Chromosome Studies on Marshall Islanders Exposed to Fallout Radiation

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~re not au) that is evident even c presence of body-color genes such I order to assess the effect of the :cne. counts of setae were carried .~n two parts of the body of ten M derived from the au and ten I the normal Texas stock, namely, nal sternite and the distal portion .ous wings which is free of veins. ‘w sternites, the setae were counted ii~ a single area 0.01 mm: in the NC of the sternite, with the aid of !Y.IS wings the setae within five . of each wing selected at random, i 0.02 mm~. were recorded with a ocular and 44x objective. : the sternites of the normal beetles :]Sean setal number in a sample of >pecimens varied from 9.4 ti 0.2 ‘~.7 & t).4 setae. In au these values .’. or approximately two to three . as many as in the normal beetles. .he membrwtous wings the mean :ber of setae in the sample of 50 \urements in each strain was I 0.84 ~~ for the normal and 11.10 & 1.66 {he mutant. The difference in the I means is not statistically significant ‘he micrographs obtained with the nning electron microscope are shown eig. 1. At the top, on the lefi is w part of the head and the pro- ~tx of the normal beetle, and on right that of the au mtitant. Clearly, number of pits and associated brisicre greatly increased in these two m of the body. The cervical bristles rux are also greatly increased in 1he micrographs of the abdominal mites (in the middle of Fig. 1) show what extent the number of pits and .tles is increased in the mutant. ~lnal. on the bottom of the figure are two md eye of the normal and the mu- ! In the normal beetle (left) there ordy single bristles between the omlidia, while in the mutant (right) interornmatidial bristles are often rhe cytogenetic basis of most mu- [s other than those from Drosophila tnelanogaster is not known. In Drowing (Hw) increases the number of hairs present in the normal wing. For ber is increased by about 17 hairs, in Hw/Hw by about 21 and, combined by about 33 extra hairs. The increase in hair number was less marked in Cytological examination and genetic data revealed that the increase in hair number resulted from a duplication. The duplication essentially doubled the number of extra hairs on the wing (4). The autosomal recessive “hairy,” which increases the numbers of hairs on the wings and other parts of the body, interacted with Hw to increase the number of hairs on the wings even In Triboliurn the cytogenetic basis of the aureate mutation has not been investigated because, even with the most powerful compound microscope, the chromosomes are too small to detect cations or deletions. As techniques are developed in conjunction with the scanning electron microscope, it should be possible to examine cytological material and determine whether chromosomal aberrations (such as duplications in Drosophila) are responsible for the Be that as it may, our data indicate that the aureate mutation in its effect appears to be unique so far, not only for the genus Tribolium but for the order Coleoptera (6). Furthermore, because the scanning electron microscope gives micrographs of high resolving power even at high magnifications, it has been possible to obtain a detailed record of the phenotype of normal and mutant to a degree not previously atDepartment of Genetics, University of California, Berkeley, and Natural Sciences Division, California State College, San Bernardino University of California, Berkeley Electronics Research Laboratory, University of California, Berkeley Department of Genetics, Univer.rity of California, Berkeley

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Table L Summary of cytogcnctic findinbs in Marshall Isltmders exposed to fallout radiation. Pcrcenmges appcw in parcnihcses. ExPo- Cells Chromosome aberrations Totals for exposed subjects TotaIs for unexposed subiects who were exposed to fallout radiation These people were accidentally exposed to radioactive fallout on Rongelap Island in 1954: the wind shifted unpredictably after the detonation of a km away; 64 inhabitants were exposed gamma rays. Eighteen other Rongelap Islanders, away fishing on a neighboring island, were exposed to 70 rads orary depression of blood cells, skin bums from beta radiation, and internal absorption of radionuclides, the most important being iodine and strontium. It was estimated that the thyroid glands of the adults received 150 rads—those of the children, as much as 1,000 rads tion to the whole-body exposure to gamma rays. Recent development of pathological changes in the thyroid is believed to have resulted from thts ex. Chromosome preparations were obtained from 5 i individuals during the annual medical review of the Marshall kl~nders in 1964; 30 of them had been exposed to 175 rads; 13, to 70 rads of gamma rays. Eight Marshall Islrmders who had escaped exposure were in. eluded for comparison. , The exposed group comprised 20 males and 23 females, their ages ranging from embryo irt u~ero to 67 years at the time of exposure. Twenty-one were under 20 and 22 over, with males and females about equal in number. The comparison group included three males and five females ranging in age from 10 to 71 years; Unfortunately it was impossible to examine more unexposed individuals matched to the exposed with respect to age and sex. Cultures of peripheral blood lymphocytes were made by use of a modification of the meti’od of Moorhead et al. (11 ); they were harvested at 48 and 72 hours (12). The slides were stained with aceto-orcein and examined by phase microscopy. Because comparison of “the 48- and 72-hour cultures from five individuals showed no differences in aberration rates, all examinations but one were made on 50 cells of each individual from the 72-hour cultures. Al. together 2150 cells were examined from the 43 exposed subjects; 400 cells from the eight unexposed. Aberrations were scored as follows (i) aneuploidy, including polyploid cells, chromatid aberrations. Karyotypes were made in cases” where the counts were equivocal or where chromosomes of questionable morphology were seen. Only aberrations that were agreed on by several observers were included in the final tabulation. Additional karyotypes were made from eight euploid cells, showing ‘no gross abnormalities, from each of four subjects showing more aberrations than most others; this was done in an effort to detect minor structural abnormalities such as small deletions or inversions that could easily escape notice under the microscope. made from two exposed subjects and one control; only one preparation from an exposed subject contained enough suitable cells in metaphase to warProportionally the largest number of aberration-positive persons appeared in cannot explain this paradox and so treat all the exposed subjects as one group in comparing them with theltnfound in 23 of the 43 exposed peopie and in five of the eight unexposed. The high incidence of acentric fragments in the latter group was unusual and unexpected, and we cannot account for it. The total numbers of all types of chromosome aberrations in the two groups were 43 and 9—rates of 2.0 and T percent, respectively. Thus superfic~ there appears to be no difference tween the two groups, but we sh point out that in the exposed gJ half of alI chromosome aberrations sisted of two-break aberrations SUC, dicentric chromosomes, translocat: and a ring, the remainder being ace: fragments. In contrast, no two-b aberrations were found in the L, posed group in which all aberr:t were acentric fragments. Only sevc the exposed people resembled the aberrations either alone or in coc, tion with fragments. The clifferenc tween the exposed and unex, groups in the relative incidence oi break aberrations and acentric ments was significant at the 1-pi Aberrations ranged from one t., per person. Sixteen of the 23 esl and two of the five unexposed pc stances no more than one aber~ ‘ was noted per cell. Aneuploidy higher in the exposed than in the ~ posed group, but the difference not significant; in most cells, arteu; was due to loss of chromosomes number of chromatid and isochrm gaps and breaks, although som higher in the exposed group, was in normal limits. Polyploid ccli groups. No aberrations were fou the karyotypes that had been from the 32 grossly intact cells the four subjects showing relative[? rates of aberration. Likewise, n. normalities were seen in 50 cells the bone marrow of one of the es. We pointed out earlier that th that among the highly exposed. \\

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ot explain this paradoxical effect, but ‘e should mention that Bloom et al. Iso failed to detect a relation between hromosomally abnormal survivors in Iiroshima and Nagasaki and their estimted doses of radiation (8). Chromoome aberrations did not correlate with ,ge or sex, nor could any relation ~e established between the occurrence We cannot account for the unusualIy ]igh incidence of acentric fragments in he unexposed and their relative deficit n the exposed people. Radiation from ngested radionuclides is not likely to be a factor, since all Marshall Islanders have been exposed to low levels of residual radionuclides, such as Cs1g7, Znos, and Sr~O since their return to Rongelap in 1957, and since body bur-. dens of these elements were about the same in both groups. Although viruses and some chemicals, in addition to ionizing radiations, are known to produce breaks in human chromosomes in vivo and in vitro (13), the kind and distribution of the fragments seen in the cells of the unexposed group do not suggest this origin. If these agents were implicated, it is not clear why they faiIed to produce a similar effect in the exposed people who have lived on the same island under identical environmental conditions since 1957. Similar chromosome aberrations have been reported in the Japanese fishermen exposed to radiation from the same fallout (7); the incidence of abexations, excluding aneuploid cells, was 2.1 percent and thus identical with our finding: most were two-b~eak aberrations. The incidence of acentric fragments in a control population of Japanese studied by the same authors was 0.11 percent —20 times less than our finding among tire eight unexposed Marshall Islanders. In ‘a controlled cytogenetic study of sampled survivors of the atomic bombings of Hiroshima and Nagasaki, Bloom et al. have found exchange-type chromosome aberrations in 33 persons —3s percent of 94 survivors examined 20 years after exposure (8); the incidence of aberrations was 0.6 percent in the exposed group-less than half our finding among the Marshall Islanders. Bloom et al. found only a single acentric fragment in 8847 cells from the 94 controls, an incidence of 0.01 percent; for the 33 aberration-positive individuals, the dose ranged from 237 to 891 rads, and for none of the entire group was it less than 200 rads. All persons examined were 30 years of age or younger at the time of the The results of our study demonstrate that a small but significant number of chromosome aberrations persists in blood lymphocytes of some Marshall [slanders 10 years after exposure to fallout radiation. The conclusion that at least some of these aberrations were caused by the radiation, and not by other factors, rests on the finding that exchange-type aberrations were found only in the exposed people and not among the controls. The biologic significance of persistent chromosome aberrations in blood lymphocytes of hematologically normal persons many years after exposure to ionizing radiation is not known.’ In particular, any role that aberrations of this type may play in the pathogenesis of radiation-induced leukemia can only be surmised at present. It is problematical and indeed doubtful whether chromosome aberrations in lymphocytes can serve to indicate abnormalities in other tissues, except perhaps inferentially. Indeed, of the ten individuals who have developed thyroid lesions since our examinations were made, only three show Cancer Research Institute, New England Deaconess Hospital, and Department of Pathology, Haward Medical School, National Laboratory Upton, Long Island, New York 11973 Refarances and Notes 26, 798 (1%5): hi. Bacschingcr and 0. HUL Veomett. Radiation Res. 23, 282 (1964). 14. Stmmrted by AEC (Brookbaven National with New En@nd Deaconesa Hospital, and of New Enslmd Deaconess Hospital, Boston rind W. Waithe of Bruokhaven National Laboratory. We shank S. M. Shea for help with the statistics, and msny individusts in AEC and the Tmst Territory of the Pacific Islsnds for their support. Heterozygous Diploid Sttains “” Abstract. Zygotes of the unicelhdar green alga Chlamydomonas reinhardi rise to stable diploid vegetative strains. As well as by ~heir mode of origin; these strains are distinguished front hap[oids by cell and nuclear size, DNA content per nucleus, and chromosome number. Diploid strains heterozygous for a variety of mutant genes are phenotypicaliy wild type and mating-type minus. Thus these mutant genes are recessive to their wild-type alleies, and the mating-type-minus is dominant over The life cycle of the unicellular green alga Chlamydomonas reinhardi, as typically described, involves the fusion of two haploid gametes to form a diploid zygote which then develops into a thickMature zygotes undergo meiosis and germinate to produce four or eight haploid cells that are capable of vegetaRecently it has been observed that newly formed zygotes may follow an alternate course of development (IY rather than differentiating into mature zygotes, some may divide mitotically to give rise to diploid cells that remain vegetative. I now describe a selective method -for obtaining diploid strains, and some of the characteristics that dktinguish them from haploid strains. AMy method for recovering dlploid strains is based upon the inability of mutant auxotrophic haploid cells to grow under conditions that allow prototrophic CCIISto proliferate rapidly. These conditions arc met by plating a mating mixture of two different auxotrophs on a minimal agar medium lacking the required growth factors.