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Stable and unstable aberrations in lymphocytes of Chernobyl accident clearance workers carrying rogue cells
Applied Radiation and Isotopes 52 (2000) 1153±1159
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Stable and unstable aberrations in lymphocytes of Chernobyl accident clearance workers carrying rogue cells E.V. Domracheva*, N.B. Rivkind, E.A. Aseeva, T.N. Obukhova, L.V. D'achenko, A.I. Vorobiov Karyology Laboratory, National Research Center for Hematology, Russian Academy of Medical Sciences, Novozykovski pr., 4a, 125167 Moscow, Russia
Abstract Cells with multiple chromosomal aberrations, the so-called rogue cells, were found in blood samples from more than 100 Chernobyl accident clearance workers. A comparative analysis of frequencies of stable and unstable chromosomal aberrations in two worker groups Ð those with or without rogue cells was made. A higher level of unstable aberrations in persons carrying rogue cells was observed. No dierence in the level of stable aberrations between the groups was seen. The possibility of low dose alpha irradiation causing the chromosomal damage is raised. 7 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Cells with numerous (r3) exchange-type chromosomal aberrations, i.e. di-, tri-, polycentrics, rings, as well as acentric fragments, remain a matter of interest for radiation cytogeneticists. Such cells are reported to be a common feature in the blood after acute accidental exposure to high doses of gamma radiation (3±5 Gy), or as a result of incorporating alpha-emitting radionuclides (Vorobiov et al., 1974; Pyatkin et al., 1989). One of the basic tenets of radiation cytogenetics is that the aberration frequency increases as some function of radiation dose, so that multiaberrant cells (MAC) are more likely to be induced by higher doses. However, not all researchers subscribe to the view of the radiation causation of rogue cells. They have been recorded at quite high frequencies in various cohorts
* Corresponding author. Fax: +7-212-42-52. E-mail address: [email protected] (E.V. Domracheva).
of presumably only background irradiated persons (Tawn et al., 1985). Indeed, they were ®rst described in a study of a newly discovered stone-age tribe of Amazonian indians (Bloom et al., 1973), and this was followed by others, such as deep sea divers (Fox et al., 1984), and controls for the study of A-bomb survivors and their ospring (Awa and Neel, 1986). This led to the suggestion of a viral origin (Awa and Neel, 1986), and some supporting evidence implicating polyoma virus has been presented by Neel et al. (1996). After the Chernobyl accident, the number of studies of chromosomal aberrations in lymphocytes of residents of contaminated areas and of clearance workers has grown dramatically. In some of these studies rogue cells were observed. Lymphocytes with multiple chromosomal aberrations have been reported from residents of contaminated areas (Domracheva et al., 1991; Neel et al., 1992; Sevan'kaev et al., 1993; Salomaa et al., 1997; Verchaeve et al., 1993; Bochkov and Katasova, 1994), and in Chernobyl clearance workers (Rivkind et al., 1996; Lazutka, 1996). Similar cells were
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seen in residents of Altai region who were exposed to radioactive contamination after the nuclear tests in Semipalatinsk (Shevchenko and Snigiryova, 1996), and in residents of the Chelyabinsk region near the Techa River. This river has been severely contaminated by radionuclides from the Mayak plutonium plant (Snigiryova et al., 1997; Kossenko and Degteva, 1994). Hence, rogue cells were observed in most radiation exposed cohorts studied, and this may indicate a radiation origin but, at the same time, cannot be considered as conclusive. The gamma-radiation doses, which clearance workers and residents of contaminated areas were exposed to, are substantially lower than the low LET radiation doses that are considered capable of inducing multiaberrant cells. However, contamination of large areas by plutonium and other high LET emitters after the Semipalatinsk nuclear tests, and Chernobyl and Mayak accidents have been admitted (Pavlotskaya and Goryachenkova, 1992; Alexakhin, 1993; Marenny et al., 1994). Over 6 years, we have studied samples from clearance workers who undertook duties at Chernobyl from 1986 to 1988 and who normally reside in the Bryansk region, and have observed in them a considerable number of multiaberrant cells. Such cells were not seen in a control group, and this strengthens our view that these rogue cells have a radiation causation (Rivkind et al., 1996). In the present study, we have tried to establish a connection between rogue cells and more commonly accepted radiaton cytogenetic markers, i.e. dicentrics and rings, and the level of stable chromosomal aberrations. A ¯uorescence in situ hybridization (FISH) study was performed within the framework of a Bryansk Branch of the International Consortium on Health Eects of Low Dose Radiation.
2. Materials and methods Peripheral blood lymphocytes were routinely cultured in the Karyology Lab of the National Research Center for Hematology and in the Biodosimetry Lab of the Bryansk Diagnostic Center. 200±300 metaphases were analysed from each of 1307 clearance workers aged 21±54 years. They had been engaged in dierent activities; comprising persons who built the sarcophagus around the damaged reactor; those who worked elsewhere inside the 30 km exclusion zone, and those who performed dosimetry measurements. The durations of working inside the zone varied from 8 days to 1.5 months, the average being 22 days. Personal physical dosimetry was performed for the majority of the subjects and recorded radiation doses ranged from 0.03 to 0.24 Gy. The body content of radiocaesium was also measured, but did not exceed background
level varying from 14 to 157 nCi. The cohort could be considered generally healthy, although some had minor malfunctions of the nervous system. The control group comprised 47 healthy persons with ages ranging from 20 to 47 years. They apparently had not been exposed to radiation and lived in non-contaminated areas of Bryansk and the Bryansk Region. In addition to routine cytogenetic analysis of unstable aberrations, the frequency of stable aberrations was evaluated by FISH in 74 subjects comprising of both workers and controls. Whole chromosome probes for Nos. 1, 2 and 4 (Vysis, USA) were used. These comprise 22.3% of genome and lead to 34.4% eciency in detecting translocations (Tucker and Senft, 1994). Generally, 400±500 metaphases were analysed per sample, and the observed aberration frequency was converted to full genome equivalence Fg using the equation Fg Fp =2:05fp 1 ÿ fp , where Fp is the frequency of stable aberrations observed and fp is the fraction of the genome painted (Lucas et al., 1992). Additionally, G-banding for analysis of samples from 28 persons was undertaken. On an average, 50 metaphases per sample were studied. The correlation rate between the frequency of stable aberration estimated by G-banding and by FISH was calculated by the Spearman method. A value of p < 0.05 was considered as statistically signi®cant. A comparative analysis of the frequencies of dicentrics and rings was performed by a non-parametrical criterion of distribution similarity (Kolmogorov±Smirnov test). Again, a value of p < 0.05 was considered as indicating statistical signi®cance.
3. Results and discussion Cells with multiple chromosomal aberrations were observed by routine cytogenetic analysis in samples from 103 out of 1307 persons engaged in the study (7.4%). These 103 persons comprised a subgroup designated 1B. Table 1 shows the frequencies of dicentrics and rings in groups 1 and 2; the workers and controls, respectively, with group 1 being subdivided into 1A and 1B; respectively, without and with rogue cells, and then being further subdivided by the year in which they undertook duty at Chernobyl. The results are as follows: 1. Despite 8±10 years having elapsed between radiation exposure and blood sampling, the frequency of dicentrics and rings observed in lymphocytes of the clearance workers exceeded by two to three times the background level scored in the control group. After completing their duties in the exclusion zone, these workers lived in non-contaminated areas and were not exposed to additional radiation.
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2. The frequency of dicentrics plus rings was higher in group 1B as compared with group 1A: 1.14 2 0.18 and 0.93 2 0.1, respectively ( p < 0.05). It should be noted that these values do not include the aberrations observed in rogue cells. 3. There is no connection between the frequency of rogue cells and the year in which a particular worker was engaged in clearance activities, and this frequency ranges from 1.83 to 1.94 per 1000 cells. The level of stable aberrations in clearance workers included in 1A and 1B groups was studied. Twentyfour samples from group 1A and 32 from 1B were analysed. Eighteen persons were also selected from the control group 2. Tables 2±4 show these results, which were principally obtained by the FISH method. Additionally, all 18 controls were examined by G- banding and samples from 10 of the clearance workers with rogue cells. The use of G-banding permitted a comparison with FISH for evaluating the accuracy of calculating the stable aberrations per genome, when probes for only three pairs of chromosomes were used. A statistically signi®cant correlation between results of both methods was found. The mean frequency level of stable aberrations expressed as full genome equivalent in the control group was 0.65 per 100 cells, and this agrees well with other studies (Straume et al., 1992; Tucker et al., 1994). The level of stable aberrations in most of the samples studied from groups 1A and 1B was several times higher than in the control subjects. In group 1A this range was 2.2±13.7 per 100 cells with a median of 7.6, and in group 1B, it was 0±19.7 with a median of 6.6. No statistically signi®cant intergroup dierence in the frequencies of stable aberrations was found.
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In addition to stable aberrations, all other chromosomal anomalies that involved chromosomes 1, 2 and 4 were recorded, such as dicentrics, rings, deletions, fragments, and marker chromosomes. An intergroup dierence in the ratio between dicentrics and rings was noted. In group 1A the dicentrics:rings ratio was 4:1, and in group 1B it was 1:2. It appears therefore that there is an increased frequency of rings in clearance workers carrying rogue cells. There is no obvious explanation for this but it is noteworthy that the same trend was described by Salomaa et al. (1997) in residents of radionuclide contaminated areas of Bryansk Region. The frequency of cells with trisomies, fragments, and markers involving Nos. 1, 2 and 4 was also higher in group 1B. By FISH, two types of rogue cells were observed. The ®rst contained multiple one- and two-coloured polycentrics with fragments. These cells were similar to those observed during conventional block-stained analysis. In the second type, there was no unstable damage seen, although numerous (r8) less complex rearrangements of chromosomes were noted. It is not feasible to detect reliably cells of this second type by routine analysis because all abnormalities still comprised monocentric chromosomes. In summarising the results from the FISH study, it could be concluded that the level of stable aberrations did not dier between the groups, but there was a wide range of frequencies within the two groups. By contrast, unstable aberrations including deletions, marker chromosomes, and fragments were more frequent in clearance workers with rogue cells and there was also an increase in the number of ring chromosomes in these workers. In addition, the number of trisomic cells was doubled in this group. Although only a limited number of rogue cells were observed by FISH in group 1B subjects, it was still possible to identify the
Table 1 Frequencies of dicentrics and rogue cells in Chernobyl accident clearance workers according to their year of work Year of work Group 1 1986±88 Subgroup 1A 1986 1987 1988 Total Subgroup 1B 1986 1987 1988 Total Group 2 (control)
No. of cases
No. of cells
No. of dic+r
dic+r/1000
No. of rogue cells
Rogue cells/1000
1307
420,977
415
0.9920.05
217
0.5220.03
721 431 52 1204
186,964 104,624 13,942 305,260
174 97 12 283
0.9320.07 0.9320.09 0.8620.25 0.9320.06
0 0 0 0
72 27 4 103 47
88,164 22,399 5154 115,717 9342
85 40 7 132 4
0.9620.10 1.7920.28 1.3520.51 1.1420.10 0.4320.22
166 41 10 217 0
0 0 0 0 1.8820.15 1.8320.29 1.9420.61 1.8720.13 0
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type with multiple damage that could not be detected by conventional staining. Whether such cells also occurred in group 1A subjects is unknown as none of them were FISHed. Rogue cells proved to be very elusive in the FISH preparations, and therefore it was decided to sample 14 persons from group 1B again, 1 year later, and to score as many metaphases as possible by conventional block staining. In 13 subjects (total No. of cells 11,154) no rogue cells, of the ®rst type, were seen. The second type, of course, would not be detectable. Only in 1 clearance worker were 28 rogues in 3668 cells seen, but that is higher than the frequency observed previously in any member of the whole group 1B. It should be particularly emphasised that the worker group was divided into two subgroups based only on the presence of type-one rogue cells observed with
block staining. All clearance workers were exposed to low doses of radiation at Chernobyl, and later resided in non-contaminated areas and were not exposed to any additional radiation. Our studies of stable aberration frequencies suggest that the spectrum of radiation doses was more or less the same amongst workers in either group. Hence, it may be presumed that there is no connection between their certainly low gamma-radiation dose and the presence of rogue cells. The higher frequency of unstable chromosomal aberrations detected by FISH in clearance workers carrying rogue cells correlates well with the data on the statistically signi®cant increase of dicentrics and rings in them detected by routine analysis. It is dicult to imagine that the rate of elimination of cells with unstable aberrations was greater in one group compared to the other. It is more probable that some of the aberrations
Table 2 Cytogenetic features of peripheral blood lymphocytes in clearance workers carrying rogue cells (group 1B) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total
Routine staining
FISH
G-band
No. of cells
% of abn. cells
dic+r/100
MAC (abs.)
No. of cells
t/100 per genome
458 887 901 1206 716 517 658 742 568 616 512 388 566 612 339 358 1036 236 1739 626 1059 545 431 311 405 523 338 211 432 546 410 549 19,441
3 0.3 0.2 0.6 0.4 0.8 1.1 0.5 1.0 2.2 1.0 0 0.7 0.5 0.9 1.4 0.4 3.4 0.2 0.9 0.3 0.3 0.5 0.6 1.0 0.4 0.9 0.9 0.7 0 0.5 0.2 0.6720.06
0.2 0.1 0.2 0.1 0.4 0 0 0.3 0.2 0.5 0 0 0 0 0.3 0.3 0 0.4 0 0 0.1 0 0.5 0 0 1 1 0 0 0 0.2 0 0.220.03
1 2 1 1 1 1 1 2 1 2 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 39
342 667 810 741 1120 732 1108 427 802 608 799 711 1007 246 754 654 643 852 770 1227 498 405 854 933 1271 1281 1091 842 745 933 840 425 25,138
7.5 4.3 6.3 0 7.9 1.2 2.8 12.7 9.9 12.6 8.2 4.4 1.1 2.3 7.7 19.7 2.6 10.2 5.1 4.1 5.1 1.4 15.4 6.8 4.8 2.7 12.5 5.4 9.2 7.4 7.1 8.0 6.820.16
No. of cells
t/100
64
14.1
76 55 64 72 50
7.8 6.4 0 8.3 4.0
58 82
6.8 4.8
72 50 643
5.5 12.0 7.021.0
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Table 3 Cytogenetic features of peripheral blood lymphocytes in clearance workers without rogue cells (group 1A) No.
Routine staining
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Total
FISH
No. of cells
% of abn. cells
dic+ r/100
MAC (abs.)
No. of cells
t/100 per genome
200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 4800
0 0.5 0 1.0 0 0 0.5 0 0 0 0 0 0 0 0 0 0.5 0 1.0 0 0.5 0 0 1.0 0.2120.07
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 0 0 0 0.0620.04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
846 777 942 641 713 719 1116 993 712 1268 807 812 867 883 1191 782 1270 244 291 289 598 2187 587 416 19,951
3.4 4.0 6.0 2.2 4.2 4.6 9.7 7.7 9.7 10.5 7.1 12.0 13.7 4.0 2.8 10.8 4.2 9.1 2.8 11.4 8.8 11.4 9.7 7.5 7.420.2
Table 4 Spontaneous level of chromosome aberrations in the controls (group 2) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
Routine staining
FISH
G-band
No. of cells
% of abn. cells
dic+r/100
MAC (abs.)
No. of cells
t/100 per genome
No. of cells
t/100
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 5400
0 0.6 0 0 0.3 0 1.0 0 0 1.0 0 0 0.6 0 0 0 0 0.6 0.2420.07
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3 0.0220.02
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1000 1000 1000 1000 1000 875 950 1000 1000 726 1000 1000 1000 1000 726 690 1000 1000 16,967
0 1.1 0.6 0 0.6 0.6 0.8 0.6 1.1 0.3 1.1 0.6 0.6 0.9 0.4 0.6 1.2 0.6 0.5020.05
100 100 62 100 50 55 74 100 100 56 82 54 50 100 54 50 80 55 1322
0 1.0 0 0 0 0 1.3 0 0 0 1.2 0 2.0 1.0 0 0 1.3 0 0.4520.19
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in clearance workers carrying rogue cells are due to irradiation of hematopoietic cells. Studies of chromosomal aberrations in irradiated cultured cells demonstrate that ionizing radiation is able to cause genomic instability, although the impacts of low LET gamma and high LET radiations are biologically dierent (Kronenberg, 1994). Kadhim et al. (1992) demonstrated that a major impact of low doses of radiation on cultured bone marrow cells was the appearance of some colonies with clonal aberrations. Low doses of alpha radiation induced cells with one or more nonclonal aberrations, and these emerged after several cell cycles. After many cell divisions, Sabatier et al. (1992) observed dicentrics and broken chromosomes amongst numerous nonclonal aberrations emerging in lymphocytes and ®broblasts exposed to high LET radiation. Studies of the in¯uence of alpha particles on ®broblasts have shown that the most frequently induced aberrations were insertions and rings (Grin et al., 1995), and in some cases an evolution of aneuploidy was noted (Sabatier et al., 1994). With FISH, Marder and Morgan (1993) observed a quantitative heterogeneity of chromosome No. 4 in metaphases of one irradiated clone.
4. Conclusion When comparing these literature data with the present results in samples from group 1B, we postulate that the relative increase of trisomies, and rings, and the emergence of rogue cells, which are all types of damage that are traditionally considered to be rapidly eliminated, may be a manifestation of delayed chromosomal instability induced by low dose of incorporated alpha emitters. This, however, contrasts with the conclusion of Sevan'kaev et al. (1993) who did not consider the possibility of delayed instability. They argued mainly from the standpoint of microdosimetry and the metabolism of alpha-emitters that this could not have been the cause of rogue cells, which they observed in children resident in contaminated areas of Belarus.
References Alexakhin, R.H., 1993. Radioecological lessons of Chernobyl. Radiobiologiya 33, 3 (in Russian). Awa, A.A., Neel, J.V., 1986. Cytogenetic `rogue' cells: what is their frequency, origin and evolutionary signi®cance? Proc. Natl. Acad. Sci. USA 83, 1021. Bloom, A.D., Neel, J.V., Tsuchimoto, T., Meilinger, K., 1973. Chromosome breakage in leukocytes of South American indians. Cytogenet. Cell Genet. 12, 175. Bochkov, N.P., Katasova, L.D., 1994. Analysis of multiaberrant cells in lymphocytes. Mutat. Res. 323, 7.
Domracheva, E.V., Kuznetsov, S.A., Shklovski-Kordi, N.E., Vorobiov, AÁ.EÁ., 1991. Cells with multiple chromosomal aberrations in residents of Chernobyl area (in Russian). Gematologiya i transfuziologiya 11, 36. Fox, D.P., Robertson, F.W., Brown, T., Whitehead, A.R., Douglas, J.D.M., 1984. Chromosome aberrations in divers. Undersea Biomedical Research 11, 193. Grin, C.S., Marsden, S.J., Stevens, D.L., Simpson, P., et al., 1995. Frequencies of complex chromosome exchange aberrations induced by 238Pu-particles and detected by ¯uorescence in situ hybridization using single chromosomespeci®c probes. Int. J. Radiat. Biol. 67 (4), 431. Kadhim, M.A., Macdonald, D.A., Goodhead, D.T., Lorimore, S.A., Marsden, S.J., Wright, E.G., 1992. Transmission of chromosomal instability after plutonium alpha-particle irradiation. Nature 355, 738. Kossenko, M.M., Degteva, M.O., 1994. Cancer mortality and radiation risk evaluation for Techa river population. Sci. Tot. Environment 142, 73. Kronenberg, A., 1994. Radiation-induced genomic instability. Int. J. Radiat. Biol. 66, 603. Lazutka, J.R., 1996. Chromosome aberrations and rogue cells in lymphocytes of Chernobyl clean-up workers. Mutat. Res. 350, 315. Lucas, J.N., Awa, A.A., Straume, T., et al., 1992. Rapid translocation frequency analysis in humans decades after exposure to radiation. Int. J. Radiat. Biol. 62, 53. Marder, B.A., Morgan, W.R., 1993. Delayed chromosomal instability induced by DNA damage. Mol. Cell. Biol. 13, 6667. Marenny, A.M., Kovalev, E.E., Shoikhet, J.N., Vlasov, P.A., Lepilov, A.V., Tretyakova, S.P., 1994. Radionuclides in the lung tissues of oncological patients in Altai region. Proceedings of scienti®c program `Semipalatinsk test station-Altai' 4, 73. Neel, J.V., Awa, A.A., Kodama, Y., Nakano, M., Mabuchi, K., 1992. Rogue lymphocytes among Ukranians not exposed to radioactive fall-out from Chernobyl accident. The possible role of this phenomenon in oncogenesis, teratogenesis and mutagenesis. Proc. Nat. Acad. Sci. USA 89, 6973. Neel, J.V., Major, E.O., Awa, A.A., Glover, T., Burgess, A., Traub, R., Curfman, B., Satoh, C., 1996. Hypothesis: Rogue cell-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncogenesis. Proc. Nat. Acad. Sci. USA 93, 2690. Pavlotskaya, F.I., Goryachenkova, T.A., et al., 1992. Characteristics of 239Pu, 240Pu in the contaminated soil after the accident at South Urals in1957 (in Russian). Atomnaya energiya 73 (1), 32. Pyatkin, E.K., Nugis, V.Yu., Chirkov, A.A., 1989. The estimation of absorbed dose on the basis of cytogenetic studies of lymphocytes obtained from victims of Chernobyl accident (in Russian). Meditsinskaya radiologiya 34 (6), 54. Rivkind, N.B., Staritsina, L.P., Skvortsova, G.V., 1996. Multiaberrant cells in clearance workers of Chernobyl accident (in Russian). Terapevticheskii arkhiv 7, 77. Sabatier, L., Dutrillaux, B., Martin, M.B., 1992. Chromosomal instability. Nature 357, 548.
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1153±1159 Sabatier, L., Lebeau, J., Dutrillaux, B., 1994. Chromosomal instability and alterations of telomeric repeats in irradiated human ®broblasts. Int. J. Radiat. Biol. 66, 611. Salomaa, S., Sevan'kaev, A.V., Zhloba, A.A., Kumpusalo, E., Makinen, S., Lindholm, C., Kumpusalo, L., Kolmakow, S., Nissinen, A., 1997. Unstable and stable chromosomal aberrations in lymphocytes of people exposed to Chernobyl fallout in Bryansk. Russia. Int. J. Radiat. Biol. 97, 51. Sevan'kaev, A.V., Tsyb, A.F., Lloyd, D.C., Zhloba, A.A., Moiseenko, V.V., Skrjabin, A.V., Climov, V.M., 1993. Rogue cells observed in children exposed to radiation from the chernobyl accident. Int. J. Radiat. Biol. 63, 361. Shevchenko, V.A., Snigiryova, G.P. 1996. Cytogenetic features of the impact of ionizing radiation on human population. In: Burlakova, E.B. (Ed.), Consequences of Chernobyl accident: Human health. Russian Academy of Sciences, Moscow, pp. 24±29 (in Russian). Snigiryova, G.P., Novitskaja, N.N., Sidorova, V.F., Khazins, E.D., Elisova, T.V., Iofa, E.L., Shevcheko, V.A., 1997. Cytogenetic damage of the population from the neighbourhood of the radionuclide-contaminated Techa river. In:
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Stable and unstable aberrations in lymphocytes of Chernobyl accident clearance workers carrying rogue cells E.V. Domracheva*, N.B. Rivkind, E.A. Aseeva, T.N. Obukhova, L.V. D'achenko, A.I. Vorobiov Karyology Laboratory, National Research Center for Hematology, Russian Academy of Medical Sciences, Novozykovski pr., 4a, 125167 Moscow, Russia
Abstract Cells with multiple chromosomal aberrations, the so-called rogue cells, were found in blood samples from more than 100 Chernobyl accident clearance workers. A comparative analysis of frequencies of stable and unstable chromosomal aberrations in two worker groups Ð those with or without rogue cells was made. A higher level of unstable aberrations in persons carrying rogue cells was observed. No dierence in the level of stable aberrations between the groups was seen. The possibility of low dose alpha irradiation causing the chromosomal damage is raised. 7 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Cells with numerous (r3) exchange-type chromosomal aberrations, i.e. di-, tri-, polycentrics, rings, as well as acentric fragments, remain a matter of interest for radiation cytogeneticists. Such cells are reported to be a common feature in the blood after acute accidental exposure to high doses of gamma radiation (3±5 Gy), or as a result of incorporating alpha-emitting radionuclides (Vorobiov et al., 1974; Pyatkin et al., 1989). One of the basic tenets of radiation cytogenetics is that the aberration frequency increases as some function of radiation dose, so that multiaberrant cells (MAC) are more likely to be induced by higher doses. However, not all researchers subscribe to the view of the radiation causation of rogue cells. They have been recorded at quite high frequencies in various cohorts
* Corresponding author. Fax: +7-212-42-52. E-mail address: [email protected] (E.V. Domracheva).
of presumably only background irradiated persons (Tawn et al., 1985). Indeed, they were ®rst described in a study of a newly discovered stone-age tribe of Amazonian indians (Bloom et al., 1973), and this was followed by others, such as deep sea divers (Fox et al., 1984), and controls for the study of A-bomb survivors and their ospring (Awa and Neel, 1986). This led to the suggestion of a viral origin (Awa and Neel, 1986), and some supporting evidence implicating polyoma virus has been presented by Neel et al. (1996). After the Chernobyl accident, the number of studies of chromosomal aberrations in lymphocytes of residents of contaminated areas and of clearance workers has grown dramatically. In some of these studies rogue cells were observed. Lymphocytes with multiple chromosomal aberrations have been reported from residents of contaminated areas (Domracheva et al., 1991; Neel et al., 1992; Sevan'kaev et al., 1993; Salomaa et al., 1997; Verchaeve et al., 1993; Bochkov and Katasova, 1994), and in Chernobyl clearance workers (Rivkind et al., 1996; Lazutka, 1996). Similar cells were
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seen in residents of Altai region who were exposed to radioactive contamination after the nuclear tests in Semipalatinsk (Shevchenko and Snigiryova, 1996), and in residents of the Chelyabinsk region near the Techa River. This river has been severely contaminated by radionuclides from the Mayak plutonium plant (Snigiryova et al., 1997; Kossenko and Degteva, 1994). Hence, rogue cells were observed in most radiation exposed cohorts studied, and this may indicate a radiation origin but, at the same time, cannot be considered as conclusive. The gamma-radiation doses, which clearance workers and residents of contaminated areas were exposed to, are substantially lower than the low LET radiation doses that are considered capable of inducing multiaberrant cells. However, contamination of large areas by plutonium and other high LET emitters after the Semipalatinsk nuclear tests, and Chernobyl and Mayak accidents have been admitted (Pavlotskaya and Goryachenkova, 1992; Alexakhin, 1993; Marenny et al., 1994). Over 6 years, we have studied samples from clearance workers who undertook duties at Chernobyl from 1986 to 1988 and who normally reside in the Bryansk region, and have observed in them a considerable number of multiaberrant cells. Such cells were not seen in a control group, and this strengthens our view that these rogue cells have a radiation causation (Rivkind et al., 1996). In the present study, we have tried to establish a connection between rogue cells and more commonly accepted radiaton cytogenetic markers, i.e. dicentrics and rings, and the level of stable chromosomal aberrations. A ¯uorescence in situ hybridization (FISH) study was performed within the framework of a Bryansk Branch of the International Consortium on Health Eects of Low Dose Radiation.
2. Materials and methods Peripheral blood lymphocytes were routinely cultured in the Karyology Lab of the National Research Center for Hematology and in the Biodosimetry Lab of the Bryansk Diagnostic Center. 200±300 metaphases were analysed from each of 1307 clearance workers aged 21±54 years. They had been engaged in dierent activities; comprising persons who built the sarcophagus around the damaged reactor; those who worked elsewhere inside the 30 km exclusion zone, and those who performed dosimetry measurements. The durations of working inside the zone varied from 8 days to 1.5 months, the average being 22 days. Personal physical dosimetry was performed for the majority of the subjects and recorded radiation doses ranged from 0.03 to 0.24 Gy. The body content of radiocaesium was also measured, but did not exceed background
level varying from 14 to 157 nCi. The cohort could be considered generally healthy, although some had minor malfunctions of the nervous system. The control group comprised 47 healthy persons with ages ranging from 20 to 47 years. They apparently had not been exposed to radiation and lived in non-contaminated areas of Bryansk and the Bryansk Region. In addition to routine cytogenetic analysis of unstable aberrations, the frequency of stable aberrations was evaluated by FISH in 74 subjects comprising of both workers and controls. Whole chromosome probes for Nos. 1, 2 and 4 (Vysis, USA) were used. These comprise 22.3% of genome and lead to 34.4% eciency in detecting translocations (Tucker and Senft, 1994). Generally, 400±500 metaphases were analysed per sample, and the observed aberration frequency was converted to full genome equivalence Fg using the equation Fg Fp =2:05fp 1 ÿ fp , where Fp is the frequency of stable aberrations observed and fp is the fraction of the genome painted (Lucas et al., 1992). Additionally, G-banding for analysis of samples from 28 persons was undertaken. On an average, 50 metaphases per sample were studied. The correlation rate between the frequency of stable aberration estimated by G-banding and by FISH was calculated by the Spearman method. A value of p < 0.05 was considered as statistically signi®cant. A comparative analysis of the frequencies of dicentrics and rings was performed by a non-parametrical criterion of distribution similarity (Kolmogorov±Smirnov test). Again, a value of p < 0.05 was considered as indicating statistical signi®cance.
3. Results and discussion Cells with multiple chromosomal aberrations were observed by routine cytogenetic analysis in samples from 103 out of 1307 persons engaged in the study (7.4%). These 103 persons comprised a subgroup designated 1B. Table 1 shows the frequencies of dicentrics and rings in groups 1 and 2; the workers and controls, respectively, with group 1 being subdivided into 1A and 1B; respectively, without and with rogue cells, and then being further subdivided by the year in which they undertook duty at Chernobyl. The results are as follows: 1. Despite 8±10 years having elapsed between radiation exposure and blood sampling, the frequency of dicentrics and rings observed in lymphocytes of the clearance workers exceeded by two to three times the background level scored in the control group. After completing their duties in the exclusion zone, these workers lived in non-contaminated areas and were not exposed to additional radiation.
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2. The frequency of dicentrics plus rings was higher in group 1B as compared with group 1A: 1.14 2 0.18 and 0.93 2 0.1, respectively ( p < 0.05). It should be noted that these values do not include the aberrations observed in rogue cells. 3. There is no connection between the frequency of rogue cells and the year in which a particular worker was engaged in clearance activities, and this frequency ranges from 1.83 to 1.94 per 1000 cells. The level of stable aberrations in clearance workers included in 1A and 1B groups was studied. Twentyfour samples from group 1A and 32 from 1B were analysed. Eighteen persons were also selected from the control group 2. Tables 2±4 show these results, which were principally obtained by the FISH method. Additionally, all 18 controls were examined by G- banding and samples from 10 of the clearance workers with rogue cells. The use of G-banding permitted a comparison with FISH for evaluating the accuracy of calculating the stable aberrations per genome, when probes for only three pairs of chromosomes were used. A statistically signi®cant correlation between results of both methods was found. The mean frequency level of stable aberrations expressed as full genome equivalent in the control group was 0.65 per 100 cells, and this agrees well with other studies (Straume et al., 1992; Tucker et al., 1994). The level of stable aberrations in most of the samples studied from groups 1A and 1B was several times higher than in the control subjects. In group 1A this range was 2.2±13.7 per 100 cells with a median of 7.6, and in group 1B, it was 0±19.7 with a median of 6.6. No statistically signi®cant intergroup dierence in the frequencies of stable aberrations was found.
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In addition to stable aberrations, all other chromosomal anomalies that involved chromosomes 1, 2 and 4 were recorded, such as dicentrics, rings, deletions, fragments, and marker chromosomes. An intergroup dierence in the ratio between dicentrics and rings was noted. In group 1A the dicentrics:rings ratio was 4:1, and in group 1B it was 1:2. It appears therefore that there is an increased frequency of rings in clearance workers carrying rogue cells. There is no obvious explanation for this but it is noteworthy that the same trend was described by Salomaa et al. (1997) in residents of radionuclide contaminated areas of Bryansk Region. The frequency of cells with trisomies, fragments, and markers involving Nos. 1, 2 and 4 was also higher in group 1B. By FISH, two types of rogue cells were observed. The ®rst contained multiple one- and two-coloured polycentrics with fragments. These cells were similar to those observed during conventional block-stained analysis. In the second type, there was no unstable damage seen, although numerous (r8) less complex rearrangements of chromosomes were noted. It is not feasible to detect reliably cells of this second type by routine analysis because all abnormalities still comprised monocentric chromosomes. In summarising the results from the FISH study, it could be concluded that the level of stable aberrations did not dier between the groups, but there was a wide range of frequencies within the two groups. By contrast, unstable aberrations including deletions, marker chromosomes, and fragments were more frequent in clearance workers with rogue cells and there was also an increase in the number of ring chromosomes in these workers. In addition, the number of trisomic cells was doubled in this group. Although only a limited number of rogue cells were observed by FISH in group 1B subjects, it was still possible to identify the
Table 1 Frequencies of dicentrics and rogue cells in Chernobyl accident clearance workers according to their year of work Year of work Group 1 1986±88 Subgroup 1A 1986 1987 1988 Total Subgroup 1B 1986 1987 1988 Total Group 2 (control)
No. of cases
No. of cells
No. of dic+r
dic+r/1000
No. of rogue cells
Rogue cells/1000
1307
420,977
415
0.9920.05
217
0.5220.03
721 431 52 1204
186,964 104,624 13,942 305,260
174 97 12 283
0.9320.07 0.9320.09 0.8620.25 0.9320.06
0 0 0 0
72 27 4 103 47
88,164 22,399 5154 115,717 9342
85 40 7 132 4
0.9620.10 1.7920.28 1.3520.51 1.1420.10 0.4320.22
166 41 10 217 0
0 0 0 0 1.8820.15 1.8320.29 1.9420.61 1.8720.13 0
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type with multiple damage that could not be detected by conventional staining. Whether such cells also occurred in group 1A subjects is unknown as none of them were FISHed. Rogue cells proved to be very elusive in the FISH preparations, and therefore it was decided to sample 14 persons from group 1B again, 1 year later, and to score as many metaphases as possible by conventional block staining. In 13 subjects (total No. of cells 11,154) no rogue cells, of the ®rst type, were seen. The second type, of course, would not be detectable. Only in 1 clearance worker were 28 rogues in 3668 cells seen, but that is higher than the frequency observed previously in any member of the whole group 1B. It should be particularly emphasised that the worker group was divided into two subgroups based only on the presence of type-one rogue cells observed with
block staining. All clearance workers were exposed to low doses of radiation at Chernobyl, and later resided in non-contaminated areas and were not exposed to any additional radiation. Our studies of stable aberration frequencies suggest that the spectrum of radiation doses was more or less the same amongst workers in either group. Hence, it may be presumed that there is no connection between their certainly low gamma-radiation dose and the presence of rogue cells. The higher frequency of unstable chromosomal aberrations detected by FISH in clearance workers carrying rogue cells correlates well with the data on the statistically signi®cant increase of dicentrics and rings in them detected by routine analysis. It is dicult to imagine that the rate of elimination of cells with unstable aberrations was greater in one group compared to the other. It is more probable that some of the aberrations
Table 2 Cytogenetic features of peripheral blood lymphocytes in clearance workers carrying rogue cells (group 1B) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Total
Routine staining
FISH
G-band
No. of cells
% of abn. cells
dic+r/100
MAC (abs.)
No. of cells
t/100 per genome
458 887 901 1206 716 517 658 742 568 616 512 388 566 612 339 358 1036 236 1739 626 1059 545 431 311 405 523 338 211 432 546 410 549 19,441
3 0.3 0.2 0.6 0.4 0.8 1.1 0.5 1.0 2.2 1.0 0 0.7 0.5 0.9 1.4 0.4 3.4 0.2 0.9 0.3 0.3 0.5 0.6 1.0 0.4 0.9 0.9 0.7 0 0.5 0.2 0.6720.06
0.2 0.1 0.2 0.1 0.4 0 0 0.3 0.2 0.5 0 0 0 0 0.3 0.3 0 0.4 0 0 0.1 0 0.5 0 0 1 1 0 0 0 0.2 0 0.220.03
1 2 1 1 1 1 1 2 1 2 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 39
342 667 810 741 1120 732 1108 427 802 608 799 711 1007 246 754 654 643 852 770 1227 498 405 854 933 1271 1281 1091 842 745 933 840 425 25,138
7.5 4.3 6.3 0 7.9 1.2 2.8 12.7 9.9 12.6 8.2 4.4 1.1 2.3 7.7 19.7 2.6 10.2 5.1 4.1 5.1 1.4 15.4 6.8 4.8 2.7 12.5 5.4 9.2 7.4 7.1 8.0 6.820.16
No. of cells
t/100
64
14.1
76 55 64 72 50
7.8 6.4 0 8.3 4.0
58 82
6.8 4.8
72 50 643
5.5 12.0 7.021.0
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Table 3 Cytogenetic features of peripheral blood lymphocytes in clearance workers without rogue cells (group 1A) No.
Routine staining
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Total
FISH
No. of cells
% of abn. cells
dic+ r/100
MAC (abs.)
No. of cells
t/100 per genome
200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 4800
0 0.5 0 1.0 0 0 0.5 0 0 0 0 0 0 0 0 0 0.5 0 1.0 0 0.5 0 0 1.0 0.2120.07
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 0 0 0 0.0620.04
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
846 777 942 641 713 719 1116 993 712 1268 807 812 867 883 1191 782 1270 244 291 289 598 2187 587 416 19,951
3.4 4.0 6.0 2.2 4.2 4.6 9.7 7.7 9.7 10.5 7.1 12.0 13.7 4.0 2.8 10.8 4.2 9.1 2.8 11.4 8.8 11.4 9.7 7.5 7.420.2
Table 4 Spontaneous level of chromosome aberrations in the controls (group 2) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
Routine staining
FISH
G-band
No. of cells
% of abn. cells
dic+r/100
MAC (abs.)
No. of cells
t/100 per genome
No. of cells
t/100
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 5400
0 0.6 0 0 0.3 0 1.0 0 0 1.0 0 0 0.6 0 0 0 0 0.6 0.2420.07
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3 0.0220.02
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1000 1000 1000 1000 1000 875 950 1000 1000 726 1000 1000 1000 1000 726 690 1000 1000 16,967
0 1.1 0.6 0 0.6 0.6 0.8 0.6 1.1 0.3 1.1 0.6 0.6 0.9 0.4 0.6 1.2 0.6 0.5020.05
100 100 62 100 50 55 74 100 100 56 82 54 50 100 54 50 80 55 1322
0 1.0 0 0 0 0 1.3 0 0 0 1.2 0 2.0 1.0 0 0 1.3 0 0.4520.19
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in clearance workers carrying rogue cells are due to irradiation of hematopoietic cells. Studies of chromosomal aberrations in irradiated cultured cells demonstrate that ionizing radiation is able to cause genomic instability, although the impacts of low LET gamma and high LET radiations are biologically dierent (Kronenberg, 1994). Kadhim et al. (1992) demonstrated that a major impact of low doses of radiation on cultured bone marrow cells was the appearance of some colonies with clonal aberrations. Low doses of alpha radiation induced cells with one or more nonclonal aberrations, and these emerged after several cell cycles. After many cell divisions, Sabatier et al. (1992) observed dicentrics and broken chromosomes amongst numerous nonclonal aberrations emerging in lymphocytes and ®broblasts exposed to high LET radiation. Studies of the in¯uence of alpha particles on ®broblasts have shown that the most frequently induced aberrations were insertions and rings (Grin et al., 1995), and in some cases an evolution of aneuploidy was noted (Sabatier et al., 1994). With FISH, Marder and Morgan (1993) observed a quantitative heterogeneity of chromosome No. 4 in metaphases of one irradiated clone.
4. Conclusion When comparing these literature data with the present results in samples from group 1B, we postulate that the relative increase of trisomies, and rings, and the emergence of rogue cells, which are all types of damage that are traditionally considered to be rapidly eliminated, may be a manifestation of delayed chromosomal instability induced by low dose of incorporated alpha emitters. This, however, contrasts with the conclusion of Sevan'kaev et al. (1993) who did not consider the possibility of delayed instability. They argued mainly from the standpoint of microdosimetry and the metabolism of alpha-emitters that this could not have been the cause of rogue cells, which they observed in children resident in contaminated areas of Belarus.
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