Increased somatic cell mutant frequency in atomic bomb survivors

Mutation Research, 201 (1988) 39-48

39

Elsevier MTR 04610

Increased somatic cell mutant frequency in atomic bomb survivors Masayuki Hakoda 1, Mitoshi Akiyama 1, Seishi Kyoizumi 1, Akio A. Awa Michio Yamakido 4 and Masanori Otake 3

2,

Department of I Radiobiology, e Genetics and "¢Statistics, Radiation Effects Research Foundation *, 5-2 Hijiyama Park, Minami-ku Hiroshima 732 (Japan) and ~ Second Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734 (Japan)

(Received 23 November 1987) (Accepted 4 January 1988)

Keywords: Mutant T-cell frequency; HPRT; Atomic bomb survivors; Radiation dose; Chromosome aberration

Summary Frequencies of mutant T-cells in peripheral blood, which are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) activity, were determined for atomic b o m b survivors by direct clonal assay using a previously reported method (Hakoda et al., 1987). Results from 30 exposed survivors (more than 1 rad exposed) and 17 age- and sex-matched controls (less than 1 rad exposed) were analyzed. The mean mutant frequency (Mr) in the exposed (5.2 × 10-6; range 0.8-14.4 X 10 -6) was significantly higher than in controls ( 3 . 4 x 10-6; range 1.3-9.3 × 10-6), which was not attributable to a difference in non-mutant cell-cloning efficiencies between the 2 groups, which were virtually identical. An initial analysis of the data did not reveal a significant correlation between individual Mrs and individual radiation dose estimates when the latter were defined by the original, tentative estimates (T65D), even though there was a significant positive correlation of Mfs with individual frequency of lymphocytes bearing chromosome aberrations. However, reanalysis using the newer revised individual dose estimates (DS86) for 27 exposed survivors and 17 controls did reveal a significant but shallow positive correlation between T-cell M f values and individual exposure doses. These results indicate that H P R T mutation in vivo in human T-cells could be detected in these survivors 40 years after the presumed mutational event.

Correspondence: Mitoshi Akiyama, M.D., Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732 (Japan). * The Radiation Effects Research Foundation (formerly ABCC) was established in April 1975 as a private non-profit Japanese foundation, supported equally by the Government of Japan through the Ministry of Health and Welfare, and the Govermmentof the United States through the National Academy of Sciences under contract with the Department of Energy.

It has been shown that the incidence of several malignancies in atomic b o m b survivors is increased proportionally to the estimated radiation dose received by each individual at Hiroshima and Nagasaki (Monzen and Wakabayashi, 1986). These data have provided basic information for the estimation of long-term effects of ionizing radiation on h u m a n health. While the frequency of lymphocytes with chromosome aberrations has been shown to be increased in these populations

0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

40 (Awa et al., 1978), investigation of other genetic damage on somatic cells, such as specific-locus mutations, would be valuable in understanding the mechanisms involved in long-term effects of ionizing radiation. Although there had been several attempts (Atwood, 1958; De Mars and Held, 1972; Strauss and Albertini, 1979; Stamatoyannopoulos et al., 1980) to investigate somatic mutations arising in vivo, the T-cell cloning method reported by A1bertini et al. (1982) and Morley et al. (1983), is the first method in which mutant nature can be characterized by the analysis of gene products or genes themselves. In this method, mutant T-cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) activity, can be grown in the presence of interleukin 2 (IL2) by taking advantage of their resistance to the purine analogue, 6-thioguanine (TG). The frequency of mutant Tcells has been reported to be in the order of 10-6-10 -5 in healthy adults (Albertini et al., 1982; Morley et al., 1983; Vijayalaxmi and Evans, 1984; Henderson et al., 1986) and to increase in older people (Vijayalaxmi and Evans, 1984; Trainor et al., 1984) and in cancer patients who have received chemotherapy a n d / o r radiotherapy (Dempsy et al., 1985; Messing and Bradley, 1985). Elevated mutant frequencies (Mf) have also been reported using a similar T-cell cloning method in mice after intraperitoneal injection of the potent mutagen ethylnitrosourea (ENU) (Jones et al., 1985) or whole-body irradiation (Dempsy and Morley, 1986). Thus, the T-cell cloning method has been shown to be feasible for monitoring the mutagenic effect of environmental agents on somatic cells. However, as mentioned above, considerable differences in mutant T-cell frequencies have been seen among healthy adults. These differences are probably due to several factors, including differences in culture methods. It has been pointed out by Albertini (1985) and Henderson et al. (1986) that Mf tends to be overestimated when the cloning efficiency (CE) of non-mutant T-cells is low. We have modified the originally reported method to permit mutant T-cells and non-mutant T-cells to be cultured under similar conditions (Hakoda et al., 1988). This modification resulted in an efficient cloning of non-mutant T-cells. Recently, a new method for detecting variant

erythrocytes, lacking expression of one allele of glycophorin A on the cell surface, was developed using monoclonal antibodies and a cell sorter (Langlois et al., 1986). The variant frequency has been shown to be increased in cancer patients who received chemotherapy (Langlois et al., 1986) and also in atomic bomb survivors (Langlois et al., 1987). The regression of variant frequency as a function of radiation dose estimates in the survivors was found to be similar to those of other studies of radiation-induced specific-locus mutations. The mutant nature of the variant cells, however, has not been examined due to the lack of DNA or proliferation capacity in erythrocytes. Here we have applied the T-cell cloning method to investigate the mutagenic effect of atomic bomb radiation on somatic cells, and to provide mutant cells for the future study of determining the nature of the gene changes involved at the DNA level. Materials and methods

Study cohort Sampling sources were atomic bomb survivors who were participants in the Radiation Effects Research Foundation (RERF) Adult Health Study (Beebe and Usagawa, 1968) at Hiroshima, for whom tentative dose estimates (T65D) (Milton and Shohoji, 1968) were available, and for whom chromosome aberration frequencies in peripheral blood lymphocytes had been previously measured (Awa et al., 1978). The exposed subjects in this study were selected by a stratified random sampling based on 4 dose groups: 1-99 rad, 100-199 rad, 200-299 rad and 300+ rad. Eight cases were randomly selected from each dose group except for the 300+ rad group, from which 16 cases were selected. Thus, 40 cases were selected from the exposed survivors. Twenty control subjects were randomly selected from those who were exposed distally to the bomb and whose T65D estimates were less than 1 rad. Age and sex of controls were matched to the exposed subjects as much as possible. Measurement of mutant frequency Several modifications were added to the method originally reported by Albertini et al. (1982) and the full details of the assay used have been de-

41 scribed previously ( H a k o d a et al., 1988). Briefly, lymphocytes were separated from defibrinated peripheral blood using Ficoll-Hypaque density centrifugation and cloned in the wells of microtiter plates with a flat bottom. An average of 1 or 105 fresh cells per well were inoculated with feeder cells into non-selected wells or thioguanine (TG)selected wells, respectively. Autologous mononuclear cells (105/well) were also X-irradiated and added to the non-selected wells as feeder cells to obtain similar culture conditions for both nonselected and selected wells. This modification resulted in an efficient cloning of non-selected cells ( H a k o d a et al., 1988). TG-selected wells contained 2.5 /xg/ml TG. The cells were cultured with the medium containing phytohemagglutinin (PHA) and 20% conditioned medium (culture supernatant of PHA-stimulated spleen cells) as a source of interleukin 2 (IL 2). After 15 days of culture, each well was observed using an inverted microscope to determine the presence or absence of colonies. Incorporation of [3H]thymidine (0.5 #Ci/well) was also used for verification of the presence of colonies. Wells in which cells had incorporated more than 800 cpm [3H]thymidine were scored as colony-positive, which was determined by the bimodal distribution of the counts, and usually good consistency with the microscopic observation existed. Cloning efficiency (CE) was calculated from the proportion of colony-negative wells, assuming a Poisson distribution of the cells with the ability to form colonies. Mutant frequency was obtained by dividing the CE of non-selected cells by the CE of TG-selected cells. As described previously (Hakoda et al., 1988), repeated experiments using blood from the same individuals revealed that the frequency of mutant cells measured by this method was almost constant for each individual even though the CEs of non-selected and TG-selected cells varied somewhat from experiment to experiment.

analysis in order to avoid the possible overestimation of mutant frequency. When those 4 samples were included, correlation between Mf and CE became negative although it was not significant at the 5% level. Henderson et al. (1986) also pointed out that samples which have CEs of less than 0.25 need to be treated with caution. The results from this study are presented in Table 1. As shown in Fig. 1, the mean Mf of 17 controls was 3.4 x 10 -6 (range: 1.3-9.3 x 10 -6) and that of 30 exposed survivors was 5.2 × 10 -6 (range: 0.8-14.4 x 10-6). The control value was similar to that of 4 normal volunteers of similar age in our laboratory (mean: 3.7 x 10 -6, range: 1.8-7.3 x 10-6). The t-test on the difference of the means between control and exposed groups was done. It is well known that the t-statistic has a robustness

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Of the 60 survivors selected, peripheral blood was obtained from 51 individuals. Nine people declined to come to R E R F . Results for 4 individuals whose CE of non-selected lymphocytes was less than 0.25 were not included in the present

CONTROL

EXPOSED

Fig. 1. Mutant frequency of controls (less than 1 rad exposed) and exposed survivors (mote than 1 rad exposed). Mean value for controls was 3.4x 10 -6 and that for exposed survivors was 5.2x10 -6 as indicated by small bars, the difference being statistically significant.

42 TABLE 1 FREQUENCY Number

OF MUTANT Age

T-CELLS IN THE PERIPHERAL Sex

2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17

52 52 53 53 53 54 55 55 55 56 57 58 59 69 70 71 14

M F M F M F F F M F M F F F M F M

18 19 20 21

53 59 51 58

22 23 24 25 26 2-l 28

1

Exposure dose (rad)

BLOOD OF ATOMIC Aberration frequency

T65D

DS86

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0 0

(W)

0

0

0

0

0

0

0

1

BOMB SURVIVORS CE ‘of non-selected cells

Mutant frequency (X 10-6)

0.49 0.55 0.36 0.65 0.44 0.41 0.45 0.54 0.39 0.51 0.30 0.42 0.49 0.35 0.41 0.30 0.31

2.1 1.7 6.1 4.0 9.3 1.3 1.9 2.5 3.4 1.3 3.0 3.8 2.3 4.9 1.8 4.4 2.3

0

0

0

0

0

0

2 3 0

M F F F

5 33 35 45

NDb 34 35 37

0 0 2 2

0.27 0.58 0.63 0.47

4.9 1.8 8.5 4.2

58 58 70 14 14 58 51

M F F F M F M

123 131 132 171 173 193 198

90 72 125 173 101 114 136

14 8 5 12 8 8 10

0.46 0.32 0.54 0.30 0.31 0.33 0.54

2.1 0.8 5.1 2.1 11.4 5.9 4.6

29 30 31 32 33

59 53 50 60 52

F F M F M

218 237 245 265 276

165 121 125 206 215

11 21 3 13 26

0.26 0.49 0.74 0.35 0.40

2.1 6.2 3.4 3.9 14.4

34 35 36 37 38 39 40 41 42 43 44 45 46 47

58 55 54 56 53 54 53 54 71 75 57 59 58 54

M M M M M F M M F F M M F F

305 314 315 324 336 337 361 390 404 413 434 504 522 882

230 156 230 279 315 316 246 243 201 277 304 ND ND 445

11 22 24 31 43 22 19 18 13 27 43 14 7 45

0.69 0.42 0.29 0.33 0.38 0.55 0.59 0.32 0.63 0.59 0.40 0.36 0.52 0.29

3.7 7.5 2.3 1.9 7.8 3.1 10.0 4.6 6.2 4.3 5.0 3.1 3.1 5.1

’ Cloning efficiency. b Not determined.

43 15

trois ( p < 0.05). Mf is plotted against corresponding CE in Fig. 2 and no correlation was observed. The mean CE of the controls and exposed group was virtually identical: 0.43 and 0.44, respectively, indicating that the observed elevation of Mf in the exposed survivors is not attributable to a lower CE of non-mutant cells. The relationship between radiation exposure and observed Mf was analyzed using total T65D dose (including both gamma rays and neutrons) for each donor (Fig. 3). Mf did not show a significant correlation with dose at the 5% level. However, the T65D estimates have been replaced by new revised estimates (DS86) based on improved calculations of the yield of neutrons and gamma rays from the bomb and shielding effects (Christy and Tajima, 1987). DS86 doses were available for 44 individuals, 17 controls and 27 exposed survivors. Using the DS86 dose estimates, a significant ( p < 0.05) positive correlation between Mf and dose was observed (Fig. 4). The relationship between H P R T mutant frequency and the frequency of peripheral lymphocytes bearing chromosome aberrations was also analyzed (Fig. 5). (Both stable and unstable aberrations were included in the analysis.) Individual

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Cloning

Efficiency of

Non-selected

Cells

Fig. 2. Relationship between mutant frequencyand the cloning efficiency of peripheral blood lymphocytes in non-selected wells, o, control (less than 1 rad exposed); e, exposed survivors (more than 1 rad exposed). No correlation was observed by least-squares regression analysis (r = -0.026).

even in the cases where the distribution is a little skewed to the right from normality, such as the data used here. The mean Mf of the exposed group was significantly higher than that of con-

15

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10

z

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0.22 (0.05
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0

0

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i

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100

200

300

400

500

600

T65D

DOSE

(,ad)

Fig. 3. Relationship between mutant frequencyand the tentative radiation dose estimates (T65D). The correlation was not significant at the 5% level by least-squares regression analysis.

44

15 A I 0 P X

>" 10 Z IdJ

r = 0.30 (P<0.05)

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5

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100

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200 300 DS86 DOSE (rads)

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400

500

Fig. 4. Relationship between mutant frequency and the revised dose estimates (DS86). Least-squares regression analysis shows a significant linear increase in mutant frequency with increase in DS86 radiation dose ( r = 0.30; p < 0.05), with a best fitting straight line corresponding to y = 3.7 + 7.5 × 10-3x.

15 © Control • Survivor

I

.= x

10 z

•

r = 0.34

(P<0.05)

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P E R C E N T OF C E L L S

i 30

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(%)

WITH C H R O M O S O M E A B E R R A T I O N

Fig. 5. Relationship between mutant frequency and the frequency of peripheral lymphocytes with chromosome aberration. Least-squares regression analysis shows a significant linear increase in mutant frequency with increase in chromosome aberration frequency ( r = 0.34; p < 0.05) with a best fitting straight line corresponding to y = 3.7+0.08x.

45 chromosome aberration frequency has been shown already to increase significantly with increasing T65D dose (Awa, 1984) and has been regarded as a useful marker for biological dosimetry. A significant ( p < 0.05) positive correlation between Mf and aberration frequency was observed and a slightly higher y value was observed than in the case where the relationship between Mf and radiation dose was analyzed. The relationship between Mf and aberration frequency will be studied further by collecting samples that show outlying values of aberration frequency compared with exposure doses. N o effect of age or sex on Mf was observed in this study. Discussion

An elevation of the frequency of mutant T-cells was observed in the peripheral blood of atomic bomb survivors more than 40 years after the exposure. Although there was not a significant increase in Mf with increasing T65D dose estimates, there was a positive correlation when revised dose estimates (DS86) were used. The Mf was also significantly correlated with the frequency of peripheral lymphocytes bearing chromosome aberrations. Although Mf correlated significantly with the revised dose estimates, the slope of the regression line was very shallow when compared with the results from other studies of radiation-induced mutagenesis. Elevated frequencies of lymphocytes with mutations at the H P R T locus have been reported after in vivo or in vitro radiation exposure of humans (Vijayalaxmi and Evans, 1984; Sanderson et al., 1984; Messing and Bradley, 1985) and in vivo exposure of mice (Dempsy and Morley, 1986). The increase is 10-20 times higher than the increase reported here. In these experiments, the mutation frequency was studied 10-30 days after the irradiation. An elevated frequency of T-cells resistant to T G was also reported using short-term cultures and autoradiographic techniques, in 3 people suspected to have been exposed to 6°Co in an accident which occurred in Mexico (Ostrosky-Wegman et al., 1987) and in 2 people who were in Kiev during the accident in Chernobyl (Ostrosky-Wegrnan et al., 1987). In these people, the variant frequency was 3-20 times

higher than the control value. These findings indicate that mutations can be induced at the H P R T locus by whole-body irradiation in humans. However, in atomic bomb survivors, many of the induced mutants have presumably disappeared from the peripheral blood in the 40 years following exposure. Although the mechanism is not yet clear, selection against HPRT-deficient cells during the ontogeny of the hemopoietic system has been suggested. A m o n g Lesch-Nyhan heterozygotes, roughly 50% of somatic cells are expected to be HPRT-negative by random inactivation of the X chromosome (Lyon, 1961), and this was confirmed by the cloning of skin fibroblasts (Johnson et al., 1976). However, only 1-10% of erythrocytes (Johnson et al., 1976) or T-cells (Strauss et al., 1980; Dempsy et al., 1983) in the peripheral blood have been found to be HPRT-negative. It is suggested that a similar selection occurs on induced HPRT-negative mutant cells in adults from the observation that the elevated TG-resistant cell frequency after cyclophosphamide therapy of multiple sclerosis patients returned to the background level in 6 months (Ammenheuser et al., 1986). Disappearance of induced mutant cells from the peripheral blood of atomic bomb survivors seems, however, to be attributable to the stage of differentiation of T-cells at which mutation was induced. If mutation is preferentially induced in differentiated, mature T-cells, they will be eliminated during the process of renewal of the peripheral T-cell population (Scollay et al., 1980). It is possible to estimate whether mutations occur in prethymic, undifferentiated cells or in postthymic, differentiated T-cells by analyzing both H P R T gene alterations and T-cell receptor gene rearrangements in mutant clones (Nicklas et al., 1986). The results of such analyses of mutant T-cell colonies from normal male adults have suggested that mutational events occur preferentially in differentiated T-cells (Nicklas et al., 1986). On the other hand, we have observed very high Mf (2 x 10 -4, which is 70 times higher than the control value) in one male exposed survivor who was not involved in the random selection in this study and the mutant cells were found to be derived from a single undifferentiated stem cell (Hakoda et al., in preparation). This person is not a Lesch-

46 N y h a n heterozygote because the karyotype was normal 46 XY. The mutant colonies from this person had the same H P R T gene alteration but different T-cell receptor gene rearrangements. A similar high Mf was observed again when blood was obtained and measured 6 months later. Thus, in the case where mutations occur in undifferentiated stem cells, a high Mf will continue for a long time. Recently, a new system has been developed for detecting variant erythrocytes which lost expression of one allele of glycophorin A from the cell surface (Langlois et al., 1986). The frequency of such variants observed in atomic b o m b survivors has been reported to be 6 - 7 times higher than that observed in unexposed individuals and to be correlated with increasing dose estimates (Langlois et al., 1987). In this system, only mutations occurring in undifferentiated precursor cells are detected, because peripheral erythrocytes have no D N A and turn over every 120 days (Wintrobe et al., 1981). This is in sharp contrast to T-cells in which mutations occurring in peripheral mature cells are usually detected. This difference may be the main reason why a different dose response of the 2 assay systems was observed in the study of atomic b o m b survivors. Although significant increases in the frequency of H P R T mutant T-cells could be detected in atomic b o m b survivors 40 years after the exposure, it seems that the T-cell cloning system is more suitable for relatively short-term monitoring of the in vivo effect of mutagens. The biggest advantage of the T-cell cloning system is that both gene product and altered gene can be analyzed by propagating mutant cells in vitro. TG-selected Tcell colonies have been shown to be mutants by measuring the activity of the H P R T enzyme (A1bertini et al., 1982; Morley et al., 1983). Analyses have also been conducted at the D N A level on TG-selected colonies isolated from normal adults (Turner et al., 1985; Albertini et al., 1985; Messing et al., 1986; Bradley et al., 1987; Nicklas et al., 1987). Gross alterations (mainly deletion of some exons) were found by Southern blotting in some mutant colonies, providing evidence that these cells are genetic mutants. The frequencies of H P R T gross alterations among mutants obtained from normal individuals

have been reported to be 10-20% (Messing et al., 1986; Bradley et al., 1987; Nicklas et al., 1987) although earlier studies (Turner et al., 1985; A1bertini et al., 1985) showed higher frequencies (30-57%), in which fewer mutants were analyzed. Such a low frequency of gross alterations among spontaneous mutants may allow the definition of the type of mutagen exposure of the mutants studied, especially in the case where the mutagen preferentially induces such alterations. As ionizing radiations have been shown to frequently induce large deletions (Vrieling et al., 1985; Stankowski and Hsie, 1986; Skulimowski et al., 1986), the analysis of mutant colonies from atomic b o m b survivors may provide conclusions as to whether the observed elevation of Mf is due to the radiation exposure or not. Studies are under way to analyze D N A s of 130 mutant colonies isolated from 2 exposed survivors who showed Mfs 3 - 4 times higher than the average value of the controis.

Acknowledgements We are grateful to Drs. Richard J. Albertini and James V. Neel for providing critical commentg on the manuscript and Drs. Nori N a k a m u r a and Yuko Hirai for valuable discussions. We thank Yoshiko Watanabe, Tatsuo Mandai and Fusako Hasegawa for their technical assistance. The assistance in typing the manuscript by Michiko Takagi and Mitsue Wakasa is appreciated.

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