THE COMPARATIVE RADIATION GENETICS OF HUMANS AND MICE

James V.Neel

Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109–0618

Susan E.Lewis

Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709

KEY WORDS: mutation rates/induced/mouse, mutation rates/induced/man, genetic doubling dose/radiation/man, genetic doubling dose/radiation/mouse, low-level radiation/genetic effect

CONTENTS

   

 INTRODUCTION

 

328

   

 A PRÉCIS OF THE HUMAN DATA ON THE GENETIC EFFECTS OF ACUTE RADIATION

 

328

   

 THE GENERATION OF AN ESTIMATE OF THE DOUBLING DOSE FROM THE HUMAN DATA

 

335

   

 SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE HUMAN DOUBLING DOSE

 

337

   

 A PRÉCIS OF THE MOUSE DATA ON THE GENETIC EFFECTS OF ACUTE RADIATION

 

338

   

 THE GENERATION OF AN ESTIMATE OF THE DOUBLING DOSE FROM THE MOUSE DATA

 

352

   

 SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE MOUSE DOUBLING DOSE

 

355

   

 CONCLUSIONS

 

356

Reproduced, with permission, from the Annual Review of Genetics, vol. 24, © 1990 by Annual Reviews, Inc.



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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study THE COMPARATIVE RADIATION GENETICS OF HUMANS AND MICE James V.Neel Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109–0618 Susan E.Lewis Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709 KEY WORDS: mutation rates/induced/mouse, mutation rates/induced/man, genetic doubling dose/radiation/man, genetic doubling dose/radiation/mouse, low-level radiation/genetic effect CONTENTS      INTRODUCTION   328      A PRÉCIS OF THE HUMAN DATA ON THE GENETIC EFFECTS OF ACUTE RADIATION   328      THE GENERATION OF AN ESTIMATE OF THE DOUBLING DOSE FROM THE HUMAN DATA   335      SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE HUMAN DOUBLING DOSE   337      A PRÉCIS OF THE MOUSE DATA ON THE GENETIC EFFECTS OF ACUTE RADIATION   338      THE GENERATION OF AN ESTIMATE OF THE DOUBLING DOSE FROM THE MOUSE DATA   352      SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE MOUSE DOUBLING DOSE   355      CONCLUSIONS   356 Reproduced, with permission, from the Annual Review of Genetics, vol. 24, © 1990 by Annual Reviews, Inc.

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study INTRODUCTION The attempt by geneticists to predict the genetic consequences for humans of exposure to ionizing radiation has arguably been one of the most serious social responsibilities they have faced in the past half century. Important for its own sake, this issue also serves as a prototype for the effort to evaluate the ultimate genetic impact on ourselves of other human perturbations of the environment in which our species functions. Recently we (67–69) have been developing the thesis that according to the results of studies on the children of survivors of the atomic bombings, humans may not be as sensitive to the genetic effects of radiation as has been projected by various committees on the basis of data from the most commonly employed paradigm, the laboratory mouse (cf 12, 104, 105). In this review we attempt as detailed a comparison as space permits of the findings on humans and mice, presenting the data in a fashion that will enable those who at certain critical points in the argument wish to make other assumptions, to do so. We argue that a reconsideration that includes all the data now available on mice brings the estimate of the doubling dose for mice into satisfactory agreement with the higher estimate based on humans. Since the concept of a doubling dose is critical in these discussions, a clear definition is needed at the outset. The genetic doubling dose of radiation is the amount of acute or chronic radiation that will produce the same mutational impact on a population as occurs spontaneously each generation. To be societally relevant, that estimate is best expressed in terms of morbidity and mortality. For both spontaneous and induced mutation, the reference is customarily first generation effects. This definition in principle encompasses the impact of the gamut of all possible alterations in DNA, from single nucleotide substitutions and very small insertions and deletions to gain or loss of entire chromosomes. The definition is as simple as the concept is useful. Expressing genetic damage as a doubling dose provides a convenient societal frame of reference. Unfortunately, for neither mice nor humans is the impact of spontaneous mutation each generation as yet satisfactorily defined. Thus, in the calculation of a doubling dose, the errors inherent in estimating the magnitude of a radiation effect may be further compounded by imprecision in the definition of baseline values. We present radiation in the units employed by the investigator, but for our own studies and analyses present the results in gray (1 Gy=100 rad) and sieverts (1 Sv=100 rem). In the experimental settings reviewed herein, one r may be equated to one rad (rem). A PRÉCIS OF THE HUMAN DATA ON THE GENETIC EFFECTS OF ACUTE RADIATION This treatment of the human data is confined to the information accumulated in Hiroshima and Nagasaki over the past 40 years, because of their high

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study information content compared to other sets of human data, the relative accuracy of radiation-exposure estimates, and a study design that provides essentially nonoverlapping indicators of radiation effects. The Genetic Program that evolved in Japan has been described in detail on several occasions (4, 39, 64–67, 93), Briefly, in the immediate postwar period in Japan, the economic stringencies were such that a rationing program initiated during the war years to benefit pregnant women was continued. This involved registration of the fact of pregnancy at the completion of the fifth lunar month of gestation. By incorporating the registrants into a genetic study, a prospective investigation of pregnancy outcome in Hiroshima and Nagasaki was initiated in February, 1948, that included reference to sex ratio, congenital defect, viability at birth, birth weight, and survival of child during the neonatal period. This more clinical program, which included a physical examination of all newborn infants by a physician, was terminated in February, 1954, but the collection of data on the sex ratio and survival of live-born infants continued, with ascertainment of additional births now through city records. In addition, from the birth registrations in these two cities, the sample for the consideration of these latter two indicators was extended backward in time, to include births between May, 1946, and January, 1948. During the clinical program in Hiroshima, some 62% of all infants who were stillborn or died in the first six days of life were subjected to autopsy. A subset of some 26% of the infants born during this period was reexamined at approximately 9 months of age. Relatively few survivors beyond 2000 meters from the hypocenter were exposed to radiation from the bombs; the mortality within the 2000 meter zone was high. Accordingly, the original sample of 76,617 children plus subsequent additions contained many more born to parents at distances from the hypocenter >2000 meters ATB, who seldom received increased radiation from the explosions, than to parents within the 2000 meter radius, whose gonadal doses were relatively large. In 1959, to increase the efficiency of the study of survival, three cohorts were defined from the children born in the two cities since the bombings: The first comprised all children born between May 1, 1946, and December 31, 1958, to parents one or both of whom were within 2000 meters of the hypocenter ATB (proximally exposed); the second was composed of age, sex, and city-matched births to parents one or both of whom were distally exposed to the bombs (>2500 meters from the hypocenter); and the third, of age, sex, and city-matched children born to parents now residing in Hiroshima and Nagasaki, neither of whom were in the cities ATB. Children born to parents within the 2000–2500 meter radius of distance from the hypocenter are omitted from consideration, because of the exceptional difficulties in estimating the (low) exposures at this distance and the work involved in following children who would contribute relatively little information regarding radiation effects. Setting the earliest date for admission into the

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study cohort at May 1, 1946, effectively eliminates from the sample all children in utero ATB. These cohorts have been periodically expanded to include matched numbers of recent births, the limiting factor in the expansion being the number of births to the proximally exposed parents. In 1984, some 39 years after the birth of the first children conceived following the bombings, there were only three births to proximally exposed parents and the cohorts were closed, effective January, 1985. The cohort of children born through 1982 (the cut-off date for analyses) to parents receiving > 0.01 Gy of radiation now equals 31,150. In addition to the study of the survival of these and the “control” children, special studies have been mounted on the physical development of these children during their school years and on the incidence of cancer. In 1968, a search for cytogenetic abnormalities in those children in the cohorts over 12 years of age was initiated, and, in 1975, a search was undertaken in these same cohorts for mutation altering the electrophoretic mobility and/or function of a select battery of proteins. For both these studies, the necessary controls were drawn from the children of parents not receiving increased radiation ATB. The approach to assigning gonadal doses to the survivors underwent a drastic revision during the early 1980s, resulting in the system known as Dose Schedule 1986 (DS86) (81). All the genetic data have now been analyzed on the basis of the new dosage schedules, supplemented, where DS86 dose are not available, by an ad hoc procedure (cf 74). The radiation resulting from the explosions was predominantly gamma, but included a small neutron component. These two components have been separately estimated, and for these exposure levels, the neutron component assigned a Relative Biological Efficiency (RBE) of 20 (cf 37). This permits assigning the total dose in sieverts. The average conjoint parental gonadal exposures of course vary from study to study, as the composition of the parents of the children examined varies, but in general has been between 0.32 and 0.60 Sv; the distribution of conjoint doses is markedly skewed to the right. Given the chronology of this study, the findings must apply almost exclusively to the results of the radiation of spermatogonia and early, immature oocyte. We turn now to a consideration of the data. Untoward pregnancy outcome Because of the interrelatedness of major congenital defect, stillbirth, and neonatal death, we have treated these endpoints as an entity, termed Untoward Pregnancy Outcome (UPO), defined as an outcome resulting in a child with major congenital defect and/or stillborn and/or dying within the first two weeks of life. Between 1948 and 1954, data were collected on these outcomes for a total of 76,617 newborn infants in Hiroshima and Nagasaki, on 69,706 of whom the data were sufficiently

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study complete in all respects to permit inclusion in the analysis (cf 66). A total of 3,498 children were classified as UPOs. The congenital defects considered major are listed in Neel & Schull (66) and Neel (62). Although the data have been analyzed in many ways, we will for this and the subsequent endpoints to be considered present only the results derived from fitting a simple linear dose-response model to the occurrence of the various indicators of radiation-related damage. This model yields a regression coefficient per Sv (ß), and an intercept term, a. Table 1 presents the results of the most recent analysis, based on the use of both DS86 and the ad hoc doses (74). The regression of indicator on dose is positive, and so consistent with the hypothesis that genetic damage was produced, but well below the level of significance. “Prereproductive” deaths among live-born infants (exclusive of those resulting from a malignant tumor) The mortality data on the three cohorts of live-born children described earlier extend through 1985, when the mean age of the members of the cohorts was 26.2 years. The data are thus relatively complete for death during the prereproductive years. Details of the organization of the study have been presented elsewhere (39, 64). After exclusion of the neonatal deaths included in the analysis of the foregoing section, and cancer deaths (to be discussed in the next section), the three cohorts together include 67,202 individuals, among whom there have been 2,584 deaths. The most recent estimate of the regression of death on parental exposure, employing DS86 doses, is presented in Table 1 (63). Again, the regression is positive, as might be expected if deleterious mutations had been produced, but less than its standard error. Malignancies in the F1 A minority of certain cancers of childhood, such as retinoblastoma and Wilms' tumor, result from the presence of one defective gene inherited from a parent plus a second defective allele of this gene resulting from a somatic mutation in the child. Most cancers of childhood, however, seem to be entirely due to somatic cell events occurring in the child. Only the frequency of the former type might be expected to respond to parental radiation. The normal alleles at the loci associated with these “genetic” cancers are usually referred to as “tumor-suppressing” genes. In addition, the occurrence of proto-oncogenes is well established; in principal, radiation could introduce mutations in proto-oncogenes, although thus far there are no clear examples in humans of a genetic (inherited) predisposition to cancer (or any other trait) due to a mutant proto-oncogene. On the thesis that the frequency of tumor-suppressor and proto-oncogenes was probably such that collectively they constituted a significant target for radiation, the incidence of cancer in the populations under consideration was studied, using both death

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study Table 1 An estimate of the genetic doubling doses that can be excluded at specified confidence levels by the human data. Further explanation in text.   Regression Observed total background Mutational contribution background* Mutational component Lower confidence limit (Sv) Trait ßSV a (a) (b) (%, b÷a) 99% 95% 90% UPO 0.00264 0.03856 0.0502 0.0017– 3.4–5.4 0.14– 0.18– 0.21– ±0.00277 ±0.00582   0.0027   0.23 0.29 0.33 F1 Mortality 0.00076 0.06346 0.0458 0.0016– 3.5–5.7 0.51– 0.68– 0.81– ±0.00154 ±0.00181   0.0026   0.83 1.10 1.32 F1 Cancer –0.00008 0.00104 0.0012 0.00002– 2.0–4.0 0.04– 0.05– 0.07– ±0.00028 ±0.00033   0.00005   0.07 0.11 0.15 Sex-chromosome aneuploids 0.00044 0.00252 0.0030 0.0030 100 1.23 1.60 1.91 ±0.00069 ±0.00043             Protein variants –0.00001 0.00001 0.000013 0.000013 100 0.99 2.27 7.41 ±0.00001 ±0.00001             * per diploid locus

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study certificates and the cancer registries maintained in Hiroshima and Nagasaki (109). There were 43 cancers in the 31,150 children born to exposed parents and 49 in the 41,066 children of unexposed parents, for an incidence in the latter of 1.2/1000 persons. As shown in Table 1, the regression on exposure was very small and negative, i.e. counter hypothesis. This was true both for all cancers and for those thought most likely to fit the retinoblastoma model, i.e. retinoblastoma, Wilms' tumor, neuroblastoma, embryonal carcinoma of the testis, and sarcomas (considered an alternative expression of the retinoblastoma allele). None of these latter tumors was in this series associated with a positive family history. Frequency of balanced structural rearrangements of chromosomes and of sex-chromosome aneuploidy Cytogenetic studies on the children of survivors were initiated in the 1960s, involving members of the F1 Mortality Study described earlier (2–4). Two age- and sex-matched samples were established, one drawn from the children of proximally exposed and the other from the children of the distally exposed. To increase participation, no effort was made to obtain the venous blood samples necessary to the study from children under age 13. The study will therefore not supply reliable data on chromosomal abnormalities associated with high childhood mortality (including Down syndrome under the conditions of postwar Japan) but is thought to provide appropriate data on the occurrence of sex-chromosome aneuploidy and balanced structural rearrangements of chromosomes, the latter defined as reciprocal translocations, pericentric inversions, and Robertsonian translocations. Awa et al (4) detected 19 individuals with sex-chromosome aneuploidy in the 8322 children of proximally exposed parents who were examined, and 25 such individuals in the 7976 children of controls. Individuals with some of these abnormalities are sterile (XXY) and with others may exhibit decreased fertility (XXX); furthermore, XYY and XXX individuals only rarely have similarly affected children (19). Thus a very high proportion of these persons may be presumed to result from primary nondisjunction (i.e. mutation) in a parent, but specific family studies were not undertaken on these individuals. DS86 doses were not available to Awa et al (4); we have now distributed these children according to joint parental gonad exposure and derived the appropriate regressions (67). The regression of incidence on dose (Sv) is 0.00044 ± 0.00069, with an intercept of 0.00252 ± 0.00044 (Table 1). Awa et al (4) also encountered 18 balanced structural rearrangements in the 8322 children of proximally exposed parents and 25 in the 7976 children of controls. Family studies could be performed on about 60% of the children, and one de novo mutational event was detected in the children of the exposed

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study and one in the controls. With adjustment for the children not subjected to family studies, Awa et al (4) estimate a mutation rate for both groups of 1.0× 10-4/gamete/generation, with a 95% confidence interval between 0.2 and 4.5 ×10-4. The number of mutations was not felt sufficient to warrant a regression analysis. “Point” mutations affecting protein charge and/or function As used throughout this treatment, the term “point” mutation of necessity encompasses not only specific nucleotide changes but also small insertions/ deletions/rearrangements. Between 1972 and 1985, a battery of 30 serum transport and erythrocyte enzyme proteins were screened for the occurrence of mutations altering electrophoretic mobility, and a subset of 11 of these proteins (all enzymes) were screened for mutations resulting in loss of function (65). Three mutations altering electrophoretic mobility were encountered in the equivalent of 667,404 locus product tests on children of proximally exposed persons, and the same number (3) in the equivalent of 466, 881 tests on the children of distally exposed parents. These mutations were teased out of family studies on some 964 rare protein variants that had the potential of resulting from mutation in the parental generation. The examination of a subset of 60,529 locus products for loss of enzyme activity in the children of proximally exposed parents yielded 26 rare variants, one of which proved to be a newly arisen mutation; no mutations were encountered in the 21 variants identified in the 61,741 determinations on the children of the comparison group. When the data on the mobility and loss-of-function mutations were combined, the mutation rate in the children of proximally exposed was 0.60×10 -5/locus/generation, with 95% confidence intervals between 0.2 and 1.5×10-5/locus/generation. In the comparison group the rate was 0.64×10-5/locus/generation, with 95% intervals between 0.1 and 1.9×10-5/locus/generation. The average conjoint parental exposure for the proximally exposed was 0.41 Sv and the regression of mutation occurrence on conjoint parental gonadal exposure was –0.00001±0.00001/ Sv with an intercept of 0.00001±0.00001 (Table 1) (67). Sex ratio The relation of sex ratio to parental radiation history in the Japanese experience has been presented on several occasions. With the discovery of sex-chromosome aneuploids in 1959 and recognition of the Lyonization phenomenon in 1961, it became clear that the interpretation of any observed change in the phenotypic sex-ratio at birth would not be simple, and no further analyses have been conducted on sex-ratio since our 1966 publication. The demonstration of no significant difference in the occurrence of sex-chromosome aneuploidy in the two groups, as described earlier, now indicates this phenomenon is not a complication in our findings regarding

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study sex-ratio. Because of Lyonization, the least complicated data on sex-ratio concern the offspring of exposed mother:unexposed father marriages, where the expectation is for a decrease in the frequency of male offspring if the mutation rate had been increased. In our last analysis (92), the regression, based on the previous (T65DR) dose schedule, was 0.0027±0.0040/Sv, i.e. insignificantly counter hypothesis. Since we see no way to bring these data into the framework of a doubling-dose estimate (see below), these data have not been reanalyzed or extended with the advent of the DS86 dose system. Anthropometric studies Birthweights were obtained on the 69,706 children examined during the clinical phase of the program, and weight, body length, head circumference, and chest circumference obtained on a subset of 18,498 of these infants reexamined between ages 8 and 10 months (66). In addition, the annual school measurements (height, weight, sitting height, and chest circumference) have for a subset of Hiroshima school children been analyzed in relation to parental radiation exposure, using the T65DR schedule (22–26). No significant or suggestive differences existed between the children of proximally exposed and the control series. THE GENERATION OF AN ESTIMATE OF THE DOUBLING DOSE FROM THE HUMAN DATA The attempt to derive both the lower limits to the genetic doubling dose(s) compatible with these data and the most probable doubling dose consistent with all the data has presented a number of unique problems recently described in detail (67). On the other hand, an unusual and favorable feature of this study is that all of its various components have been based on a well-defined population subsampled in various, essentially nonoverlapping ways. The result is a multifaceted appraisal of the genetic impact of the bombs on what is essentially a single cohort. We will enumerate below some of the ways in which these studies lack the elegance that can be achieved in an experimental setting. They are, however, based on a single complex population exhibiting the full range of human heterogeneity. Furthermore, the subjects of the study have been born over a sufficiently extended time interval that we begin to approximate the effects on an entire generation rather than on a “window” of access to a very limited period in the reproductive histories of the parents. We take the position that in the studies in Hiroshima and Nagasaki we are not testing the hypothesis that radiation is mutagenic. It has been so for every properly studied species of prokaryote and eukaryote, and Homo sapiens cannot be an exception. The present data are thus the best approximation to the genetic effect of the bombs to be available for some years. To explore

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study their precise implications requires specification for each indicator of the contribution of mutation in the parental generation to the frequency of the trait. For sex-chromosome aneuploids, reciprocal chromosome exchanges, and protein variants, this frequency can and has been directly established by the appropriate family studies. For UPOs, and death and cancer among live-born infants, this is not the case; elsewhere we have attempted to generate the estimate of the contribution of spontaneous mutation in the parental generation to these events that is necessary to derive a doubling dose (67). Table 1 summarizes the results of this effort. Column 4 presents the incidence of the events to be used in subsequent calculations. For the first three items, it is total incidence; for the last two, where it can be specified, it is mutational incidence. Because of the paucity of balanced chromosomal rearrangement that can be attributed to mutation in the parental generation, this indicator is not incorporated in the calculations to come. The fifth column presents the estimated incidence of the trait as a result of spontaneous parental mutation; for the last two indicators it is of course identical with the entry in column 4. The range reflects the uncertainty in the estimate. A detailed justification of these estimates is presented elsewhere (67). Column 6 attempts to provide perspective by indicating the percent of the entry in column 4 to be attributed to spontaneous parental mutation. Note—contrary to Sankaranarayanan's (90) unfortunate erroneous perception of how the doubling dose has been estimated—that only a small fraction of these three indicators, ranging from 2–5%, is attributed to mutation in the parental generation. Finally, by a standard statistical procedure presented elsewhere (67), we have calculated (cols 7–9) for the various indicators lower confidence limits for the doubling dose at the specified probabilities. The range given for the first three indicators results from the range in the estimated mutational component for each. Such estimates as these five can be combined only on the assumption that they are drawn from a common pool of estimates, i.e. that the doubling doses for the individual phenomenon are essentially the same. An extensive literature suggests that the true doubling dose for radiation-induced sex-linked chromosome aneuploids and protein variants (resulting from nondisjunction and nucleotide substitutions, respectively) is probably higher than the doubling dose for the other three indicators (resulting predominantly from insertion/ deletion/rearrangement events and unrepaired chromosome breaks). We have therefore pooled the first three and last two estimates of Table 1, using a statistical procedure described elsewhere (67). The resulting lower confidence limits, at the 95% confidence limit, are between 0.63–1.04 Sv for the first three indicators pooled and 2.71 Sv for the last two. The data of Table 1 can also be used to derive the most probable overall doubling dose suggested by these data. For this we assume the effect on the F1

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study of any mutations resulting in protein variants (i.e. point mutations) is subsumed within the UPOs and F1 mortality. Since we are basically working with independent observations on the same cohort, we have suggested that the doubling dose can be derived by dividing the sum of the first four entries in column 4 (0.00632–0.00835) by the sum of the five regression terms (0.00375), resulting in an estimate of 1.69–2.23 Sv. There is satisfactory agreement between this estimate and the minimal 95% probability estimates. The most probable estimate cannot be compared directly with any estimate based on acutely irradiated mice because with humans, both sexes enter into the estimate, the average conjoint gonadal exposure being 0.3–0.6 Sv, depending on the precise study. Moreover, the dosage curve is highly skewed to the right, some couples having received an estimated joint gonadal dose as high as 6.0 Sv. The mouse data, on the other hand, are largely derived from gonadal doses to one sex, the male, of 3.0 or 6.0 Gy (=Sv). The difference in radiation exposures can in principle be met by converting both the human and mouse data to equivalent results from low-dose rate, low-LET radiation (LET =linear energy transfer). The many difficulties in extrapolating from acute, relatively high-level radiation effects to the effects of low-level, chronic, or intermittent exposure are detailed in Report 64 of the National Council on Radiation Protection (61), and yet the effort must be made, both to render the man-mouse comparison more valid and to supply guidelines for human exposures. Russell et al (89) have demonstrated with the 7-locus system that high-level, acute radiation exposures are at least three times more mutagenic than low-dose pulsed or chronic radiation. Other indicators, notably those on translocation frequencies, have yielded higher dose rate factors (revs, in 98, 106). Following the linear quadratic treatment of the mouse data by Abrahamson & Wolff (1), we suggest applying a dose rate factor of 2 to convert the Hiroshima-Nagasaki data into their low-dose rate, low-LET radiation equivalent. The resulting figure is 3.38–4.46 Sv. This is a conservative estimate, since it does not incorporate three sets of data that showed no hint of an exposure effect, namely, the data on the balanced chromosomal exchanges, the sex-ratio, and physical development. We find it impossible to set limits in the usual statistical sense on this estimate, because of statistical sampling errors and the uncertainty we have described in establishing the contribution of parental spontaneous mutation to the indicators in the control population. SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE HUMAN DOUBLING DOSE As we turn to a similar treatment of the accumulated mouse data, the following features of the human data must be kept in mind: None of the studies has yielded a statistically significant difference be-

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study Sex-ratio In the mouse, the sex-chromosome is about 1/20th of the total genome. Thus, in principle there could be of the order of 1000–2000 genes on the X susceptible to radiation damage, perhaps half of which could yield lethal mutations. The published data on the effect of radiation on the mouse sex-ratio are confusing (reviewed in 29 , 30 , 57). Most early studies found no effect of radiation of males on the sex-ratio of the F1 or F2; in those studies that report findings in the F2 of irradiated males consistent with the induction of sex-linked lethals, efforts to isolate these presumed lethals have failed (cf 29), leading Lüning & Sheridan (51) to conclude that changes in the sex-ratio are an “unacceptable” way to search for sex-linked recessive lethals. Lyon et al (57) met many of the early problems by developing a strain with an X-chromosome inversion encompassing about 85% of the X-chromosome. Treated (500+500 rad) males of the F1 C3H/HeH×101/H stock were mated to females heterozygous for the inversion. Two of 536 irradiated and 0/529 control X-chromosome carried a confirmed lethal, corresponding to a rate for recessive lethals of 1.9×10-6/rad/X-chromosome. The authors suggest that this points to a locus rate much below that of the 7-locus system, but in the absence of control mutations, no doubling dose can be calculated. The murine data most comparable to the least ambiguous aspects of the human data would be the proportion of males in the offspring of irradiated females. We have found no published murine data of this type. Accordingly, we have tabulated the (tertiary) sex-ratio observed at weaning in several relatively small experiments involving female radiation performed in the laboratory of one of the authors (S.E.L.) (Table 5). Note that these results are based on the radiation of mature oocytes. Two observations are noteworthy. First, in the controls (and irradiated) there is an insignificant excess of females (X2=1.658, d.f.=1, 0.10<p<0.20), whereas in humans at a corresponding stage in development, there is a slight excess of males. Second, the difference between the offspring of control and treated animals is (nonsignificantly) counter-hypothesis in sign (X2=0.124, d.f.=1, 0.70<p <0.80). Once again, loci on the X-chromosome appear resistant to the induction of lethal mutations. Table 5 The sex ratio at weaning in the offspring of females treated with 300r and mated during the week following treatment.   No. litters No. progeny at birth Average birth litter size Females Males Total M/F Ratio % Survival Treated 488 3,265 6.7 1,475 1,331 2,806 .902 85.9±0.7 Concurrent control 51 390 7.6 171 148 319 .865 81.8±2.2

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study Anthropometrics We have found no analysis of data on mouse body weight or other measurements in the first generation following parental radiation that allows for the litter-size effect. Accordingly, there do not seem to be any data suitable for comparison with the measurements on humans. THE GENERATION OF A DOUBLING DOSE FROM THE MOUSE DATA Unlike the human situation, in the various experiments with mice it has usually been possible to drive the system under study with sufficient radiation to obtain significant differences between the experimental and control material. This should increase the precision in estimation procedures and provide us in principle with two alternatives in calculating a doubling dose. On the one hand, we can simply combine the results of the various specific-locus/ specific-phenotype studies. This provides a very definite and clear-cut endpoint, which, however, does not readily translate into a morbidity-mortality figure comparable to the basis for the estimation of the human doubling dose. On the other hand, we can attempt to proceed as with the human data. This requires estimating the contribution of mutation in the parental generation to the incidence of malformed and dead fetuses, preweaning mortality, and sex-chromosome aneuploids, with the specific-locus findings represented in the calculation through their heterozygous effects on malformations and fetal deaths and preweaning mortality. While this procedure produces a more relevant figure, it requires assumptions that are probably even softer for mice than for humans. In the derivations of a mouse gametic doubling dose to be extrapolated to humans, it is essentially the first approach that has been employed in the past. On this basis, various national and international committees have set the doubling dose of acute low-LET gonidal radiation for humans at 30 to 40 r, with limits of 10 to 100 r (reviewed in 12 , 105). That estimate appears to be based primarily on the data summarized pictorially by Lüning & Searle in 1971 (50 ; see also 97), namely, data on semisterility (=reciprocal translocations), the 7-locus system, dominant visible mutations, dominant skeletal mutations, and recessive lethals. The average of these values at that time was 31 r. Our Table 3 in essence extends the approach of Lüning & Searle (50), adding new data but, for consistency in the use of specific-locus/specific-phenotype results, omitting the findings on reciprocal translations (treated elsewhere in this review). The average of the eight estimates of Table 3 is 1.35 Gy. As with the human data, these data sets are of variable information content, but none can justifiably be disregarded, and collectively they comprise a formidable body of data. Three of these estimates, however, are based on the

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study occurrence of a single mutation in the controls (cataract, skeletal defects, protein variants). One additional mutation in the controls would double the estimate of the doubling dose for these three outcomes. Thus, this estimate of 1.35 Gy carries a considerable potential for error. For the high doses of radiation employed in these studies, the appropriate dose-rate factor in converting to the effects of low-dose, chronic, or intermittent radiation is no less than 3.0 (86 , 87). This would lead to an estimate of the doubling dose for chronic radiation of approximately 4.0 Gy. The data in Table 3 are based on spermatogonial radiation, and are thus a male-oriented figure. When the failure to recover specific-locus mutations in the late litters of irradiated females (88) is considered, that doubling-dose estimate must be increased, to an indeterminate degree (but less than doubled). The “information content” of the 7-locus test in particular outweighs that of any of the seven other specific-locus/specific-phenotype systems that have been employed, and some will argue a weighting system should be employed in estimating the doubling dose. However, both empirical and theoretical reasons considerations suggest that the component loci of the 7-locus system may be unusually responsive to the mutagenic effects of radiation. In the context of this discussion, it is important to bear in mind that W.L.Russell in his earliest presentations of the results of the system he had developed expressed concern over the “representativeness” of the loci he had selected (84). Since then significant progress has been made on the subject of “representativeness.” To begin with, although the existence of mutable loci has been recognized for many years, it is now clear that loci in general show a wide range in spontaneous and induced mutability. In the mouse, five coat-color loci differed in their spontaneous mutability by a factor of 7 (but the individual estimates carry large errors) (31 , 91). Favor (20 ; Table 5) has emphasized the differing responses to radiation of the 7-locus, the cataract, and the enzyme-activity systems; data to that effect have also been presented in this review. With humans, recently we have suggested a tenfold difference in the mutation rates of loci encoding for serum transport proteins and erythrocyte enzymes, these loci having been selected for study only because of the clarity of resolution of their products with one-dimensional electrophoresis (9). We also suggest that the loci responsible for the cellular proteins visualized with two-dimensional polyacrylamide gel electrophoresis and silver staining, proteins sometimes referred to as ‘structural', are as a group less mutable than the loci encoding for erythrocyte and serum transport proteins (63). Finally, in a recent mutagenization experiment using the TK6 line of lymphocytoid cells and employing ethylnitrosourea as mutagen, the products of 263 loci were scored for the occurrence of mutation resulting in electrophoretic variants, using the two-dimensional gel technique (34). Ten of

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study these 263 loci were known from family-oriented studies on peripheral lymphocytes to be associated with genetic polymorphisms. The induced mutation rate at these 10 loci was 3.6 times greater than at the monomorphic loci, an observation with a probability <0.004. Now, a specific-locus test system such as developed by Russell can only use loci associated with genetic variants. The possibility of a bias towards mutability in selecting the components of the specific-locus system is obvious. On the other hand, there may also be bias towards lesser mutability in some of the other systems; this will presumably become apparent with the passage of time. The least prejudicial procedure at present is simply to average the results of all valid approaches. The alternative approach to developing an estimate of the doubling dose for the mouse is to attempt to analyze the mouse data in the context of radiation-induced morbidity and mortality, in keeping with the basis for the human estimate. At the outset of this review, it was indeed our intention to proceed in this direction, taking into due consideration the early studies on F1 characteristics following paternal radiation so well summarized by E.Green (30), as well as the later studies on nondisjunction, congenital malformations, and cancer. Unfortunately, as we have progressed, it has become clear that this is simply not feasible. The three dominant components of the human estimate are UPOs, prereproductive mortality among live-born offspring, and sex-chromosome aneuploids. As we have pointed out, each of the murine equivalents of these indicators differs in such important respects from the human data that a valid comparison does not seem possible. Even if this were not the case, however, there is a further reason for the inability to use the first two of these indicators. For the human data, we can estimate, albeit with considerable error, the contribution of mutation in the parental generation to the indicator; this is the necessary basis for an estimate of a doubling dose. For the mouse, no such estimates exist. Indeed, given the complex backgrounds of the various strains of experimental mice used in radiation experiments, it is not clear a normative figure comparable to the estimates for humans could be developed. Finally, relatively few of the published observations on litter size and survival in the older literature are on F1. Under the circumstances, then, we must compare a specific-locus/specific-phenotype estimate from the mouse with an estimate for humans based on the presumed genetic component in neonatal and childhood morbidity-mortality and sex-chromosome aneuploids. SOME UNUSUAL FEATURES OF THE ESTIMATE OF THE MOUSE DOUBLING DOSE (FROM THE STANDPOINT OF EXTRAPOLATION TO HUMANS) As with the human data, several features must be born in mind concerning the murine data:

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study In the several decades following the advent of the atomic bombs, when major programs on mammalian radiation genetics were funded to provide guidance for human risks, the effort was about evenly distributed between programs oriented towards specific-locus tests and towards such strain characteristics as litter size, preweaning and lifetime mortality, body weight at various stages, and sex-ratio. When the latter programs failed to reveal the expected strain deterioration even after repeated generations of massive radiation (cf 30), whereas the specific-locus test was revealing rates greatly in excess of those encountered in Drosophila (cf 85), a consensus developed that the “strain deterioration” approach—which should reflect the impact of mutation at all loci—was too blunt an instrument for the problem (cf 30 , 48). At the same time, however, investigators repeatedly suggested that the loci of the specific-locus test of Russell (84) might be unusually sensitive to the genetic effects of radiation (e.g. 42 , 45 , 48 , 80 , 84), and also recognized the many difficulties in extrapolating from mice to humans (see 43 , 48 , 53). The present analysis has attempted to meet those concerns by using all the specific-locus/specific-phenotype data available. The exclusive use of specific-locus/specific-phenotype data in reaching a murine doubling dose entails fewer assumptions than in the human estimate but also reflects a smaller sampling of the total genome. A problem with the mouse data that may never be rectified is lack of data on a variety of genetic endpoints in the late litters of irradiated females. If, for whatever reason, all other specific-locus/specific-phenotype indicators behave as do those in the 7-locus test, then the results of female radiation can be only a fraction of the results of male, and the doubling dose of chronic radiation for a normal (two-sexed) mouse population will be substantially higher than the 4.0 Gy we have estimated, and would exceed the estimate for humans, although, given the looseness of these estimates, probably not “significantly” so. CONCLUSIONS Studies of eight indicators in the children of atomic bomb survivors and suitable controls have led us to suggest that the gametic doubling dose for the spectrum of acute gonadal radiation experienced by survivors of the atomic bombings is in the neighborhood of 2.0 Sv. Inasmuch as statistically significant effects were not encountered with any of the indicators, this estimate carries a large but indeterminate error. Additional error may be introduced by the assumptions made concerning the extent to which mutation in the parental generation contributes to some of these indicators. For an extrapolation to the effects of chronic radiation, we employ for these specific exposures a dose-rate factor of 2, resulting in an estimate of 4.0 Sv.

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study A review of all of the pertinent murine data was undertaken, organized to be as comparable as possible to the human data. Because of the differences in the maturity of mice and humans at birth, plus radiation-induced litter-size effects that, as we have shown, strongly influence pre-and postnatal survival, much of the mouse data cannot be compared directly with the human data. The strongest aspect of the mouse data—which is the weakest aspect of the human data—are the specific-locus, specific-phenotype studies. Averaged, these lead to a doubling-dose estimate for acute spermatogonial radiation of 1.35 Gy. Because of the higher acute doses employed in the mouse experiments than obtain for the human experience, we suggest a dose-rate factor of not less than 3, leading to a doubling-dose estimate for chronic radiation of approximately 4.0 Gy. Although the situation requires that we express the human and mouse estimates in different units, in any practical sense the human and mouse doubling dose estimates for chronic radiation are very similar. The current general agreement between these estimates, reached by relatively independent approaches, is noteworthy, albeit, as we have repeatedly emphasized, the error in both estimates is large but essentially indeterminate. There is, of course, no reason to expect identical doubling doses in two such different species. Together, however, these estimates suggest that the genetic risk of low-dose rate, low-LET radiation for humans is less than has generally been assumed during the past 30 years. As noted in the Introduction, the attempt to develop a rounded appraisal of the genetic effects of radiation on either the mouse or human genome represents one of the most complex undertakings of modern genetics. The forthrightness with which we have dealt with certain problems with both the human and murine data should not divert attention from the great progress that has been made. Inasmuch as the general issue of genetic damage from environmental exposures will be with society for some time to come, we hope that this discussion of the problems inherent in the present data, both human and mouse, will be useful in planning the experiments and observations of the future. Space constraints unfortunately do not permit discussion of possible future developments and especially the exciting prospects inherent in the parallel study in the two species of radiation-induced mutation in DNA. This review was well along when the most recent report of the Committee on the Biological Effects of Ionizing Radiation (13) appeared. With respect to the human data, the Committee treatment was appropriate to the time, but, unfortunately, the Committee did not have access to the most recent analyses-syntheses of the Japanese data, based on the DS86 dose schedule, which, we suggest, place the human estimates on somewhat firmer footings than previously. With respect to the mouse data, where we have analyzed essentially the same findings, our approach in an effort to use the mouse data most appropriate to the human situation leads to a somewhat higher estimate of the doubling dose of chronic radiation for the mouse than the Committee's, and

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THE CHILDREN OF ATOMIC BOMB SURVIVORS: A Genetic Study lessens the apparent conflict between the two sets of data that the Committee identifies in its report. ACKNOWLEDGMENTS James V.Neel gratefully acknowledges the support of Department of Energy Grant FG02–8760533 and Susan E.Lewis similarly acknowledges the support of NIH Contract No. 1-ES-55078. We are greatly indebted to Dr. Josephine Peters for critiquing the manuscript and making available unpublished data on mutation rates in an electrophoretic system, and to Drs. Michael Shelby and Robert P.Erickson for a critical reading of the manuscript. Literature Cited 1. Abrahamson, S., Wolff, S. 1976. Reanalysis of radiation-induced specific locus mutations in the mouse . Nature 264:715–19 2. Awa, A.A. 1975. Cytogenetic study. J.Radiat. Res. 16:75–81 (Suppl.) 3. Awa, A.A., Bloom, A.D., Yoshida, M.C., Neriishi, S., Archer, P. 1968. A cytogenetic survey of the offspring of atomic bomb survivors. Nature 218: 367–68 4. Awa, A.A., Honda, T., Neriishi, S., Sufuni, T., Shimba, H., et al. 1987. Cytogenetic study of the offspring of atomic bomb survivors, Hiroshima and Nagasaki. In Cytogenetics: Basic and Applied Aspects , ed. G.Obe, A.Basler, pp. 166–83. Heidelberg: Springer-Verlag 5. Bailey, D.W. 1963. Histoincompatability associated with the X chromosome in mice. Transplantation 1:70–74 6. Bailey, D.W., Kohn, H.I. 1965. Inherited histocompatability changes in the progeny of irradiated and unirradiated inbred mice. Genet. Res. 6:330–40 7. Bailey, D.W., Usama, B. 1960. A rapid method of grafting skin on tails of mice. Transplant. Bull. 7:424–25 8. Batra, B.K., Stridharan, B.N. 1964. A study of the progeny of mice descended from X-irradiated females with special reference to the gonads. Acta Unio Int.Contra Cancrum 20:1181–86 8a. Baverstock, K.F., Stather, J.W., eds. 1989. Low Dose Radiation: BiologicalBases of Risk Assessment . London: Taylor & Francis 9. Chakraborty, R., Neel, J.V. 1989. Description and validation of a method for simultaneous estimation of effective population size and mutation rate from human population data. Proc. Natl.Acad. Sci. USA 86:9407–11 10. Charles, D.J., Pretsch, W. 1986. Enzyme-activity mutations detected in mice after paternal fractionated irradiation. Mutat. Res. 160:243–48 11. Charles, D.R., Tihen, J.A., Otis, E. M., Grobman, A.B. 1960. Genetic effects of chronic x-irradiation exposure in mice. AEC Res. Devel. Rep. UR-565 . Off. Tech. Serv., Dept. Commerce. 354 12. Comm. Biol. Effects of Ionizing Radiat. 1980. The Effects of Populations of Exposure to Low Levels of Ionizing Radiation (BEIR III) , pp. xv & 524 Washington, DC: Natl. Acad. Press 13. Comm. Biol. Effects of Radiat. 1990. Health Effects of Exposure to LowLevels of Ionizing Radiation (BEIR V) , pp. xiii & 421 Washington, DC: Natl. Acad. Press 14. de Serres, F.J. 1958. Studies with purple adenine mutants in Neurospora crassa. III. Reversion of X-ray-induced mutants. Genetics 43:187–206 14a. de Serres, F.J., Sheridan, W., eds. 1983. Utilization of Mammalian SpecificLocus Studies in Hazard Evaluation and Estimation of Genetic Risk . New York: Plenum 15. Ehling, U.H. 1966. Dominant mutations affecting the skeleton in offspring of X-irradiated male mice. Genetics 54:1381–89 16. Ehling, U.H., Charles, D.J., Favor, J., Graw, J., Kratochvilova, J., et al. 1985. Induction of gene mutations in mice: The multiple endpoint approach . Mutat.Res. 150:393–401 17. Endo, A., Watanabe, T. 1989. A case of X-trisomy in the mouse. Cytogenet. CellGenet. 52:98–99 18. Erickson, R.P. 1989. Why isn't a mouse more like a man?Trends Genet. 5:1–3

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