A Review of Forty-Five Years Study of Hiroshima and Nagasaki Atomic Bomb Survivors SEYMOUR ABRAHAMSON

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1 A Review of Forty-Five Years Study of Hiroshima and Nagasaki Atomic Bomb Survivors Summary and Conclusions SEYMOUR ABRAHAMSON Chief of Research, RERF, Department of Zoology, University of Wisconsin, Madison, Wisconsin I. Dosimetry A major portion of the research associated with the A-bomb survivor's studies on health effects in the last decade centered on a reanalysis of the A-bomb and their resultant population doses. This study culminated in an extensive publication delineating a new dosimetry system, now known as DS86. In the present review Thiessen and Kaul provide us with a concise summary of the features that distinguish DS86 from the earlier previously used T 65D dosimetry system. Under the DS86 system, calculated doses (and all doses in this system are calculated) are lower, on average, resulting from, among other elements a considerable reduction in neutron doses by about a factor of 10 in Hiroshima. Shielding factors also result in a reduction in total gamma radiation although this is to some extent offset by an increase in gamma dose estimates over the distance of major importance to population exposure in Hiroshima. Figures 2 and 3 in their paper provide a comparison of the two dosimetry systems. A detailed discussion of the components involved in the calculation procedures used in the DS86 system is provided by Kosako in the following paper. The calculations involve three major phases; 1) the transport calculations of the bomb output in terms of prompt and delayed neutrons, and gamma ray production both early and late. 2) house shielding conditions are divided into some 57 categories and finally 3) body shielding for adult, child and infant, for 15 organ systems, and for the standing, kneeling and prone body positions are factored into the dose calculations. The Thiessen and Kaul paper point out some of the limitations and areas of uncertainty remaining in the DS86 system. The systematic uncertainties derive mainly from bomb output and transport models and calculations, and the random uncertainties from the survivors recall of location and the shielding factors that would be applicable to each individual. The random uncertainties are believed to contribute the largest amount to the dosimetry errors (in the range of 25-35%) while the systematic errors are considered to contribute about 10-15% error in dosimetry. To some perhaps substantial degree (although it remains to be clarified) there is disagreement between the doses computed in the DS86 system with those derived from recently obtained direct measurements by Japanese investigators using newly developed sophisticated measuring techniques.

2 A series of papers are specifically devoted to this issue. Ceramic samples, tiles and bricks were used with thermoluminescence dosimetry to estimate gamma doses. (Nagotomo et al.) Particularly disturbing is the fact that 24 of the 29 measurements in Hiroshima obtained from samples meters from the hypocenter were higher than calculated in the DS86 system. The discrepancy is greatest at plus meters, where five of the six measured values are in excess by %. (see table). Since the authors state that the measurement procedures are also corrected for background contribution the source of the discrepancy must be resolved particularly since an appreciable part of the survivor data derives from this dose range. It should also be noted that while measurement error in the range of might explain part of the disagreement, other factors may be involved. In Nagasaki only four samples are scored from greater than a thousand meters and all the measured values are lower (about 24%) than calculated values. More samples and over a wider distance are presently being measured from Nagasaki. Maruyama et al. from the National Institute of Radiological Science have similarly carried out thermoluminescence studies on tiles and bricks and also show that three of their measurements exceed calculations by about 30% in 1400 meter range but they have not done measurements in the meter range. What remains to be shown is whether independent measurements from the two laboratories are consistent and whether their discrepancies with DS86 result from errors inherent in the DS86 system or inherent in the thermoluminescent measurement dosimetry procedures. Clearly it will take a few more years of reanalysis to resolve these issues. With respect to Neutron dosimetry, papers in this volume also deal with differences in measurement versus DS86 computed values. The paper by Nakanishi at Kanazawa University, provides a series of new Europium 152 measurements for ground distances from 800 m-out to 1328 m. All nine measurements from five points demonstrate higher measured values than DS86 calculated values, indicating that DS86 is primarily underestimating thermal neutrons by a factor 2-7. At distances near ground zero the reverse is true, calculated values are larger than measured values. (see also Hamada's cobalt 60 analysis, this volume). Hoshi et al. of Hiroshima University have independent measurements of Europium and Cobalt from granite tombstones, roof tiles and iron rings. Their data collected under 500 meters from ground zero agrees with Nakanishi in that measured values were lower than calculated values nearer ground zero. Both research groups feel that systematic errors in the DS86 system must be resolved. An important point in these analyses is that the recent discrepancies in measured values of thermal neutrons appears to be linked to fast neutrons as well, as demonstrated by the very early studies on phosphorus 32 measurements (an indicator of fast neutrons) which shows the same pattern as thermal neutrons relative to calculated values. I am not a physicist and can not readily deduce what these neutron and gamma discrepancies will mean in terms of a re-estimate of the survivors dose. One nevertheless comes away with the feeling that the dose estimates are still not that solid and revisions of greater that 50% are still indeed possible for some groups of survivors. Two papers in this volume review efforts to assess measurements of radioactive fallout particularly in the black rain areas (Sakanoue et al.; Hasai et al.) but at this juncture it does

3 not appear that this information is readily translated into an assignable dose for the survivors exposed in this area, although it is possible that combined with biological dosimetry procedures, such efforts may become fruitful. As stated earlier there are two components to the dosimetry errors, the systematic or bomb related errors to which the information discussed above is directly related and the random errors associated with survivor placement and shielding factors. The statistics unit of RERF has devoted considerable effort to adjusting for bias in these random errors. The paper by Pierce et al. in this volume summarizes the advances made. Since estimated doses depend on survivors recall at the moment of exposure, a moment in their lives of the most unimaginable horror and destruction, it should be obvious that precise information is unlikely to be available, and thus there will be statistical bias. Because of the skewed nature of the population distribution it appears that estimated doses will on average be higher than true doses. Such bias will obviously lead to an underestimate of the dose response, in particular the induced cancer rate; because in effect the higher dose groups are diluted by in fact lower dose individuals, leading to a shallower dose response curve. Corrections have been applied and indicate that for the under 4 Gy range, the cancer risk estimates require upward adjustments by 5-10% depending upon the percentage of error i.e. the coefficient of variation (CV) associated with dose estimates. Procedures for computing C.V. adjusting for the variables that may influence the response i.e. city, sex, age, ATB, dose response models are considered in this review and reference to the pertinent publications addressing them are given. Two very specific examples of random error determination are presented in the review paper by Stram and Sposto of RERF. Their analyses concern the frequency of leukemia in LSS survivors either experiencing the early effect of extensive epilation or not; and the chromosome aberration yields with dose in the Adult Health Study (AHS) population, again studied with respect to epilation manifestation. Without entering deeply into statistical analysis, the work shows that the chromosome dose response curve is much greater for the epilation group that the non epilation group. The same observations are observed with leukemia. Two interpretations are initially possible; (1) that the non-epilators are indeed a more radioresistant subset of the population. Reasons why this may not be the case will be discussed later with respect to independent studies of both Nakamura and Ban (this volume), (2) that the non-epilators are more likely to have had an estimated dose that is too high. Since neither interpretation can be dismissed at present, analyses to test both were undertaken. It is of particular interest that the statistical analyses of both sets of data, leukemia and chromosome aberrations, (+ or epilation) lead to substantially the same conclusion, if random dose error is at the root of the discrepancy namely such errors are in the (CV) range. Both the leukemia curves and chromosome aberration curves come into agreement, but not so at the usual 35% (CV) range generally employed. Other biological end points such as B and T cell mutation frequencies will be studied in the future from samples obtained from Adult Health Study survivors in the same manner and it may well turn out that such biological dosimeters will indeed provide important refinements of dose related health effect responses. Given the cytogenetic and molecular technologies that now exist, one can only regret that their availability has come too late to have provided the opportunity to develop a cell bank for all the Life Span Study population from which direct radiation effects, chromosomal and genetic could have been

4 assessed; the lesson should not be lost on those involved in Chernobyl radiation studies. To this point we have focused on the major components of the DS86 dose system that have direct implications to the survivors risk estimation. One final and unresolved issue independent of DS86 but still of importance in assessment of risk is the medically associated radiation exposure experienced by the survivors in all the subsequent years. Sawada and Kato of Hiroshima University review over 30 years of studies on the dosimetry of diagnostic medical exposures experienced by the clinically followed Adult Health Study Population. It is clear that for that part of the population which received less than 0.1 Gy, they have also received a substantial and possibly larger diagnostic exposure. A major effort will be required to determine how these doses and their distribution over time will influence the risk estimates. This effort will be complicated by the fact that the general non-exposed population has also received similar and possibly larger diagnostic doses. II. Biological Effects Most of the studies of biological effects of the A-bomb are concerned with the long term health effects, cancer morbidity, genetics, aging and prenatal development. Radiation delivered acutely was also responsible for immediate deaths, within the first two months as a result of major tissue damage of vital systems. The DS86 dosimetry system is designed to provide accurate bone marrow dose estimates to those survivors who were shielded inside of Japanese style wooden houses. Fujita et al. have used this information to recalculate the LD50/60 estimate (the acute dose required to kill 50 7o of an exposed population within 60 days of the exposure). By restricting their analysis to those records for which detailed interviews of survivors in wooden structures exist in Hiroshima, 2,518 structures containing nearly 7600 individuals were available for analysis. At least one survivor was required for each unit which contained between 2 and 8 members (cluster size). From the survivors one could then obtain information on the number of individuals present, an estimate of their location and their survivorship and when if known, death occurred. The problems are in fact horrendous. There are ascertainment biases to be dealt with, death from blast and fire, multiple injury complications, poor and differential nutritional status, age and sex differences and the occurrence of a devastating typhoon, which would individually and collectively seem to invalidate any reliable estimates of LD50. Given all these confounding factors Fujita and colleagues proceed to provide reasonable statistical approaches to examine the data. They develop crude death rates by distance and free in air dose estimates in relation to cluster size. A series of dose response models were tested and an unweighted linear model was found to give as good a fit as other models. When different ascertainment rates are used in the model remarkably similar estimates, which vary by less than 20% overall, are obtained. Based on DS86 marrow doses the LD50/60 ranges between Gy. If deaths in the first day are excluded, the estimates rise to Gy and are similar to values obtained in other studies of differently selected groups which are also reviewed by the authors. The Life Span Study population, comprising about 90,000 exposed survivors and 30,000 non-exposed, has been the major continuing epidemiological program of the Radiation Effects Research Foundation (RERF) and its predecessor the Atomic Bomb Casualty Commission. The

5 study is to determine what the long term health effects of radiation are. Any change in the health status of this exposed population relative to the non-exposed cohort is under consideration. For the most part cancer mortality has served as the major indicator of a radiation effect, but more recently cancer incidence analysis indicate still a broader range of effects which should lead to more accurate risk assessments in terms of morbidity. Moreover, the issue of a radiation induced non-cancer component is still moot. An overview of the mortality data and interpretation is provided in the paper by Shimizu, Kato and Schull. Some of the salient points of this overview are discussed below. For leukemia the dose response (employing DS86 dosimetry) appears to follow a linear quadratic relationship for doses below 2 Gy whereas for all other cancers the linear model provides an equally good fit to the data as do non-linear models, regardless of whether there is a dose range restriction. A significant increase in cancers can be demonstrated in the dose range of Gy. In addition to leukemia, lung, breast, stomach, colon, esophagus, urinary bladder, ovary and multiple myelomas are significantly increased malignancies. The latency period has been shortest for leukemia and for this type, latency may be dose dependent suggesting that fewer mutational steps are involved than for the solid cancers. The distribution of excess solid cancers increasing in proportion to the age related normal cancer rate suggests that relative risk is a more accurate risk projection model based on the accumulated mortality data, although there also appears to be a shortening of the latency period for the above 1 Gy group relative to the lower dose groups. What the data also demonstrate is a higher risk of cancer mortality in the younger age groups at the time of bomb (ATB). Both as a result of the new dosimetry and longer surveillance period the present risk estimates exceed those estimated by the BEIR 1980 report by factors of about 3 for leukemia and five for solid cancers. Tables 1-6 of this paper provide the numerical data for the authors' analysis. The issue of radiation induced non-cancer mortality is considered in the paper and the results of previously published reports on survivor data up through 1978 are discussed. As of that period a significant increase was observed only for the 4 + Gy group. However, the present paper alludes to the preliminary results of the analysis carried out through 1985 with the new dosimetry, which does suggest an excess risk from non-cancer deaths at about 2 + Gy for those in the younger ATB groups. Since the initial data is based on death certificate reports considerable effort is required to determine the accuracy of the cause of death, which can frequently be misleading. Checks of the accuracy are being performed by comparing death certificate cases to autopsy records and tumor registry records to determine misclassification rates for specific categories of diseases. Detailed medical records exist for that segment of the LSS group who are also participants in the Adult Health Program (AHS) and this information should also provide greater diagnostic accuracy. For the majority of the LSS population who have died of non-cancer related diseases, hospital records beyond seven years are unlikely to exist and have not been abstracted as was the case for cancer records. This could be done in the future and would be of great value since a considerable proportion of the surviving population is in the under 20 ATB and have yet to reach the natural period of mortality increase. This issue too would be of importance in newer studies being undertaken with respect to Chernobyl, namely maintaining a comprehensive and continuing medical disease data base on the study population with particular emphasis on high dose groups, of which the military personnel involved in the clean up program may be of special

6 significance. Within the next year it is expected that RERF will produce a major report on its cancer incidence studies involving enormous efforts by members of its epidemiology, epidemiologic pathology and statistics units. This will be a very powerful data set because it reflects not only the cancer mortality component, but cancer morbidity, for example thyroid or skin cancers have low mortality and would usually not be determined from death certificate only records. RERF working with both Hiroshima and Nagasaki medical associations has been maintaining such records since the late 1950's. This data set has already provided the base for many of the site specific cancer studies which are dealt with in other contributions in this volume. Mabuchi and Soda in this volume provide an overview of the tumor registry program, its level of reliability and the issues involved in maintaining accurate incidence estimates. At present, the number of incidence cases through 1985 is in the range of 10,000 or more, about twice as large as the number of mortality cases. This quantity enhances considerably the statistical power of analyses with respect to the radiation induced component of these diseases. This information will play a major role in risk analysis by various national and international bodies responsible for establishing both risk estimates and radiation protection guidelines for the public and occupationally employed sector; but of course its major role will be in providing more accurate risk estimates for the Japanese A-bomb survivors. A very important subset of the population under study is that group which was irradiated in utero, for the moment we will deal with only the carcinogenesis study and later return to teratogenic and intelligence effects. Yoshimoto et al. review the cancer risk information with respect to the approximately 1000 in utero exposed versus 800 non-exposed in utero subjects. There were 13 cancer cases found in the exposed versus 5 cases in the 0 dose group. Since the population has not yet reached the cancer prone age (through 1985) it remains to be seen whether this elevated relative cancer risk (3.8 at 1 Gy) continues to rise over the next decade or longer. Presently this represents the highest risk observed relative to adult group, albeit based on small numbers. Only 2 of the 13 cases were childhood associated cancers and neither of them were leukemia, unlike the excess reported from in utero diagnostic medical exposures in other epidemiologic studies. Seventy percent of deaths (from all causes) were observed before October 1950, but no cancer deaths were reported before 1950 and the vital statistics system just after the war was in some state of disarray thus it is impossible to determine whether any of these early deaths were associated with leukemia or other forms of cancer. The in utero group will continue to be an important focal point for studying radiation related health effects. The following several papers to be discussed deal with specific site cancers. Ichimaru and colleagues from Nagasaki Medical School provide the reader with a concise overview of the leukemia types observed in both Hiroshima and Nagasaki populations. Since about of all the leukemias have been reanalyzed with the new FAB (French American British) reclassification system but the reanalyses is not yet published, the present report based also on T65D dosimetry is an interim report. It mainly cites the difference in distribution between the cities of leukemia subtypes, twice as great a frequency for chronic myelogenous leukemia in Hiroshima compared to Nagasaki. The authors also describe for specific subtypes the cumulative distribution over time by dose groups which demonstrates very dramatically the acceleration in time that occurred for

7 the highest dose groups. The authors also discuss the apparent threshold ranges for some of the leukemia subtypes. Ichimaru and Mabuchi present a short update on multiple myeloma studies (B-cell lineage tumors) among A-bomb survivors. These blood cell malignancies unlike leukemias have a much long latency period, 15 or more years, show a dose related response, but the observed cases are to date, restricted to the above 20 ATB and below 60 ATB. It is primarily a disease of the elderly with very few cases observed in the 0 dose group. The paper by Kamada of Hiroshima University reviews the literature with respect to cytogenetic and molecular damage observed in survivors and non-exposed patients for different leukemic types. Both in his study of about 300 patients with acute leukemia, of whom 76 were exposed survivors and Tomonaga's study of Nagasaki patients a significantly higher number of cells with complex chromosome aberrations were found in leukemic cells from the heavily exposed individuals. This is of course consistent with the findings of chromosome aberration frequency in the lymphocytes of non-leukemic exposed individuals. Among the aberration types found in the exposed (1 Gy +) was a higher incidence of aberrations involving chromosomes 5 and 7, a characteristic often observed in secondary leukemias of patients exposed therapeutically to radiation or drugs in cancer treatment. In patients with chronic myelocytic leukemia there was no difference found in the frequency of the Philadelphia chromosome aberration between exposed and non-exposed patients. At the molecular level studies on both the ras and bcr gene mutations involvements in leukemia patients has shown no difference in those of the exposed versus non exposed patients but the studies are on a limited sample of individuals, as of now. Thyroid cancer was the first solid cancer demonstrated to be significantly increased among the A-bomb survivors. The paper by Takeichi, Ezaki and Dohi of Hiroshima University provides a very extensive review of all the major publications on this topic including case reports, epidemiologic studies, histologic classifications by earlier, pre DS86 dose assessments and other factors. There are about six times as many cases found among females than in males. The dose response is reported to show a significant linear increase for females alone or for both sexes combined. Unfortunately DS86 dose relationships have not been completed and thus more accurate risk estimates are not yet available. The majority (94010) of cancers are of the papillary type and are of the non-fatal variety as opposed to the squamous or anaplastic lethal types (3%) in a sample of 103 analyzed in a Hiroshima University study. The three case were exclusively found in the exposed survivors (see there table 1). A significant dose related increase in non-malignant thyroid diseases has also been reported (in pre publication from RERF) for females under 30 years of age ATB. A second paper by Takeichi et al. deals specifically with a review of parathyroid tumors and there is a paper by Fujiwara reviewing her studies on the prevalence of hyperparathyroidism. Until the late 1970's neither of these types of diseases had been reported to be associated with radiation, then a series of reports linked them to radiotherapy exposures of the head-neck region. The studies associated with A-bomb survivors are not full scale incidence studies since they have been restricted to the Hiroshima survivors and diagnostic procedures were poor before 1980 except during autopsy. Takeichi et al. report that 23 tumor cases were detected in the tumor registry and that there was a clearly significant dose response relationship (T65D) which is supported

8 by earlier autopsy reports where 10 of 13 adenomas detected in 4,136 autopsies performed were in exposed individuals. It would appear that an accurate incidence estimate of this class of tumor among the entire LSS population is not likely to be obtained but that continuing studies are required to get the best estimate among the surviving population. To this end the prevalence studies of Fujiwara on the clinically examined AHS population will be of considerable importance. Among 4000 subjects, screening of serum calcium followed by other analyses for those whose serum level exceeded 10.3 mg./100 ml. was carried out in the hyperparathyroidism study. There was a significant dose response relationship and an apparently strong sex difference, four times as many females affected as males. Collectively, the studies on induced parathyroid defects, tumors and hyperparathyroidism, indicate a high radiosensitivity of this gland. "Although breast cancer rates among Japanese women are normally very low compared to other countries, the excess risk associated with exposure to ionizing radiation, as observed in the LSS sample... is fully as high as that seen in medically irradiated Western populations." This begins the review of the breast cancer incidence studies of the survivors and other populations (Tokunaga et al.) and is certainly one of the most extensive incidence studies under investigation. To date the data indicate that those exposed from infancy on are at excess relative risk. The 0-9 ATB group is at higher relative risk by a factor of about 2 more than the group which is also about a factor of 2 greater than the 20+ age groups. There appears to be no acceleration of risk in time of exposure to diagnosis, the excess becoming apparent at the normal age of occurrence in the non-exposed population. Moreover, the excess risk experienced by the LSS population is apparently considerably higher than that observed in other epidemiological studies. The breast has been characterized as "the single organ most susceptible to cancer induction after exposure to ionizing radiation. The authors note that most of the breast cancers among the under 40 ATB receiving low doses would not have occurred in the absence of exposure. Other factors can affect the risk, namely age at first full-term pregnancy, age at menarche, presumably dietary and other environmental factors play a role as determined by case-control studies. Because their study already contains a large number of ascertained cases (note these are not exclusively radiation induced) the statistical power of their analyses is very great indeed and the authors venture to predict what radiation related excess cases should be found for the surviving age cohorts. Much may still be learned from the under 20 cohorts relative to their estimates, however. Recent theories implicate the stem cell populations as being the radiation target and the above cited evidence is entirely consistent with the model, namely the younger the individual the larger the pool of undifferentiated stem cells that will become mutated. Should it not soon be possible to identify the mutant cells with modern techniques, or prevent their clonal proliferation prior to final carcinogenesis at least among high risk individuals? A single paper by Dobbing and Sands (1973) on growth and development of the human brain provided the critical stimulus to reexamine the earlier data collected at ABCC and RERF on in utero induced severe mental retardation (SMR) and other intellection disorders as discussed by Otake et al. in this issue. This 1973 paper provided the critical evidence of the timing of major stages in brain development. The weeks 8-15, the period of extensive neuronal proliferation and migration within the developing cerebrum marks the period of greatest radiosensitivity as measured by a dose related linear increase in severe mental retardation, over 40% at 1 Gy. DS86 dosimetric

9 analysis does not preclude the possibility of a threshold in the range of 0.12 Gy. The week period is also a sensitive period with a significant but less steep dose related increase in SMR. IQ score depression was also found to be related to these two gestational periods, in the 8-15 week period, score decline was in the range of 30 IQ points/gy, less for the week period. School performance as might be expected follows a similar pattern of sensitivity and response. The occurrence and frequency of seizures in this exposed cohort reflects similar time and dose related patterns but the data lack significance when the mentally retarded individuals are excluded. Many other analyses can now be expected on other elements of the brain and their accessory structures and these are discussed in a second paper on future studies. What should also be pointed out is that microcephaly and small head size was the major teratological observation first recorded for the in utero exposed group. Indeed, there were about three times more such cases than that of severe mental retardation and the frequency was clearly increased by increased doses. Since not all of these individuals with smaller head size were not retarded, it would be of interest to determine whether they represent an important component of the diminished IQ cohorts. Awa (RERF) reviews the extensive cytogenetics research carried out by him and his colleagues in the Hiroshima and Nagasaki laboratories. The new DS86 dosimetry impacted extensively on the dose-response relationship both within as well as between cities. The major part of their study involves standard examination of lymphocytes preparations employing conventional Giemsa staining. Some 1245 subjects (426 exposed 362 controls, 206 exposed 251 controls from Hiroshima and Nagasaki respectively) were involved and over 94,000 metaphase cells were scored for both stable and unstable chromosome aberrations. Comparative plots of the stable aberration data by city, DS86 vs. T65D dose, kerma vs. bone marrow dose are very informative. In general the dose-response up to 2-3 Gy is curvilinear and significantly so for either kerma or marrow dose. Even with the new dosimetry which removes a substantial neutron contribution, particularly from the Hiroshima dose, there is still a surprising inter city difference although less pronounced than with T65D doses. It remains to be seen whether future neutron reevaluation as discussed in the earlier section may account for these observations or whether other bias elements (see the earlier discussion by Stram and Sposto) are also involved. Because the Giemsa staining procedure is less accurate than the G-banding method, a smaller sample study has indicated that the Giemsa procedure underestimates aberration frequency by about 30% in the 1 Gy and above group but has little effect on the lower dose scoring efficiency. An interesting and as yet unresolved question is whether the break points scored by the G-banding procedure are randomly or non-randomly distributed along the chromosomes. A non-random observation might indicate either differential sites of breakage or subsequent rejoinability on the one hand or some selection process acting on cells with a wide range of rearrangements. For example, in the in utero distally exposed survivors (presumably 0 dose group) breakpoints in chromosomes 7 and 14 at specific sites are predominant. These specific sites are known to represent the genes for T cell receptors and heavy chains of immunoglobulins, genes that are intimately involved in the activity and diversity of T lymphocyte cells. Another form of selection is also observed in a second in utero study involving aneuploidy where cells are scored for loss or gain of each chromosome in the genome. Chromosome 21 and the X aneuploidies (in females) are

10 found far more frequently than chromosome size relationship would predict. It should be noted that these observations are not the result of radiation. It is expected that very new techniques will provide more rapid and large scale screening. Certainly this area of research can provide an important biological baseline against which to assess induced damage either recently occurring or in the distant past, 40 to 50 years earlier. Kamada of Hiroshima University provides a short note based on his chromosome aberration study of 10 individuals who were early entrants in Hiroshima and their possible doses from residual radioactivity. This group entered the city one day after the bombing and worked near hypocenter for 1 week. Medical histories were taken and those with severe medical exposure were excluded. Five individuals averaged 1.8 ±0.5% aberrations, five individuals averaged 0.3 ± 0.2% aberrations and two controls (age matched) 1.3 ± 0.7% aberrations. By utilizing the equation Y =1.03 ± 7.7D (Gy) he estimated the average residual dose to the first group to be 0.1 Gy and under 0.01 Gy to the second group. As indicated by the confidence limits there may be considerable uncertainty resulting from extraneous sources that complicate the estimates; nevertheless, it is an innovative study and it would be valuable to have a larger sample and/or apply this approach to estimate residual radioactivity levels in the black rain areas. The most useful somatic cell mutation screen developed to date for measuring long term effects as described in the paper by Akiyama and his colleagues (RERF) is the red blood cell glycophorin A assay. This system examines with the use of flow cytometry the loss of either M or N phenotype in cells from heterozygote MN individuals. About 106 cells can be screened in a 15 minute period. It appears that this mutation causing loss of cell membrane antigen product is not selected against and therefore persists in stem cells throughout the life of the individual. The measured mutation rate is about 2.5 x 10-5/Gy. The limitations of the system are the requirement of heterozygotes and that the variants are detected in mature erythrocytes, devoid of genetic material, and therefore not subject to molecular analysis. Three other mutation systems involve the use of T lymphocytes, the HPRT, TCR and HLA assays. The first measures mutation of the X-linked gene producing loss of hypoxanthine phosphoribosyl transferase enzyme activity. Though the study of survivors showed a dose-related response, it was quite shallow indicating, relative to in vitro studies, that selection against such mutations had occurred in the intervening 40 plus years. The latter two systems detecting mutation controlling T cell membrane receptor molecules and T cell histocompatibility membrane antigens were unable to show a dose response relationship in survivors cells, again probably because of negative selection operating on defective cells. Of interest is the fact that the TCR system detected significant increases in mutation rate in patients treated with radioactive thorium or recently treated with Iodine 131. Thus it would likely serve as an additional biological dosimeter in accident cases such as Goyiana and Chernobyl, where time since exposure was shorter, or where radioactive uptake provides a continuing exposure source. Until such systems are developed one will not know their effectiveness and where they may be most applicable. However, it should be noted that they are very cost effective in screening enormous number of cells by automated procedures. One of the first programs initiated by the then ABCC with Japanese and American physicians and scientists was to determine the extent of genetic effects on the offspring of the survivors using biological endpoints available then and incorporating newer approaches as the techniques

11 developed. Differential mortality represents one genetic indicator. A continuing study of about 72,000 first generation children, born to the survivors or non exposed members after May 1946 through 1984 is reported by Yoshimoto and Mabuchi. DS86 doses are presently available for 64,500, 27,000 from exposed parents and over 40,000 from 0 dose parents.. Mortality from all causes was increased but by no means significantly in the F1 of exposed parents with 75% of the deaths occurring between 0-4 years of life. With respect to cancer incidence neither leukemias, nor other types are different in frequency between the two groups including those cancer types that are known to have a heritable component, such as retinoblastoma, Wilm's tumor or neuroblastoma. For all cancers appearing before the age of 20, 49 were in the 0 dose class and 43 in the children of exposed parents, (data through 1982). This extensive data set and the leukemia data specifically, is not in agreement with other studies indicating that childhood leukemia rates were increased by preconceptual irradiation either from medical or occupational sources. Mouse experiments, utilizing very high doses to the parents and highly inbred strains have shown increases in cancer of adult types in the offspring and point up the need for continuing and stepping up research in this area such as case control studies and clinical studies on some adequate sample of this population. The immune system, the nervous and endocrine systems play a prominent role in maintaining the body's dynamic equilibrium and defenses against infectious disease. Within the immune system there exists a diverse array of cell types with a variety of response functions against both infectious diseases and neoplastic changes in cells. Since these cells are initially derived from the bone marrow which is a radiosensitive organ it becomes clear that studying the survivors with respect to long term immunological damage as measured by change in specific cellular subtypes may be a critical prognosticator of the diseases of aging and cancer. Moreover, the T cell components require maturation within the thymus to complete development and differentiation into the variety of subtypes and functions that are now known. The thymus involutes and becomes progressively less functional in early adulthood. Akiyama and his colleagues provide an overview of their recent studies in this area. Using an array of monoclonal antibodies to the different T cell subtypes their studies, consistent with that of others, shows a significant decline in the absolute number of T cell lymphocytes bearing CD4, CD5, or CD8 antigens with age. The changes in the ratio of subtypes is more dramatic in females. For survivors exposed to more than 0.5 Gy and over 30 years ATB, there was a significant decrease in CD5 and CD4 antigen positive cells measured 40 years later relative to unexposed controls. The changes in T cell subtype numbers is also reflected in their capacity to respond to either phytohemagglutinin and allo-antigens, with decreasing response observed with increasing dose. A special T cell class, believed not to require the thymus for development and therefore an anomalous type, the CD57 + is found to increase with both age and high dose. B cell lymphocyte studies are still in a preliminary stage. There appears to be a significant age related but not radiation related decrease in the subtype classes. Since a very substantial proportion of the population are infected with EB virus and antibody titers reflect reactivation of the virus in vivo, exposed individuals manifest a significant increase in antibody to one of the virus antigen types relative to the 0 dose group.

12 The natural killer cell component (NK cells) actually increase in absolute number with increasing age but not dose, more so in males than in females. The authors propose various age-dependent models to explain their observations and describe future cellular and molecular studies which will be undertaken. The Adult Health Study (AHS) begun in 1958 examines clinically (originally 20,000) selected members of the Life Span Study group on a biennial basis and is the largest on-going study of its kind in Japan. In addition to its major focus on radiation induced effects on health and life span, it provides an extensive data base for studies on the natural aging processes. Sasaki et al. review the work in this program with particular emphasis on the last 15 years of studies and also provide an excellent summary of the current models on radiation induced non-specific aging, i.e. unrelated to cancer. Pathological changes of muscular, nervous and circulatory systems have, as expected, been positively correlated with the normal aging processes but not with radiation dose. There was also a loss of physical strength and function in a series of tests correlating with age and possibly a high dose effect on a few of the measures studied. What is particularly interesting is a follow up study 15 years later of those originally evaluated. For specific age cohorts, say for example year olds, those who measured as physiologically older than their chronological age had higher mortality rates, not associated with cancer, than did the physio logically young identically aged cohort members. Again it must be emphasized that this was not a radiation related effect. Work on this subject is continuing. As would also be expected, a large class of diseases increase with age, but in addition, stroke and coronary heart disease was significantly increased in Hiroshima women with radiation dose and very recent results for Nagasaki women are showing the same trend. These observations may provide the needed insights in the study of noncancer related radiation related mortality. With an abundant array of different clinical measurements it is now possible to study the temporal sequence of changes for each AHS individual over their lifetime. This work is now in progress and should have important ramifications. In other sections, we have referred to immunological, cytogenetic, and other studies that are also part of this AHS program. One additional area of interest is that of age cohorts. Those born in will have experienced a different environment and lifestyle than those born in and will reflect a different aging and disease pattern as is shown by the vertebral fracture incidence data and age. Both from the standpoint of wealth of clinical and epidemiological data accruing in the AHS study and the new techniques available to examine stored serum and frozen cell samples, one can predict a very important future for this program. The first demonstrable late effect of A-bomb radiation was cataract, Otake and Schull in their paper provide a reanalysis of the radiation induced cataract data (posterior lenticular opacities) in light of the new dosimetry information on nearly 2000 or 93% of the survivors analyzed in the earlier study. Several important observations appear. At doses above 2 Gy, the risks for both cities in rate of opacities is about twice that from T65D estimates. Hiroshima survivors in this dose range show greater frequencies than Nagasaki survivors and this may be attributable to the four fold difference in neutron contribution to total dose. While neutron levels have been reduced in the new dose system they are not insignificant at high total doses. The dose response curve is sigmoid which is consistent with experimental studies which indicate a threshold dose

13 below which cataracts do not occur. The data were fitted to a variety of models with and without threshold for both gamma and neutrons. While there are considerable uncertainties in various aspects of the data and analyses, there were also certain consistencies that appear in several models with respect to thresholds, namely for gamma rays the threshold range falls somewhere between 0.7 to 1.4 Gy and between 0.06 to 0.6 Gy for neutrons. RBE's can also be calculated from this data and they range from about 100 at 0.07 Gy to 33 at 1.00 Gy of neutrons dose. Such a range in RBE with dose would suggest that the gamma ray effect was not a simple linear response. However, at the lower 95% confidence limits a constant RBE of 11.8 is possible. Two papers in this series address the issue of possible differential radio sensitivity among the A-bomb survivors. This is an issue of central importance in the development of risk estimates because if a substantial proportion of the population who perished as a result of acute radiation injury were uniquely radiosensitive, then the surviving population would provide a biased underestimate of the true risk. While it may never be possible to answer this question to everyone's satisfaction, there are experimental approaches which collectively may marshall a body of information for or against the differential radiosensitivity issue. Nakamura et al. review their in vitro studies on lymphocyte survival after radiation exposure. The experimental procedures are presented in this and other published papers and will not be discussed here. The basic approach is to expose Go lymphocyte cells to a range of doses and plot cell survival. Thirth eight survivors whose DS86 dose estimates were 1.5 Gy or more, 61 survivors with less than Gy and one donor (14 independent tests) were studied. From the survival curves, the D10 dose is computed, this is the dose required to reduce survival by 90%. The mean survival values for the three different groups showed no significant differences and the mean D10 values for the 99 survivors was 3.33 ± 0.24 Gy. The interindividual variation was very small, 7.2% coefficient of variation, and thus no evidence for differential cell radiosensitivity was demonstrated in these experiments. Two additional points are also worthy of note. When cells of ataxia telangiectasia (AT) patients were so studied they demonstrated significantly lower D10 dose values indicating extreme radiosensitivity. AT is a very rare recessive genetic disease and the cells of such patients are defective in normal DNA repair processes. Secondly, within the groups of high dose exposed survivors, there were both those who were classified as epilators and non-epilators, and although it was a small sample there was no difference in cell survival response between the groups (Nakamura, personal communication) suggesting that the difference in responses discussed in the paper by Stram and Sposto was likely to result from dosimetry error rather than radiosensitivity. Ban et al. studied skin fibroblasts survival in vitro after exposure to known doses. The fibroblasts cultures were obtained from 55 women and one man, and there were four categories for study, A-bomb exposed with and without breast cancer and non-exposed with and without breast cancer. While there was greater interindividual variation seen in the skin fibroblast study than in the lymphocyte study, (an observation also noted by Nakamura who did matched fibroblast-lymphocyte studies from the same individuals), there were no significant differences in the mean D10 values for each of the groups studied or in the variances. Thus taken together the two studies on different cell types do not provide support for the view that the survivors were more radioresistant than those who did not survive. Obviously more endpoints and larger

14 samples will have to be followed before the evidence can be considered conclusive. Radiation is the common denominator that distinguishes the A-bomb survivors from most other populations with respect to cancer risk. There are other risk factors which are also known to affect cancer risk. The best example is, of course, cigarette smoking. One of the important aspects of the epidemiological research on the survivors is to disentangle the role played by other risk factors with regard to cancer incidence in this population. One important consequence of learning how these other risk factors interact with the radiation risk is the possibility of mitigating the final outcome. For example, if a high fat diet were to increase breast cancer risk by a factor of five and radiation alone at a given dose also increased the risk by 5, but jointly the combined risk was 25 fold greater, then a major reduction in breast cancer might be achieved through dietary fat restriction. Akiba describes past and on-going studies at RERF concerned with smoking, occupation, dietary habits and obstetrical-gynecological histories of the survivors. Much of the studies take the form of case-control investigations nested within the LSS population where dose matched individuals with a disease are studied against non-affected individuals to determine how the other factors may be involved. Preliminary analysis indicates that a multiplicative interaction exists for breast cancer between radiation and factors relating to age at first pregnancy and number of pregnancies thus implicating a hormonal involvement in the development of cancer. An addition interaction of lung cancer risks between smoking and radiation has appeared in five studies at RERF and Akiba presents an interesting discussion of the differences existing in the RERF study and that of uranium miners study. As techniques of molecular biology become assimilated into the epidemiology program, utilizing stored cell lines and sera, the role of hereditary risk factors are likely to be enumerated. In 1947 the genetic studies on the offspring (F1) of the A-bomb survivors were initiated. It should be noted that at the time, experimental radiation genetics had already demonstrated a linear dose response relationship for mutation in plant and animal systems while at the same time there was no evidence that cancer was similarly dose related. Most workers then, and many even today believed that cancer induction had a threshold dose. Even so, in 1947 most authorities in the genetics field considered it unlikely that a significant increase in genetic disorders would be found among the F1 of the A-bomb survivors, probably because the sample size would be small by experimental standards and the human genetic endpoints then available were likely to be confounded by non-genetic factors, unlike laboratory based experiments on usually clear cut genetic endpoints. Nevertheless, it was felt that full scale studies should be undertaken. J.V. Neel and W.J. Schull were the two leaders in this project then which turned out to be the largest continuing genetics study ever undertaken. They have, along with many other colleagues, continued their involvement with this study and in this Journal, Neel et al. report the status of this, program, reevaluated with DS86 doses. Their emphasis in this paper (reprinted from the American Journal of Human Genetics) is to determine the doubling doses for radiation induced genetic damage based on the various indicators that have been pursued over this period. Some 31,000 children of proximally exposed parents in DS86 and 41,000 children of distally exposed (0 dose) parents have been tracked, back to 1946 and through It can be stated that there has been no significant increase in any of the endpoints studied which could be directly associated with an increase in dominant mutations, chromosome changes or recessive mutations. The major studies

15 involve 1) Untoward Pregnancy outcomes of 76,000 children. This includes stillbirths, major congenital abnormalities and deaths within the first two weeks after birth. 2) Studies on mortality of the liveborn children through 1985 excluding group 1 deaths. 3) Malignancies in the F1 (reviewed earlier in this section). 4&5) Frequencies of balanced chromosome rearrangements and sex chromosome aneuploids in children 12 years of age or older. These two endpoints should not have been selectively eliminated by virtue of their causing premature death. 6) Frequencies of mutations causing electrophoretic change in mobility of 30 different serum and red blood cell proteins. Such changes in mobility are the result of amino acid substitutions in protein structure resulting in turn from single nucleotide changes in DNA structure. Two additional studies include change in sex ratio and growth and development of the children. These latter two studies while showing no radiation effect are not included in the analysis of doubling dose because they do not lend themselves to the necessary statistical analysis. Each study is not of the same size but all are components of the same cohort. The analyses are based on linear regression with dose. As stated above none of the slope regression coefficients are significantly different from zero. An estimate of the mutational component of each trait is required to establish the spontaneous mutation rate for these traits and the doubling dose for each trait would then be that value divided by the regression coefficient (slope). The individual 95% confidence limits of the doubling doses are described in Table 5 and range from 0.05 Sv to 2.24 Sv. The cumulative regression coefficients provides the single overall doubling dose range of 1.69 Sv 2.23 Sv, this is a central estimate. If these values are multiplied by 2 to correct for low dose rate estimates the doubling dose range is Sv, a far larger estimate than generally applied by BEIR and UNSCEAR committees, indicating a much lower relative risk per unit dose. The authors note that these estimates are time and place specific and are clearly influence by post-war conditions. To which could also be reiterated they are endpoint specific. For example the electrophoretic studies were primarily studies of nucleotide base change mutations, but the majority (perhaps vast majority) of radiation induced mutations result in nucleotide deletions and rearrangements, causing loss of enzyme rather than mobility change. The data for this type of change has been collected but remains to be analyzed and I would personally urge that effort studying enzyme loss be completed. It is true that the program is mounting a major DNA sequence study but that may be a decade away from providing definitive answers (see Satoh this publication). It should also be mentioned that Abrahamson et al. 1985, and independently Ehling 1985 using the more radiosensitive mouse derived risk estimates to calculate the number of F1 genetic disorders resulting from the A-bomb, concluded that fewer than 45 dominant, sex linked or aneuploid types would have been expected on the basis of T65D dose estimates. With DS86 this number would now be about 30 cases indicating that even at the higher relative risks it would have been unlikely to find an increased genetic disease burden. To a great extent this results from the distribution of joint gonadal dose. Assuming an RBE of 20, for example, of the children were born to parents with a mean dose of 0.03 Sv, to parents with a mean dose of 0.24 Sv, to parents with a mean gonadal dose of 0.70 Sv and less than 10% exceeded 1 Sv. (Data from Otake et al RERF TR 13-89).

16 Finally what is more important, is that for the survivors and their offspring and their offspring, the threat of increased genetic damage should never be manifest. We, however, must use all the technology available to insure that we have thoroughly explored for all possible outcomes in this population. They are now reaching the age where complexly inherited diseases appear and while fortunately these are believed to be much less radiosensitive than other traits, we should continue clinical and disease incidence surveillance of the F1 population. Over the past three years RERF has held four workshops on immunology, radiation sensitivity, cancer and aging in which invited experts from Japan and the United States participated with RERF staff in discussing new approaches, technical and analytical that could be undertaken in the development of future research programs. An extensive list of recommendations were developed by these review committees, some of these have been implemented and others await future development. In addition, the Scientific Council annually reviews the progress of RERF's programs and also provides recommendations regarding both on going and future programs. This external group of eminent scientists develop an extensive knowledge of the program resulting from their many years of association with RERF and thus their recommendations play a major role in shaping future research directions. In the final papers of this issue members of RERF present a prospectus of their future research plans as seen from their present vantage point. Mabuchi and Preston discuss the future of Epidemiologic and Statistics programs. In addition to the continuing efforts of updating risk assessments on the Life Span Study cohort and the temporal changes to be seen, extensive collaborative studies with the clinical program will provide a more detailed analysis of non-cancer related morbidity using both longitudinal and cross sectional data and new statistical approaches will be developed to assess such information. Because RERF has become a reservoir of both autopsied and living material, molecular-epidemiologic techniques are now proliferating which should expand the opportunities of defining the conditions involved in the pathogenesis of both cancer and non-cancer diseases. Comparative studies on breast cancer and thyroid diseases between the LSS data and other populations are underway and the expertise at RERF is being shared with those working on the Chernobyl disaster. As the role of other risk factors and methods to reduce bias are introduced, and expectation for more precise risk estimation for radiation induced disease is very likely to be met. Schull and Otake's presentation focuses on studies that will involve the in utero exposed population specifically. With respect to neurological disorders there are a number of questions that have been unveiled by the findings of stages of extreme sensitivity. They suggest the need for research with animal models to determine the interaction of radiation on neuron proliferation, neuron migration and programmed cell death and specifically dose response relationships of these independent phenomena. A critical and unanswered question is whether a threshold will exist for the induction of this class of neurological damage. They detail the possible avenues of research that might be followed. Because the brain and central nervous system have direct impact on the functioning of all organ systems, much remains to be done from an epidemiological and clinical standpoint on the health status of this cohort. They describe a series of non-invasive clinical procedures that might be introduced into the clinical examinations to determine if subtle behavioral and functional changes have resulted from the prenatal exposure and if such changes result in

17 an accelerated aging of various sensory organs, such as vision hearing, smell, and of course, general metabolic functions that result from the neuro-endocrinological functions of the brain. In addition, other organ systems that were exposed during organogesis stages may also show signs of increased dysfunction with age. Life-time studies on this cohort can be expected to provide considerable insights into the normal phases of growth, development, maturation, and the processes associated with senescence. The virtual explosion of new methods in nucleic acid research in the last few years permits the amplification and sequencing of specific genes or DNA fragments of genes, as well as the demonstration of nucleotide base mismatches between a normal DNA or RNA sequence and a mutant version of that gene. These techniques are rapidly being employed in studying both the survivors and germinal mutations manifest in the offspring. The question of whether radiation induced mutations in the germ cells of the survivors, can thus be resolved at the ultimate molecular level of the gene structure in both a qualitative and quantitative fashion. Satoh, reviews the work in progress to analyze the DNA of the F1. Ultimately permanent cell lines from at least 500 families, (mother-father-child) will be maintained, in which the parents (either one or both) were exposed to varying levels of radiation. An equivalent number of cell lines from control families will also be established. About 50% of these cell lines have been established to date. The use of polymerase chain reaction allows the amplification of the target genes DNA by five hundred or more fold. DNA DNA duplexes (500 bases long) or DNA RNA duplexes are produced and analyzed using denaturing gradient gel electrophoresis techniques. Mismatched duplexes denature more rapidly than perfectly matched duplexes, and both base substitutions and insertion deletion classes are detectable. Satoh describes the technology and the pilot studies underway and the logistics that will be required to carry out an extensive genomic survey. Approximately 1/10 of the total genomic DNA of an individual could be analyzed by a technician per year with current techniques. Through careful selection of important functional and representative gene targets to be done over the next few years, it should be possible to begin a full scale study. With a permanent live cell bank repository, it will always be possible to add additional tests as conditions warrant. The potential for understanding the relationship between genes, mutations and genetically determined health status is enormous and goes far beyond the first issue of radiation induced DNA damage. Well over 95% of genetic ill health is attributed to multifactorial, complexly inherited multiple gene-environment interactions, which also tend to become active after adulthood. Determining if specific gene patterns or combinations are responsible for given disease states, diabetes, psychoses of various types, arthritis, epilepsy to name just a few is within the grasp of such a program if the DNA information is associated with clinical studies of the F1 (or a well determined sample thereof) over time. Such knowledge will lead to a variety of strategies that intervene between predisposition to a disease and the disease itself. I would be remiss if I did not point out that so much of this important work depends on the remarkable cooperation provided by the Survivors to the program. Certainly none of the Adult Health Study program and the basic biology studies would have been possible without their continued support. We must not forget that we are documenting a history of personal tragedies and predicting future tragedies as well. We speak of Survivors, but regardless of exposure levels they are all victims, afraid of future unforseen health problems to themselves and their