Further Chromosomal Studies on Irradiated Human

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1 Jap. Jour. Genet. Vol. 38, No. 2, (1963) Further Chromosomal Studies on Irradiated Human Leukocytes in vitrol) Akio AWA, Yasushi OHNUKI, and C. M. POMERAT Zoological Institute, Hokkaido University, Sapporo, Japan, and Pasadena Foundation for Medical Research, Pasadena, California Received April, 15, 1963 Recent improvements of culture technique for obtaining active multiplication of leukocytes from peripheral blood give promise of advancing hematological and cytological research. In combination with the methods for producing well-spread chromosome preparations, such procedures may facilitate more detailed cytological as well as cytogenetic analyses. In previous work, studies on leukocytic chromosome damage of man irradiated in vitro from a cobalt-60 source have been reported (Ohnuki et al. 1961). Additional data have been accumulated with the study of X-ray-induced chromosome aberrations in human leukocytes under the identical experimental conditions to those described. The present report was designed to compare X-ray and gamma irradiation data. Half of the experiments were carried out with the use of rhesus monkey peripheral blood in order to have comparative studies on leukocytic chromosome injury of man and of experimental animals, especially primates, irradiated both in vitro and in vivo. The techniques were not satisfactory. It is to be hoped that this report may facilitate future work. The leukocytes examined included: Materials and Methods (1) Two 18 to 30 ml of blood samples collected from each of three human adults (2 males and 1 female). (2) Three samples of approximately equal volume were obtained from each of three rhesus monkeys, Macaca mulatta (2 males and 1 female). (3) Blood specimens also were derived from two additional male monkeys for supplementary culture studies. A routine technique for obtaining leukocytes from peripheral blood has been reported by Ohnuki et al. (1961). Variations of this method were attempted in an effort to obtain satisfactory results. Some 4-day-sister cultures (3 to 5 T-30 flasks) were exposed to 400 r X-irradiation at a dosage rate of 67 r per minute. The X-ray tube was run at 200 kvp and 18 ma with 1.0 mm Al plus 0.25 mm Cu filtration at a focal distance of 48.5 cm. After irradiation the flasks were re- 1) This study was supported in part by fund provided under Contract AF 41 (657)-357 with the School of Aerospace Medicine, USAF Aerospace Medical Center (ATC), Brooks Air Force Base, Texas.

2 FURTHER CHROMOSOMAL STUDIES ON IRRADIATED HUMAN 107 incubated without change of nutrient fluid until fixation. In subsequent experiments total body irradiation at a dosage of 400 r was given to the three monkeys whose leukocytes were employed later for the in vitro tests. Venous blood was collected from these animals approximately one and 48 hours post-irradiation and cultured. After 5 or 6 days of incubation the cells were pretreated by adding 0.05 ml of a 1 : 10,000 dilution of colchicine solution per 10 ml of medium for a three-hour period. Well-spread chromosome preparations were then obtained from these cultures with the use of our routine squash technique (Ohnuki et al. 1961). For chromosome observations phase contrast microscope was employed with the aid of an oil immersion objective. Results Chromosome injuries in human leukocytes resulting from X-irradiation: Chromosome studies were made on both control and irradiated human leukocytes two days postirradiation, and consequently a total of 6 days of incubation. Table 1. The distribution of ploidy in control and irradiated human leukocytes As seen in Table 1, there was a predominant distribution of diploid cells in the controls (99.73 per cent) derived from three different individuals. The percentage of cells other than diploids was accordingly low showing only 0.27 per cent. These elements were chiefly tetraploids involving endoreduplication of chromosomes. These were found in one-third of the tetraploid cells (12 out of 33 cells). Only two haploids (0.02 per cent) were counted, and no other polyploids could be seen in the control series.

3 108 A. AWA, Y. OHNUKI, AND C. M. POMERAT The increase in the percentage of polyploid cells was remarkable in the X-irradiated series. There were triploid, tetraploid and possibly octoploid cells of which the tetraploids were the most dominant. As shown in Table 1, 317 cells out of 10,765 (2.95 per cent) were in the tetraploid range. Cells with endoreduplication of chromosomes were frequently noted. Triploid and octoploid elements which could not be seen in the controls were observed in the irradiated series. However, both were very low; 0.06 per cent for triploids and 0.01 per cent for octoploids. The increase in the percentage of haploid cells after irradiation might have been associated with the increase of polyploidy, especially of triploidy. Table 2. The distribution of chromosome numbers in control and irradiated human leukocytes Exact chromosome counts and morphological analysis of the chromosomes were made from well-spread metaphase figures in both control and irradiated specimens. In controls, euploid cells with 46 chromosomes were predominant in each individual. As seen in Table 2, the normal human chromosome number 46 was found in 175 cells out of 180 metaphase plates. Only a single cell showed 45 chromosomes. The remaining 4 metaphases had 92 ± 1 chromosomes which belonged to the tetraploid range. Leukocytes cultivated in vitro showed low numerical chromosome variation. Chromosome aberrations were detected frequently in cells receiving X-irradiation. As presented in Tables 2 and 3, numerical fluctuation and morphological anomalies of chromosomes became evident after irradiation. There were 64 out of 100 metaphases which showed chromosomal injuries of varying degrees of severity. These abnormalities were demonstrated in every sample except for Case H 1 (P) where poor mitoses could be observed, and consequently, no detailed morphogical analysis was achieved. Among the types of damage, chromosome and chromatid fragmentations were commonly seen. Complex aberrations such as dicentrics, rings and translocation of

4 FURTHER CHROMOSOMAL STUDIES ON IRRADIATED HUMAN 109 Table 3. Distribution of normal and by 400 r of X-irradiation abnormal cells in duced the chromosomes were also frequently noted. Since fragments were found in many cells after irradiation, there was a wide variation in the chromosome number (Table 2). Technical variations for obtaining monkey leukocyte cultures: In the general procedure employed, to each 10 ml sample of heparinized blood (containing 0.05 ml of heparin) 0.20 to 0.25 ml of Bacto-phytohemagglutinin (PHA) (Difco Laboratories, Detroit 1) was added. The PHA was uniformly distributed in the blood by agitating with a syringe, transferring into conical tubes and allowed to stand for approximately one hour at 4 C in ice water. An appropriate amount of plasma containing white blood cells was collected by gentle centrifugation (250 to 300 rpm. for 10 minutes). Approximately 1 to 2 ml of the supernatant were transferred into T-30 flasks to which 5 to 6 ml of Eagle's medium, enriched with 10 per cent horse serum, were added. The volume of medium was dependent upon the ratio of cells to plasma. Such cultures were incubated without change of nutrient fluid until fixation. the medium equilibrated to approximately 7.5 to 7.6. The initial ph of The technical variations involved in the present investigation are summarized as follows: (1) the amount of phytohemagglutinin (PHA) together with the use of different nature such as Exp. 4, 6 and P, (2) the isolation of leukocytes from the blood, (3) effects of various sera on leukocyte growth, or the ratio of host serum to the culture medium. Several combination of procedures developed on the basis of these experiments were examined in the present investigation. Successful results were obtained by all of these combinations with the use of human blood materials. In contrast, no satisfactory modification of techniques could be developed for monkey material. A very small number of mitotic figures were observed in all monkey preparations irrespective of the combinations which were tested. pycnotic and inadequate for chromosome analysis. However, metaphases were rather

5 110 A. AWA, Y. OHNUKI, AND C. M. POMERAT Discussion A considerable amount of information concerning radiation-induced chromosome breakages has been accumulated by many investigators up to date. The significance of the present study was the information obtained from the X-irradiation experiments using human materials which added to the previous data presented by the authors (Ohnuki et al. 1961). Gamma-irradiation from a Co60 source at a dose level of 400 r produced a marked increase in the percentage of polyploid cells and of chromosomal aberrations. Approximately 56 per cent of the metaphases in the irradiated samples were damaged and showed various morphological irregularities. A similar tendency was found for cells irradiated with an X-ray source at the same dose level and maintained under the identical culture conditions as in the previous study (Ohnuki et al. 1961). There was damage to 64 per cent of metaphase cells. Among these abnormalities, fragmentations of chromosomes or chromatids were commonly seen. Dicentrics, rings and sometimes translocations of the chromosomes were also noted. Therefore, these abnormalities as well as the increase in the number of polyploid cells were common phenomena as a result of both Co60 and X-ray at 400 r. Such chromosome injuries were commonly found in cells of various sort of organisms received ionizing radiation. Since the samples were examined 48 hours of postirradiation, most leukocytic cells in the population in vitro might not have been capable of dividing further at this period. Therefore, it is very likely to interpret that the mitotic cells, which seemed to be normal in both morphology and number, might have remained unaffected. Even they were affected, they might still have been viable. Some may degenerate after cell division due to its genetically imbalanced condition of chromosomes as a result of radiation treatment. Conen (1961) found structurally abnormal chromosomes in cells from the bloodculture of an infant after extensive diagnostic X-ray exposure. Bender and Gooch (1961) studied blood cultures of a number of subjects who had been accidentally exposed to gamma and fission neutron irradiation, and they found abnormalities 29 months after exposure. They also have investigated the types and rates of X-rayinduced chromosome aberrations in human blood cells irradiated in vitro (1962). A similar experimental result has been obtained from study which indicated that main aberration type was not chromatid-type but chromosome-type, although the former type of aberration could be observed in a few cells in the present observation. A similar result was obtained by Buckton et al. (1962) who reported in many cases of patients suffered from ankylosing spondylitis that various chromosomal injuries persistent after a long period of radiation therapy. This indicates a possibility that some of the cells showing structural changes of chromosomes may be self-perpetual and, to some extent, viable against in vivo or in vitro circumstance. Assuming these data, a further chromosome study of the effect of irradiation upon human cells will be interesting in relation to advances in medical as well as radiobiological research. No combination of the technical variables gave satisfactory results for the pro-

6 FURTHER CHROMOSOMAL STUDIES ON IRRADIATED HUMAN 111 duction of leukocytic mitoses from the peripheral blood of the monkey, Macaca mulatta. Only a few mitotic figures were seen in all samples following a wide variety of techniques. These dividing cells, however, did not furnish adequate numbers for detailed chromosome analysis and particularly for experimental studies. Parallel procedures and culture conditions yielded successful results with human material. The negative findings from these monkey specimens were possibly due either to technical factors other than those involved in the present study or to the physiological and chemical differences between human and monkey blood. In relation to the latter possibility, Cooper suggested that polymorphonuclear leukocytes perhaps has an inhibitory effect on leukocytic proliferation (personal communication). This suggestion might be reinforced by the fact that during the isolation of erythrocytes, monkey specimens required a much higher centrifugal force than human materials. Many investigators have insisted upon the indispensability of the PHA for leukocytic growth in tissue culture (Moorhead et al. 1960, Nowell 1960, Osgood and Krippaehne 1955, and many others). The present negative findings appeared rather unusual. They might be valuable for future studies, even if the technical variations employed here were not adequate as far as the monkey blood was concerned. Summary Human leukocyte cultures received X-irradiation at a dose of 400 r on the 4th day and fixed on the 6th day. There was an increase from 0.25 to 3.01 per cent in the number of polyploid cells. These were predominantly tetraploids. Microscopic examination revealed that 64 per cent of metaphases observed showed damage. The abnormalities were due to chromosomal breakages, particularly fragments and dicentrics. Counts and detailed analysis of chromosomes in human cells used as controls demonstrated the predominant distribution of euploid elements with 46 chromosomes. These values closely parallel those reported by Ohnuki et al. (1961). In order to develop a satisfactory culture procedure for rhesus monkey (Macaca mulatta) leukocytes, several technical variations of the routine method were investigated. None of these variations gave satisfactory results for monkey samples, but all were useful for human cells. Such an unusual situation might possibly be induced by differences in the physicochemical and cellular properties of monkey blood and that of man. Acknowledgements The experimental work was done at the Radiobiological Laboratory, Balcones Research Center, Austin, Texas. The authors are indebted to Captain Loren C. Logie and his associates for their kind cooperation and for supplying materials which made this work possible. Grateful acknowledgement is also made to Professor J. J. Biesele and his staff at the University of Texas, Austin, Texas, for their valuable encouragement, culture media preparations, and other materials. The phytohemagglutinin was kindly provided by the Difco Laboratories, Detroit, Michigan.

7 112 A. AWA, Y. OHNUKI, AND C. M. POMERAT Literature Cited Bender, M. A. and P. C. Gooch 1961 Somatic chromosome aberrations in normal and irradiated humans. Rad. Res. 14: 451. and 1962 Types and rates of X-ray-induced chromosome aberrations in human blood irradiated in vitro. Proc. Nat. Acad. Sci. 48: Buckton, K. E., P. A. Jacobs, W. M. Court Brown and R. Doll 1962 A study of the chromosome damage persisting after X-ray therapy for ankylosing spondylitis. Lancet. ii : Conen, P. E Chromosome damage in an infant after diagnostic X-irradiation. Lancet., 47. Cooper, H. L. Personal communication. Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M. Batipps and D. A. Hungerford 1960 Chromosome preparations of leukocytes cultured from human peripheral blood. Exptl. Cell Research 20: Nowell, P. C Phytohemagglutinin : An initiator of mitosis in culture of normal human leucocytes. Cancer Research 20: Ohnuki, Y., A. Awa and C. M. Pomerat 1961 Chromosomal studies on irradiated leukocytes in vitro. Ann. N. Y. Acad. Sci. 95: Osgood, E. E, and M. L. Krippaehne 1955 The gradient tissue culture method. Exptl. Cell Research 9: Tough, I. M., K. E. Buckton, A. G. Baikie and W. M. Court Brown 1960 X-ray-induced chromosome damage in man. Lancet Ii:

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Radiation Research Society is collaborating with JSTOR to digitize, preserve and extend access to Radiation Research. Persistent Chromosome Aberrations in Irradiated Human Subjects Author(s): M. A. Bender and P. C. Gooch Reviewed work(s): Source: Radiation Research, Vol. 16, No. 1 (Jan., 1962), pp. 44-53 Published by: