Genetic effects from radiation have been observed in a number of species to date. However, observations in humans are nearly nonexistent. In this review, possible reasons for the paucity of positive observations in humans are discussed. Briefly, it appears likely that radiation sensitivity for the induction of mutations varies among different genes, and that the specific genes that were used in the past with the specific locus test utilizing millions of mice may have simply been very responsive to radiation. In support of this notion, recent studies targeting the whole genome to detect copy number variations (deletions and duplications) in offspring derived from irradiated spermatogonia indicated that the mutation induction rate per genome is surprisingly lower than what would have been expected from previous results with specific locus tests, even in the mouse. This finding leads us to speculate that the lack of evidence for the induction of germline mutations in humans is not due to any kind of species differences between humans and mice, but rather to the lack of highly responsive genes in humans, which could be used for effective mutation screening purposes. Examples of such responsive genes are the mouse coat color genes, but in human studies many more genes with higher response rates are required because the number of offspring examined and the radiation doses received are smaller than in mouse studies. Unfortunately, such genes have not yet been found in humans. These results suggest that radiation probably induces germline mutations in humans but that the mutation induction rate is likely to be much lower than has been estimated from past specific locus studies in mice. Whole genome sequencing studies will likely shed light on this point in the near future.

Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic 60 Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the 60 Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated 60 Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.

Ionizing radiation can induce mutations, and the majority of radiation-induced mutations in mammalian cells are deletions. The most critical types of radiation-induced DNA damage are DNA double-strand breaks, and these breaks are repaired by either the homologous recombination (HR) pathway or the non-homologous end joining (NHEJ) pathway. The HR pathway is not as mutagenic as the NHEJ pathway, and it is expected that radiation-induced deletions would usually have little sequence similarity around the deletion junction points. Here we report sequence data from the regions around the rejoined junctions of 33 de novo copy-number mutations (27 deletions and 6 duplications) obtained from offspring sired by male mice that were irradiated at the spermatogonia stage and from nonirradiated controls. The results indicate that deletions can be classified into three major groups. In group 1, nine deletions were found to share long blocks of similar sequences (200–6,000 bp) at the junctions and the deletion size varied extensively (1 kb to 2 Mb) (e.g., illegitimate recombination). In group 2, five deletions shared short identical sequences (0–7 bp) at the junctions, and the deletion sizes were shorter than 200 kb (e.g., micro-homology-mediated repair). Additional three-deletion candidates of this group were also found but turned out to be inherited from mosaic parents. They are therefore not included in germline mutations. In group 3, twelve deletions shared little sequence similarity (only 0–2 bp) at the junctions (likely due to NHEJ repair) and deletion sizes were longer than 200 kb. Group 1 consisted of deletions found in both spontaneous and irradiated genomes and thus, were probably caused by spontaneous events during meiosis or DNA replication. Group 2 consisted mainly of deletions found in nonexposed genomes. Group 3 consisted primarily of deletions that occurred in the irradiated genomes. Among the duplications, we found no indication of any association with radiation exposures. These results indicate that large size (>200 kb) and little sequence similarity around the rejoined sites are likely to be a hallmark of radiation-induced deletions in mice.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a “2M array” with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10 –2 /Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10 –5 /locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction.

In both humans and mice, fetal exposure to radiation fails to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level response to radiation exposure is counterintuitive in view of the generally accepted belief that a fetus is sensitive to radiation. To determine if this is a general phenomenon, both mammary epithelial cells and spleen cells were studied in rats. Fetuses of 17.5 days postcoitus were irradiated with 2 Gy of gamma rays, and mammary tissues were removed 6–45 weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH method. The results showed that translocation frequencies were not only elevated in rats irradiated as fetuses, but were also almost as high as those in rats that were irradiated as adults (12 weeks old, pregnant mothers or young virgins) and examined 6–45 weeks later. There was no evidence of higher sensitivity in fetal cells with respect to the induction of translocations. In contrast, translocation frequencies in spleen cells were not elevated in adult rats irradiated as fetuses but were increased after irradiation of adults as previously seen in mouse spleen cells and human T lymphocytes. In the case of irradiation of adult rats, the induced translocation frequencies were similar between spleen cells and mammary epithelial cells. If we take translocation frequency as a surrogate marker of potential carcinogenic effect of radiation, the current results suggest that fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but this does not contribute to the increased risk of cancer since it has been reported that irradiation of fetal rats of the SD strain does not increase the risk of mammary cancers. Possible reasons for this discrepancy are discussed.

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled 32 P Not I sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2–13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 Not I sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

We previously reported that mouse fetuses or neonates exposed to 2 Gy of X rays showed an unexpectedly low incidence of chromosome damage in lymphocytes, bone marrow, and spleen cells when the mice were subsequently examined at 20 weeks of age. However, cells bearing translocations were occasionally observed that, on the basis of 2-color whole chromosome painting appeared to be clonal descendants. Unfortunately, this approach typically did not permit unequivocal confirmation of their clonality. To overcome this problem, multi-color FISH (mFISH) was employed, which assigns all 21 individual chromosome types of the mouse a unique color. After mFISH analyses of the same cell samples studied previously, it was confirmed that spleen cells of 20-week-old mice irradiated either as 15.5-day fetuses or as 3- to 4-day-old neonates showed translocation frequencies close to zero. Translocations previously suspected as being clonal in nature were confirmed as such by mFISH, which also revealed the presence of an additional clone not previously detected or suspected. Since no evidence of clonality was observed in the irradiated mother, we concluded that in both fetuses and neonates, there exists a small fraction of stem cells that are distinct from the bulk of the stem cell compartment in terms of their ability to acquire and transmit radiation-induced chromosome damage through clonal expansion.

Asakawa, J., Nakamura, N., Katayama, H. and Cullings, H. M. Estimation of Mutation Induction Rates in AT-Rich Sequences Using a Genome Scanning Approach after X Irradiation of Mouse Spermatogonia. Radiat. Res. 168, 158–167 (2007). We have previously used Not I as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 × 10 −5 per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used Afl II (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (± 1.9) × 10 −5 and 2.3 (± 1.0) × 10 −5 per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (± 1.2) × 10 −5 per locus per Gy. The induction rate would be reduced to 0.9 × 10 −5 per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 × 10 −5 per locus per Gy or less, which is close to 1.08 × 10 −5 per locus per Gy, the current estimate for the mean mutation induction rate in mice.

Nakamura, N. A Hypothesis: Radiation-Related Leukemia is Mainly Attributable to the Small Number of People who Carry Pre-existing Clonally Expanded Preleukemic Cells. Radiat. Res. 163, 258–265 (2005). Human leukemia frequently involves recurrent translocations. Since radiation is a well-known inducer of both leukemia and chromosomal translocations, it has long been suspected that radiation might cause leukemia by inducing specific translocations. However, recent studies clearly indicate that spontaneous translocations specific to acute lymphocytic leukemia (ALL) actually occur much more frequently than do leukemia cases with the same translocations. Moreover, the ALL-associated translocation-bearing cells are often found to have clonally expanded in individuals who do not develop ALL. Since radiation-induced DNA damage is generated essentially randomly in the genome, it does not seem likely that radiation could ever be responsible for the induction of identical translocations of relevance to ALL in multiple cells of an individual and hence be the primary cause of radiation-related leukemia. An alternative hypothesis described here is that the radiation-related ALL risk for a population is almost entirely attributable to a small number of predisposed individuals in whom relatively large numbers of translocation-carrying pre-ALL cells have accumulated. This preleukemic clone hypothesis explains various known characteristics of radiation-related ALL and implies that people who do not have substantial numbers of preleukemic cells (i.e. the great majority) are likely at low risk of developing leukemia. The hypothesis can also be applied to chronic myelogenous leukemia and to young-at-exposure cases of acute myelogenous leukemia.

Kodaira, M., Izumi, S., Takahashi, N. and Nakamura, N. No Evidence of Radiation Effect on Mutation Rates at Hypervariable Minisatellite Loci in the Germ Cells of Atomic Bomb Survivors. Radiat. Res. 162, 350–356 (2004). Human minisatellites consist of tandem arrays of short repeat sequences, and some are highly polymorphic in numbers of repeats among individuals. Since these loci mutate much more frequently than coding sequences, they make attractive markers for screening populations for genetic effects of mutagenic agents. Here we report the results of our analysis of mutations at eight hypervariable minisatellite loci in the offspring (61 from exposed families in 60 of which only one parent was exposed, and 58 from unexposed parents) of atomic bomb survivors with mean doses of >1 Sv. We found 44 mutations in paternal alleles and eight mutations in maternal alleles with no indication that the high doses of acutely applied radiation had caused significant genetic effects. Our finding contrasts with those of some other studies in which much lower radiation doses, applied chronically, caused significantly increased mutation rates. Possible reasons for this discrepancy are discussed.

Asakawa, J., Kuick, R., Kodaira, M., Nakamura, N., Katayama, H., Pierce, D., Funamoto, S., Preston, D., Satoh, C., Neel, J. V. and Hanash, S. A Genome Scanning Approach to Assess the Genetic Effects of Radiation in Mice and Humans. Radiat. Res. 161, 380–390 (2004). We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal Not I DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1–9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 ± 0.09) × 10−5/locus Gy–1. The estimate is considerably lower than 1 × 10−5/locus Gy–1, the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.

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 cancerfree 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.

This study was intended to test for a possible early selective loss of relatively radiosensitive individuals from those atomic bomb survivors exposed to high doses using an in vitro X-ray dose-survival assay of peripheral blood lymphocytes. The assay was reasonably reproducible since the coefficient of variation (CV) was 8.2% for the mean $D_{10}$ (the dose required to kill 90% of cells) of 3.39 Gy after 15 repeat tests for one control donor during the study period. The CV for single tests for 201 survivors was essentially the same, i.e., 7.7% with a mean $D_{10}$ of 3.37 Gy, indicating very little heterogeneity of lymphocyte radiosensitivity among individuals. Linear regression analysis of $D_{10}$ on the DS86 dose showed no evidence for the consistent change in average $D_{10}$ values among the survivors exposed to high doses. The results might imply that the G 0 lymphocytes do not express full variations in radiosensitivity and may not be suitable for quantitative measurements of relative radiosensitivity. Alternatively, the very small variation in lymphocyte radiosensitivity may be real and detection of the rare individuals with altered radiosensitivity may require much larger-scale testing. Therefore, no conclusive answer to the question of population bias was provided for the survivors.

A recently developed dose-survival assay using human G 0 T lymphocytes from peripheral blood was employed to assess possible interindividual variation of cellular radiosensitivity by comparing variability between a single test for different individuals and repeated tests for a single donor. The surviving fraction at each X-ray dose level fluctuated similarly between the two groups, and the X-ray dose required to kill 90% of the cells ( $D_{10}$ ) was 3.59 ± 0.18 Gy (mean ± SD) for 31 different individuals and 3.66 ± 0.21 Gy for 28 repeated tests of one individual. Analysis of variance to compare the two sets of data showed that variation in the $D_{10}$ value was not significantly greater in the former group. Analysis of D 50 and D 90 showed similar results. These results support the hypothesis that interindividual variation in cellular radiosensitivity is quite small, if it exists at all, as far as can be determined by the loss of colony-forming ability of irradiated G 0 lymphocytes.

The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study (M. Hakoda et al., Mutat. Res. 197, 161-169, 1988) showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4 + and CD8 + cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4 + and CD8 + cells were different from each other. In the present study, CD4 + (helper/inducer T) and CD8 + (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the $D_{10}$ (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells [3.13 ± 0.10 Gy (mean ± SD) for CD4 + , 3.34 ± 0.50 Gy for CD8 + and 3.14 ± 0.17 Gy for the unsorted cells], supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.

Dose-survival curves were obtained for matched samples of peripheral T-lymphocytes and skin fibroblasts from a total of 22 patients who underwent various surgical procedures using loss of colony-forming ability as the end point. The results showed that the mean $D_{10}$ (dose required to kill 90% of cells) ± SD was 3.58 ± 0.21 Gy for T-lymphocytes irradiated in G 0 and 3.19 ± 0.37 Gy for skin fibroblasts irradiated in log phase. The coefficients of variation were found to be 6 and 11%, respectively. Contrary to the expectation, regression analysis of $D_{10}$ values for the two types of cells revealed no significant correlations. The absence of correlation most probably derives from the fact that the apparent interindividual variability of dose-survival curves is caused primarily by random experimental fluctuations at least in the case of lymphocytes. Possible reasons for the greater variability observed in the fibroblast assay are discussed.

Cultured human thyroid cells were X-irradiated in vitro and assayed for resistance to 6-thioguanine. The average mutant frequency was <tex-math>$1.69\pm 1.34\times 10^{-5}$</tex-math> (mean ± SD) in controls, <tex-math>$3.74\pm 2.21\times 10^{-5}$</tex-math> in cells exposed to 1 Gy, and <tex-math>$7.19\pm 5.37\times 10^{-5}$</tex-math> in cells exposed to 2 Gy. The positive association between mutant frequency and dose was statistically significant. The estimated mutation induction rate was <tex-math>$2.54\pm 0.71\times 10^{-5}$</tex-math> per gray, which is in close agreement with published results for human skin fibroblasts and mammary epithelial cells. These results extend and confirm earlier reports that mutation induction rates for fibroblasts and epithelial cells after exposure to X rays are similar.

Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and exposed to X irradiation to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5%, the average mean lethal dose D 0 was 97.6 cGy for HY cells and 96.7 and 94.3 cGy, respectively, for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average D 0 of 85.0 cGy and PC cells a significantly (P = 0.05) lower average D 0 of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (P = 0.82) or sex (P = 0.10). These results suggest that malignant thyroid cells may be especially radiosensitive.