments before 1984. The data on therapeutic radiation doses are indispensable for studying the relationship between radiation treatments and subsequent cancer occurrences. In this study, the radiation treatments were reproduced experimentally to determine the scattered radiation doses. The experiments were conducted using a female human phantom and various radiation sources, including a medium-voltage X-ray machine and a (60)Co gamma-ray source. Doses were measured using thermoluminescence dosimetry and ionization chambers. Radiation doses were determined for the salivary glands, thyroid gland, breast, lung, stomach, colon, ovary and active bone marrow. The results have been used for documenting the organ doses received by patients in previous surveys. The contribution of therapeutic irradiation to the occurrence of chromosome aberrations was studied using data on doses to active bone marrow from both radiation treatments and atomic bomb exposures in 26 RERF Adult Health Study participants. The results suggest that radiation treatments contributed to a large part of their frequencies of stable-type chromosome aberrations. The therapeutic radiation doses determined in the present study are available for investigating the effects of therapeutic irradiation on the subsequent primary cancers among atomic bomb survivors who received radiation treatments.

The F value for chromosome aberrations in atomic bomb survivors does not provide evidence for a primary contribution of neutrons to the dose in Hiroshima.

Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) proposed that the ratio of interchromosomal to intrachromosomal exchanges, termed the F value, can be a cytogenetic fingerprint of exposure to radiations of different linear energy transfer (LET). Using published data, they suggested that F values are over 10 for low-LET radiations and approximately 6 for high-LET radiations. Subsequently, as F values for atomic bomb survivors were reported to be around 6, Brenner suggested that the biological effects of atomic bomb radiation in Hiroshima are due primarily to neutrons. However, the F values used for the survivors were means from individuals exposed to various doses. As the F-value hypothesis predicts a radiation fingerprint at low doses, we analyzed our own data for the survivors in relation to dose. G-banding data for the survivors showed F values varying from 5 to 8 at DS86 doses of 0.2 to 5 Gy in Hiroshima and around 6 in Nagasaki with no evidence of a difference between the two cities. The results are consistent with our in vitro data that the F values are invariably around 6 for X and gamma rays at doses of 0.5 to 2 Gy as well as two types of fission-spectrum neutrons at doses of about 0.2 to 1 Gy. Thus, apart from a possible effect at even lower doses, current data do not provide evidence to support the proposition that the biological effects of atomic bomb radiation in Hiroshima are caused mainly by neutrons.

A close correlation between electron spin resonance (ESR) dosimetry from tooth enamel and cytogenetic dosimetry from lymphocytes of Hiroshima atomic-bomb survivors.

PURPOSE: To estimate gamma-ray doses received by Hiroshima atomic bomb survivors using electron spin resonance (ESR) of tooth enamel and to compare the results with cytogenetic data. MATERIALS AND METHODS: Tooth enamel ESR was performed for 100 teeth donated from 69 Hiroshima survivors, and conventional cytogenetic examinations were conducted for 61 of the tooth donors. To evaluate possible contamination from dental X-ray exposure, which primarily affects the tooth's buccal surface, each tooth was divided into buccal and lingual parts for subsequent independent enamel isolation and ESR measurement. RESULTS: Almost 20 teeth showed considerably larger buccal doses than lingual doses, but most of these discrepant teeth were incisors and canines. The results are probably attributable to solar light exposure. In contrast, the buccal and lingual doses found in molars were similar. Conventional translocation data of lymphocytes and ESR-estimated doses of 40 donors of molars showed the dose-response for translocations to be almost the same as that expected from in vitro gamma-ray irradiation experiments. CONCLUSIONS: Both tooth enamel ESR and lymphocyte cytogenetics are useful measures for individual biodosimetry of acute radiation exposure, even half a century after the exposure occurred.

Estimation of minimal size of translocated chromosome segments detectable by fluorescence in situ hybridization.

Apparent non-reciprocal translocations are commonly observed using fluorescence in situ hybridization. We hypothesize that these are 'hidden' reciprocals due to one translocated segment being too small to detect. Assuming that the translocation breakpoints distribute randomly, the proportion of reciprocal to non-reciprocal exchanges can be used to estimate the minimal detectable size of translocated segments. To estimate segment size in this study, cytogenetic data for 120 A-bomb survivors were used. Among 2295 aberrant metaphases, 1629 exhibited reciprocal translocations and 666 non-reciprocal. Of the non-reciprocal translocations, 501 showed only a painted chromosome segment, translocated to an unpainted chromosome with centromere, and 165 showed only an unpainted chromosome segment, translocated to a painted chromosome with centromere. On the basis of the above two assumptions, we obtained the most likely estimates for minimal detectable sizes: 11.1 +/- 0.8 Mb for the painted and 14.6 +/- 0.6 Mb for the unpainted chromosomes. The implications of these findings are discussed.

Somatic cell mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors: implications for radiation carcinogenesis.

To clarify the relationship between somatic cell mutations and radiation exposure, the frequency of hemizygous mutant erythrocytes at the glycophorin A (GPA) locus was measured by flow cytometry for 1,226 heterozygous atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. For statistical analysis, both GPA mutant frequency and radiation dose were log-transformed to normalize skewed distributions of these variables. The GPA mutant frequency increased slightly but significantly with age at testing and with the number of cigarettes smoked. Also, mutant frequency was significantly higher in males than in females even with adjustment for smoking and was higher in Hiroshima than in Nagasaki. These characteristics of background GPA mutant frequency are qualitatively similar to those of background solid cancer incidence or mortality obtained from previous epidemiological studies of survivors. An analysis of the mutant frequency dose response using a descriptive model showed that the doubling dose is about 1.20 Sv [95% confidence interval (CI): 0.95-1.56], whereas the minimum dose for detecting a significant increase in mutant frequency is about 0.24 Sv (95% CI: 0.041-0.51). No significant effects of sex, city or age at the time of exposure on the dose response were detected. Interestingly, the doubling dose of the GPA mutant frequency was similar to that of solid cancer incidence in A-bomb survivors. This observation is in line with the hypothesis that radiation-induced somatic cell mutations are the major cause of excess cancer risk after radiation exposure. Furthermore, the dose response was significantly higher in persons previously or subsequently diagnosed with cancer than in cancer-free individuals. This may suggest an earlier onset of cancer due to elevated mutant frequency or a higher radiation sensitivity in the cancer group, although the possibility of dosimetry errors should be considered. The findings obtained in the present study suggest that the GPA mutant frequency may reflect the cancer risk among people exposed to radiation.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

Biodosimetry: chromosome aberration in lymphocytes and electron paramagnetic resonance in tooth enamel from atomic bomb survivors.

One hundred enamel samples isolated from extracted teeth donated by atomic bomb survivors were subjected to free radical measurement by means of electron paramagnetic resonance (ESR). Results comparing ESR with the chromosome aberration frequency in lymphocytes of the tooth donors, and with the physically estimated DS86 dose suggested that ESR data correlated more closely with chromosome data than with the estimated DS86 doses, probably because DS86 may depend on erroneous memory in some cases.

Mutant frequency at the HPRT locus in peripheral blood T-lymphocytes of atomic bomb survivors.

The mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase locus in peripheral blood lymphocytes was measured for 254 atomic bomb survivors (171 exposed and 83 control survivors) by a colony assay using recombinant human interleukin-2. Weak but significant effects were detected for atomic bomb radiation dose and smoking status at the time of examination but not for age and sex. However, the slope of the dose-response curve is quite small, and the smoking effect would not have been significant without the inclusion of data from just three individuals with extremely high mutant frequencies. The weakness of the dose response is at least partly due to the time lapse of 50 years since radiation exposure. Among the 254 survivors, 23 had chromosome aberration data in lymphocytes and the dose response was highly significant. However, the correlation between the mutant frequency and the proportion of cells with aberrations was not significant. It was concluded that the lymphocyte mutation assay is presently not sensitive enough for biological dosimetry of radiation exposure in the survivors.

A proposed system for scoring structural aberrations detected by chromosome painting.

The advent of chromosome painting has brought the realization that structural aberrations can be far more complicated than previously imagined. Various investigators have devised their own nomenclature systems to deal with this difficulty, with the result that the terminology has become inconsistent and confusing. Recently, an international group of cytogeneticists experienced in chromosome painting gathered to address this issue. Results of the meeting are presented in this report, which provides a nomenclature system capable of describing chromosome aberrations that occur between painted and unpainted chromosomes, as well as aberrations involving only painted chromosomes. The nomenclature is flexible enough to describe accurately even the extensively rearranged chromosomes. As a consequence of this flexibility, the scheme upon which the nomenclature is based differs substantially from other systems of aberration classification. We call this system the Protocol for Aberration Identification and Nomenclature Terminology (PAINT).

Frequent involvement of visible chromosomal deletion in X-ray-induced mutants at the HLA-A locus in human T-lymphocytes.

Mutant T-lymphocytes at the HLA-A locus were isolated using a recently developed flow-cytometric assay either immediately after drawing blood (in vivo mutants) or after X-irradiation in vitro. Mutants were subsequently propagated clonally for cytogenetic and molecular analyses. Among the 38 in vivo mutants, none contained an abnormal chromosome 6 on which the HLA-A locus resides (6p21.3). In contrast, mutants recovered after in vitro irradiation frequently carried abnormalities in the short arm of chromosome 6: 11/19 and 5/5 independent mutants for the 1-Gy and 2-Gy groups, respectively. Characteristically, the majority of the aberrations were deletions, commonly involving chromosome 6p21-p23. Because chromosomal deletions involving the selected gene are rare among radiation-induced mutants at the hypoxanthine phosphoribosyltransferase (chromosome X) and thymidine kinase (chromosome 17) loci, the HLA-A locus can be considered as highly prone to chromosomal deletions after radiation exposure. It is generally believed that ionizing radiation randomly breaks DNA, and the higher frequency of chromosomal deletions at the HLA-A locus is unlikely to be due to preferential induction but more likely to the better survivability of the deletion-bearing mutants. Consequently, the results suggest that the human genome is quite heterogeneous with regard to the survivability of cells bearing a chromosomal deletion including different loci.

Aneuploidy in somatic cells of in utero exposed A-bomb survivors in Hiroshima.

Cytogenetic data on cultured lymphocytes of the in utero exposed A-bomb survivors in the RERF Adult Health Study cohort have been analyzed using the G-banding technique to determine the frequency of aneuploid cells. The data consist of blood samples collected between 1985 and 1987 from 264 Hiroshima individuals for whom DS86 maternal uterine dose estimates are available: 124 proximally exposed (74 males and 50 females) with an estimated dose of 0.005 Sv or more, and 140 distally exposed (76 males and 64 females) with a dose estimate of 0 Sv, assuming the neutron relative biological effectiveness (RBE) of 10. A main feature of aneuploidy was that aneuploid frequency in autosomes depended generally on chromosome length; aneuploidies were significantly more frequent in shorter chromosomes than in longer chromosomes. The frequency of aneuploidies also depended on type, with chromosome loss approximately five times more frequent than chromosome gain. However, chromosome 21, as well as the sex chromosomes, were notable in that aneuploidy was much more frequent for these chromosomes than would be predicted from a simple relationship with length. X chromosome aneuploidies were significantly more frequent in females than in males. There was no dependence of aneuploid frequencies on dose when measured 40 years after the exposure.