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Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident
Mutation Research 395 Ž1997. 249–254
Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident L. Padovani
a,)
, L. Stronati a , F. Mauro a , A. Testa a , M. Appolloni a , P. Anzidei a , D. Caporossi b, B. Tedeschi b, P. Vernole b
a
b
EnÕironment Department, CR ENEA Casaccia, Via Anguillarese 301, 00060 Rome, Italy Department of Public Health and Cell Biology, UniÕersity of Rome ‘‘Tor Vergata’’, Rome, Italy Received 21 May 1997; revised 10 July 1997; accepted 18 July 1997
Abstract In a previous paper we reported that a group of children exposed to ionizing radiation following the Chernobyl accident exhibited an appreciable number of chromosome breaks and rearrangements reflecting the persistence of a radiation-induced damage. The results suggested that the children were still exposed to radioactive contamination through consumer foodstuff and life styles. In the present paper, 31 exposed children have been considered together with a control group of 11 children with the aim to confirm previous results. All children underwent whole-body counter ŽWBC. measures and conventional cytogenetic analysis. The frequency of chromosome aberrations detected by conventional cytogenetics in the group of children chronically exposed to low doses of ionizing radiation resulted in significant differences with respect to the control group. The present work suggests that, for these groups of children, even if the frequency of aberrations is very low and the observation of statistically significant differences is consequently a problem, a persistently abnormal cytogenetic picture is still present several years after the accident. q 1997 Elsevier Science B.V. Keywords: Chernobyl; Cytogenetics; Chromosome aberration
1. Introduction In 1986, due to climatological circumstances, the accident in one of the Chernobyl nuclear power plants resulted in a non-uniform, widely different contamination of many regions of the former USSR and Europe w1x. This accident represents a unique and large-scale problem in human ecology, health monitoring and control. Thus, various cytogenetic
)
Corresponding author.
studies were performed in order to obtain some insight on the level of the human hazard due to such accidental exposure, both on Chernobyl clean-up workers w2–9x and on people exposed to relatively low doses of ionizing radiation w10–15x. Among all these, some studies have been carried out on children w16–19x. In many cases an increased frequency of chromosomal aberrations was reported. In a previous study, we also carried out an analysis of chromosomal aberrations in the peripheral blood lymphocytes of children coming from differently contaminated areas of Belarus, Ukraine and Russian Federation and exhibiting varying amount of
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L. PadoÕani et al.r Mutation Research 395 (1997) 249–254
137
Cs internal contamination w20x. Results obtained showed a persistence of a radiation-induced genomic damage suggesting a continuation of the study. Besides, a control group composed of individuals from an uncontaminated area of the former USSR was not available for that study. In the present study, we analysed a total of 42 Russian children, including 11 coming from the Smolensk area, which is reported to be uncontaminated, and thus suitable as a control. The aim of the present study is to extend the cytogenetic analysis to another group of chronically or temporally exposed subjects in comparison with the local control group in order to confirm the persistence of a chromosomal damage 10 years after the accident. It is well known that the induction of chromosomal aberrations is intimately related to other genetic changes such as mutation and mutation toward cancer, and thus provides a measure of genetic hazards. On the other hand, the knowledge of the actual health condition of the individuals exposed to relatively low doses of ionizing radiation after the Chernobyl accident is interesting in consideration of the fact that they represent the largest population, and that they have been investigated less with respect to the highly exposed people such as the clean-up workers. 2. Materials and methods 2.1. Subjects A total of 42 children Ž10–12 years old., came to Italy in 1993–1994 for a 1-month recreation stay. The children were selected by local humanitarian organizations which organized their trip and requested a series of physical, biological and medical analyses. The medical check-ups carried out in collaborating hospitals indicated no evident signs of pathologies which could be attributed to ionizing radiation. 137Cs internal contamination was measured by Whole Body Counter ŽWBC. in the laboratories of ENEA ŽNational Agency for New Technology Energy and the Environment.. These measures were performed during the first week of the children’s stay in Italy. The children were distributed into three groups. Group A. Twenty-four subjects, 9 males and 15 females, from Novosybkov in the Brianskya region
of the Russian Federation Žground contamination 148 = 10 10 Bqrkm2 as reported by IAEA maps., who exhibited an internal contamination by WBC in the range 780–30 000 Bq. Group B. Seven subjects, 1 male and 6 females, from the uncontaminated area of Smolensk ŽRussian Federation. but living in Pripjat ŽUkraine. at the moment of the accident and thus exposed to the initial ‘‘acute’’ dose of ionizing radiation; these children, evacuated to Smolensk 36 h after the accident, showed an internal contamination in the range 70–128 Bq. Control group C. Eleven subjects, 5 males and 6 females, coming from the uncontaminated area of Smolensk region ŽWBC - 70 Bq.. 2.2. Culture conditions Blood samples were collected from all children at the time of the WBC analysis. Whole blood was cultured at 378C, 5% CO 2 for 48 h, in RPMI medium ŽFlow., supplemented with 10% fetal calf serum ŽGibco., 2% phytohemagglutinin ŽMurex. and 1.5% penicillin–streptomycin Ž5000 IUrml–5000 mgrml. ŽFlow.. For each subject, three different cultures were set up for independent cytogenetic scoring. 0.15 mgrml Colcemid ŽSigma. was added 90 min before harvesting. Metaphases were obtained according to standard procedures. 2.3. Cytogenetic chromosome analysis The slides were coded and analyzed by four different observers. For most of the subjects, a total of 600 unbanded Giemsa-stained metaphases were scored and the frequency of chromosome aberrations typically induced by ionizing radiation Žsuch as dicentrics, acentrics, fragments and rings. were evaluated w21,22,10,23x. As far as translocations are concerned, this method allowed us to evaluate only those that produced chromosomes with evident morphological alteration. 2.4. Statistics Statistical analyses were performed to assess significant differences between the mean frequencies of chromosome aberrations observed in each group.
L. PadoÕani et al.r Mutation Research 395 (1997) 249–254 Table 1 Frequency of structural chromosome aberrations in groups of children coming from differently
251
137
Cs-contaminated areas
Subjects Sex Age WBC Cell Chromosome aberrations ŽBq. scored Ace q DM %
Dic q CR %
Trl %
Total %
Group A 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
F F F M M F M F F F M F F F M F F M M F F M M F
9 10 8 12 11 11 9 9 12 10 9 9 11 10 11 9 11 11 11 9 10 12 11 9
780 1365 1633 1950 1955 2366 2847 3237 3680 3900 4326 4640 4836 5239 5980 6070 7254 9039 30 000 ND ND ND ND ND
550 610 650 550 550 400 450 600 600 550 600 650 574 600 600 600 600 650 600 600 600 607 502 520 13 813
0 4 0 4 1 1 1 0 4 2 4 3 0 4 3 1 1 5 1 0 1 5 1 3 49
0 0.65 0 0.73 0.18 0.25 0.22 0 0.66 0.36 0.66 0.46 0 0.66 0.5 0.16 0.16 0.77 0.16 0 0.16 0.82 0.2 0.57 0.35 ŽES 0.06.
1 1 1 0 0 0 3 0 0 0 0 1 0 2 0 0 0 1 0 1 1 1 2 0 15
0.18 0.16 0.15 0 0 0 0.66 0 0 0 0 0.15 0 0.33 0 0 0 0.15 0 0.16 0.16 0.16 0.39 0 0.11 ŽES 0.03.
1 0 2 2 2 2 3 0 0 3 0 1 0 0 1 0 0 0 2 0 1 1 0 1 22
0.18 0 0.3 0.36 0.36 0.5 0.66 0 0 0.54 0 0.15 0 0 0.16 0 0 0 0.33 0 0.16 0.16 0 0.19 0.15 ŽES 0.04.
5 3 6 3 3 7 0 4 5 4 5 0 6 4 1 1 6 3 1 3 7 3 4 86
0.81 0.46 0.11 0.54 0.75 1.54 0 0.66 0.9 0.66 0.76 0 0.99 0.66 0.16 0.16 0.92 0.49 0.16 0.48 1.14 0.6 0.76 0.622 ŽES 0.08. a
Group B a b c d e f g Total
M F F F F F F
11 10 11 10 9 10 9
- 70 - 70 - 70 95 128 ND ND
600 600 600 264 580 600 600 3844
2 2 3 1 2 1 1 12
0.33 0.33 0.5 0.37 0.34 0.16 0.16 0.31 ŽES 0.04.
0 0 1 1 0 1 0 3
0 0 0.16 0.37 0 0.16 0 0.08 ŽES 0.05.
1 1 0 0 1 0 1 4
0.16 0.16 0 0 0.17 0 0.16 0.1 ŽES 0.03.
3 3 4 2 3 2 2 19
0.5 0.5 0.66 0.75 0.51 0.33 0.33 0.49 ŽES 0.06.
Group C I II III IV V VI VII VIII IX X XI Total
M F M M M M F F F F F
11 10 10 10 9 10 11 9 12 10 11
- 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70
650 550 650 600 600 570 550 448 530 553 600 6301
3 2 0 0 2 1 1 1 1 4 0 15
0.46 0.36 0 0 0.33 0.17 0.18 0.16 0.18 0.72 0 0.22 ŽES 0.07.
0 0 0 1 0 0 1 0 0 1 0 3
0 0 0 0 0 1 0.16 0 0 0 0 3 0.18 0 0 0 0 0 0.18 0 0 0 0.045 ŽES 0.02. 4
0 3 0 2 0.15 1 0 1 0 2 0.52 4 0 2 0 1 0 1 0 5 0 0 0.061 ŽES 0.05. 22
0.46 0.36 0.15 0.16 0.33 0.7 0.36 0.16 0.18 0.9 0 0.33 ŽES 0.08.
Group A, contaminated group; Group B, evacuated group; Group C, Russian control group; Ace q DM, acentric fagmentsq double minute; Dic q CR, dicentricsq centrics rings; Trl, translocations; ND, not determined. a Two-tailed Student’s t-test, statistically significant Ž p - 0.05. compared to control group.
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The two-tailed Student’s t probability test for independent samples was performed; both p - 0.05 and confidence limits at 95% were evaluated.
3. Results Results obtained by the conventional cytogenetic and the WBC measures for each subject are given in Table 1. As a general trend, data show that both group A and group B exhibit higher frequencies of chromosomal aberrations than the control group C. The aberrations, when present, were homogeneously distributed among the metaphases for each individual and only one aberration per cell was found. Statistical analyses were performed on both specific and total chromosome aberrations Žacentrics, dicentrics and translocations.. A significant difference has been revealed only for the mean frequency of the total chromosome aberrations between the contaminated group ŽA. and the Russian control group ŽC. Ž p 0.05; confidence limits at 95%, 0.47–0.77.. No statistically significant differences were observed for all the other comparisons. It is also evident that there is no correlation between chromosome aberrations of each subject of group A and their values of internal contamination. This is not surprising, considering that 137Cs mean life in human body is 20–110 days Žin children. w24,25x and it is not possible to evaluate the pattern of the internal contamination value for the last 10 years because of time elapsed after the accident, changes in diet habits and effects of countermeasures.
4. Discussion Chromosome aberrations can be regarded as a very important and useful indicator of radiation exposure. In this study, a cytogenetic analysis was carried out on a group of children differently exposed to the fallout of the Chernobyl accident. One group of exposed children Žgroup A. were living in a presumably still radioactive environment, while the other Žgroup B. had been supposedly exposed only to an initial acute dose. The cytogenetic analysis shows a statistically significant increase of
aberrations in children belonging to the first group with respect to the controls. This confirms that chromosome damage can persist in individuals exposed to relatively low chronic doses, in agreement with other authors w26,27,16,20x. These data reinforce those obtained in a previous work w20x and suggest that the increase of chromosomal aberrations in subjects living in contaminated areas could actually be due to the exposure to low but chronic doses of ionizing radiation possibly related to still existing problems in diet and lifestyle. Actually, the Cs WBC measures, too, appear to indicate a persisting contamination in group A. Elevated, but not statistically different frequencies of aberrations, are also detected in children of group B, which accordingly show much lower WBC values than children of group A. It can be supposed that, due to the long time elapsed since the initial exposure, a good part of the chromosome damage has been lost. As it is well known that in case of past exposure it is advised to analyse stable rather than unstable aberrations, which decay with time, a pilot study on these subjects is in progress using the chromosome painting technique Ždata not shown.. In the previous study, 10 healthy Italian children characterized by the same age but different ethnic, lifestyle and dietary background from Russian children were used as controls. Interestingly, a higher background of chromosomal aberrations was found in the Russian control group with respect with those Italian controls. Furthermore, when the data relative to the exposed groups of the present study were compared with the frequency of aberrations detected in the Italian children, the statistical analysis revealed a significance of p - 0.01 not only for group A but also for group B. On the basis of these data we put forth two possible hypotheses about differences arising from the comparison with group B and the two, Italian and Russian, control groups. Ža. Both groups B and C are exposed to some environmental < confounding factor 4 Ži.e. air pollution in the area of origin., so that they show a higher but similar background of chromosome aberrations and differences between them are not apparent. In this case, the Russian control group is the only one acceptable to be used as control. Žb. There is a bias due to a wrong selection of subjects Ži.e. they were not really excluded from the fall-out., therefore it is advisable,
L. PadoÕani et al.r Mutation Research 395 (1997) 249–254
when subjects are not directly selected by the investigators, to have as much information as possible about them using personal health questionnaires. In conclusion, the present work confirms that for these groups of children, even if the frequency of aberrations is very low and no pathologies have been observed, the cytogenetic picture is not back to normal. This abnormal situation is clearly due to the effects of the Chernobyl accident. The elevated abnormal frequency of aberrations observed in unexposed Russian subjects used as control may also suggest a further investigation.
w8x
w9x
w10x
w11x
Acknowledgements The authors thank Dr. F. Pacchierotti and Dr. M. Spano` for their critical suggestions.
w12x
References
w13x
w1x L.R. Ansphaugh, R.J. Catlin, M. Goldman, The global impact of the Chernobyl reactor accident, Science 242 Ž1988. 1513–1519. w2x M.A. Pilinskaya, A.M. Shemetun, Yu. Bondar’a, S.S. Dybskii, Cytogenetic effect in the somatic cells of persons exposed to radiation after the Chernobyl accident Žin Russian., Vestn. Akad. Med. Nauk. SSSR 8 Ž1991. 40–43. w3x M.A. Pilinskaya, A.M. Shemetun, M.N. Eremeeva, D.V. Red’ko, V.G. Fedorenko, S.E. Shepelev, Effect on peripheral blood lymphocytes of person with acute radiation sickness as a result of the accident at the Chernobyl atomic power plant Žin Russian., Tsitol. Genet. 25 Ž1991. 17–21. w4x M.A. Pilinskaya, A.M. Shemetun, D.V. Red’ko, S.E. Shepelev, Dynamics of the cytogenetic effect noted during repeated examinations of people who participated in irradiation Žin Russian., Tsitol. Genet. 25 Ž1991. 3–9. w5x E.K. Pyatkin, V.Yu. Nugis, A.A. Chirkov, Absorbed dose estimation according to the results of cytogenetic investigations of lymphocyte cultures of persons who suffered in the accident at the Chernobyl nuclear power station, Ukrainian SSR, USSR Žin Russian., Med. Radiol. 34 Ž1989. 52–57. w6x E.K. Pyatkin, A.A. Chirkov, V.Yu. Solov’ev, V.Yu. Nugis, Direct use of cytogenetic indices for plotting prognostic curves of neutrophil count dynamics after acute relatively uniform gamma-irradiation according to data on persons affected in the accident at the Chernobyl atomic power station, Ukrainian SSR, USSR Žin Russian., Med. Radiol. 35 Ž1990. 29–32. w7x A.V. Sevan’kaev, D.C. Lloyd, H. Braselmann, A.A. Edwards, V.V. Moiseenko, A.A. Zhloba, A survey of chromo-
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somal aberrations in lymphocytes of Chernobyl liquidators, Radiat. Protect. Dos. 8 Ž2. Ž1995. 85–91. F. Granath, F. Darroudi, A. Auvinen, L. Ehrenberg, T. Hakulinen, A.T. Natarajan, M. Rahu, T. Rytomaa, M. Tekkel, ¨ T. Veidebaum, Retrospective dose estimates in Estonian Chernobyl clean-up workers by means of FISH, Mutation Res. 369 Ž1996. 7–12. V.A. Schevchenko, E.A. Akayeva, I.M. Yeliseyeva, T.V. Yelisova, E.L. Yofa, I.N. Nilova, A.B. Symov, W. Burkart, Human cytogenetic consequences of the Chernobyl accident, Mutation Res. 361 Ž1996. 29–34. G. Stephan, U. Oestreicher, An increased frequency of structural chromosome aberrations in persons present in the vicinity of the Chernobyl during and after the reactor accident. Is this effect caused by radiation exposure?, Mutation Res. 223 Ž1989. 7–12. J. Pohl-Ruling, Chromosome aberrations of blood lympho¨ cytes by low level doses of ionizing radiations, in: G. Obe ŽEd.. Advances in Mutagenesis, Vol. 2, Springer, Berlin, 1990, pp. 155–190. J. Pohl-Ruling, O. Haas, A. Brogger, G. Obe, H. Lettner, F. ¨ Daschill, C. Atzmuller, D. Lloyd, R. Kubiak, A.T. Natarajan, The effect on lymphocyte chromosomes of additional radiation burden due to fallout in Salzburg ŽAustria. from the Chernobyl accident, Mutation Res. 262 Ž1991. 209–217. N.P. Bochkov, L.D. Katosova, Cytogenetic investigations in the evaluation of the consequences of Chernobyl disaster, Am. J. Hum. Genet. 49 ŽSuppl. 4. Ž1991. 490. H. Braselmann, E. Schmid, M. Bauchinger, Chromosome analysis in a population living in a area of Germany with the highest fallout deposition from the Chernobyl accident, Mutation Res. 283 Ž1992. 221–225. L. Verschaeve, E.V. Domracheva, S.A. Kuznetsov, V.V. Nechai, Chromosome aberrations in inhabitants of Byelorussia: consequences of the Chernobyl accident, Mutation Res. 267 Ž1993. 253–259. S. Salomaa, A. Koivistoinen, W. Paile, T. Rahola, T. Ritomaa, Chromosomal aberrations of Byelorussian children exposed to Chernobyl fall-out, Proceedings IRPA 8 Congress, International Radiation Protection Association, Montreal, Vol. 1, 1992, pp. 801–804. M.A. Pilinskaya, S.S. Dybskii, Frequency of chromosomal aberrations in peripheral blood lymphocytes from children living in regions with different radioecology situations, Tsitol. Genet. 26 Ž1992. 11–17. M.A. Pilinkaya, S.S. Dybsky, E.V. Dybskaya, The cytogenetic effect in lymphocytes from the peripheral blood of children living in population centers contaminated with radionuclides in the Ovruts district of the Zhitomir region of Ukraine, Tsitol. Genet. 26 Ž1992. 10–14. M.A. Pilinskaya, The results of selective cytogenetic monitoring of Chernobyl accident victims in the Ukraine, Health Phys. 71 Ž1. Ž1996. 29–33. L. Padovani, D. Caporossi, B. Tedeschi, P. Vernole, B. Nicoletti, F. Mauro, Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident, Mutation Res. 219 Ž1993. 55–60.
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w21x D.C. Lloyd, R.J. Purrott, G.W. Dolphin, D. Bolton, A.A. Edwards, M.J. Corp, The relationship between chromosome aberrations and low LET radiation dose in human lymphocytes, Int. J. Radiat. Biol. 28 Ž1975. 75–90. w22x L.G. Littlefield, The analysis of radiation-induced chromosome lesions in lymphocytes as a biological method for dose estimation, in: Proceedings NATO Working Group Meeting on Assessment of Injury from Ionizing Radiation in Warfare, Armed Forces Radiobiology Research Institute, Bethesda, MD, 1982, pp. 351–390. w23x IAEA, IAEA Technical Report 260, Biological Dosimetry – chromosomal aberration analysis for dose assessment, IAEA, 1986. w24x R.W. Leggett, K.F. Eckerman, D.E. Dunning, M. Cristy, D.J.
Crawford-Brown, L.R. Williams, On estimating dose rates to organs as a function of age following internal exposure to radionuclides. Oak Ridge National Laboratory, Oak Ridge, TN, ORNL-TM-8265, 1984. w25x J. Lipsztein, L. Bertelli, C.A.N. Oliveira, B.M. Dantas, Studies of Cs retention in the human body related to body parameters and Prussian Blue administration, Health Phys. 60 Ž1991. 57–61. w26x H.J. Evans, K.E. Buckton, G.E. Hamilton, A. Carothers, Radiation-induced chromosome aberrations in nuclear dockyard workers, Nature 277 Ž1979. 531–534. w27x A.T. Ramalho, C.H. Nascimiento, The fate of chromosomal aberration in 137Cs-exposed individuals in the Goiania radiation accident, Health Phys. 60 Ž1991. 67–70.
Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident L. Padovani
a,)
, L. Stronati a , F. Mauro a , A. Testa a , M. Appolloni a , P. Anzidei a , D. Caporossi b, B. Tedeschi b, P. Vernole b
a
b
EnÕironment Department, CR ENEA Casaccia, Via Anguillarese 301, 00060 Rome, Italy Department of Public Health and Cell Biology, UniÕersity of Rome ‘‘Tor Vergata’’, Rome, Italy Received 21 May 1997; revised 10 July 1997; accepted 18 July 1997
Abstract In a previous paper we reported that a group of children exposed to ionizing radiation following the Chernobyl accident exhibited an appreciable number of chromosome breaks and rearrangements reflecting the persistence of a radiation-induced damage. The results suggested that the children were still exposed to radioactive contamination through consumer foodstuff and life styles. In the present paper, 31 exposed children have been considered together with a control group of 11 children with the aim to confirm previous results. All children underwent whole-body counter ŽWBC. measures and conventional cytogenetic analysis. The frequency of chromosome aberrations detected by conventional cytogenetics in the group of children chronically exposed to low doses of ionizing radiation resulted in significant differences with respect to the control group. The present work suggests that, for these groups of children, even if the frequency of aberrations is very low and the observation of statistically significant differences is consequently a problem, a persistently abnormal cytogenetic picture is still present several years after the accident. q 1997 Elsevier Science B.V. Keywords: Chernobyl; Cytogenetics; Chromosome aberration
1. Introduction In 1986, due to climatological circumstances, the accident in one of the Chernobyl nuclear power plants resulted in a non-uniform, widely different contamination of many regions of the former USSR and Europe w1x. This accident represents a unique and large-scale problem in human ecology, health monitoring and control. Thus, various cytogenetic
)
Corresponding author.
studies were performed in order to obtain some insight on the level of the human hazard due to such accidental exposure, both on Chernobyl clean-up workers w2–9x and on people exposed to relatively low doses of ionizing radiation w10–15x. Among all these, some studies have been carried out on children w16–19x. In many cases an increased frequency of chromosomal aberrations was reported. In a previous study, we also carried out an analysis of chromosomal aberrations in the peripheral blood lymphocytes of children coming from differently contaminated areas of Belarus, Ukraine and Russian Federation and exhibiting varying amount of
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 3 7 - X
250
L. PadoÕani et al.r Mutation Research 395 (1997) 249–254
137
Cs internal contamination w20x. Results obtained showed a persistence of a radiation-induced genomic damage suggesting a continuation of the study. Besides, a control group composed of individuals from an uncontaminated area of the former USSR was not available for that study. In the present study, we analysed a total of 42 Russian children, including 11 coming from the Smolensk area, which is reported to be uncontaminated, and thus suitable as a control. The aim of the present study is to extend the cytogenetic analysis to another group of chronically or temporally exposed subjects in comparison with the local control group in order to confirm the persistence of a chromosomal damage 10 years after the accident. It is well known that the induction of chromosomal aberrations is intimately related to other genetic changes such as mutation and mutation toward cancer, and thus provides a measure of genetic hazards. On the other hand, the knowledge of the actual health condition of the individuals exposed to relatively low doses of ionizing radiation after the Chernobyl accident is interesting in consideration of the fact that they represent the largest population, and that they have been investigated less with respect to the highly exposed people such as the clean-up workers. 2. Materials and methods 2.1. Subjects A total of 42 children Ž10–12 years old., came to Italy in 1993–1994 for a 1-month recreation stay. The children were selected by local humanitarian organizations which organized their trip and requested a series of physical, biological and medical analyses. The medical check-ups carried out in collaborating hospitals indicated no evident signs of pathologies which could be attributed to ionizing radiation. 137Cs internal contamination was measured by Whole Body Counter ŽWBC. in the laboratories of ENEA ŽNational Agency for New Technology Energy and the Environment.. These measures were performed during the first week of the children’s stay in Italy. The children were distributed into three groups. Group A. Twenty-four subjects, 9 males and 15 females, from Novosybkov in the Brianskya region
of the Russian Federation Žground contamination 148 = 10 10 Bqrkm2 as reported by IAEA maps., who exhibited an internal contamination by WBC in the range 780–30 000 Bq. Group B. Seven subjects, 1 male and 6 females, from the uncontaminated area of Smolensk ŽRussian Federation. but living in Pripjat ŽUkraine. at the moment of the accident and thus exposed to the initial ‘‘acute’’ dose of ionizing radiation; these children, evacuated to Smolensk 36 h after the accident, showed an internal contamination in the range 70–128 Bq. Control group C. Eleven subjects, 5 males and 6 females, coming from the uncontaminated area of Smolensk region ŽWBC - 70 Bq.. 2.2. Culture conditions Blood samples were collected from all children at the time of the WBC analysis. Whole blood was cultured at 378C, 5% CO 2 for 48 h, in RPMI medium ŽFlow., supplemented with 10% fetal calf serum ŽGibco., 2% phytohemagglutinin ŽMurex. and 1.5% penicillin–streptomycin Ž5000 IUrml–5000 mgrml. ŽFlow.. For each subject, three different cultures were set up for independent cytogenetic scoring. 0.15 mgrml Colcemid ŽSigma. was added 90 min before harvesting. Metaphases were obtained according to standard procedures. 2.3. Cytogenetic chromosome analysis The slides were coded and analyzed by four different observers. For most of the subjects, a total of 600 unbanded Giemsa-stained metaphases were scored and the frequency of chromosome aberrations typically induced by ionizing radiation Žsuch as dicentrics, acentrics, fragments and rings. were evaluated w21,22,10,23x. As far as translocations are concerned, this method allowed us to evaluate only those that produced chromosomes with evident morphological alteration. 2.4. Statistics Statistical analyses were performed to assess significant differences between the mean frequencies of chromosome aberrations observed in each group.
L. PadoÕani et al.r Mutation Research 395 (1997) 249–254 Table 1 Frequency of structural chromosome aberrations in groups of children coming from differently
251
137
Cs-contaminated areas
Subjects Sex Age WBC Cell Chromosome aberrations ŽBq. scored Ace q DM %
Dic q CR %
Trl %
Total %
Group A 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
F F F M M F M F F F M F F F M F F M M F F M M F
9 10 8 12 11 11 9 9 12 10 9 9 11 10 11 9 11 11 11 9 10 12 11 9
780 1365 1633 1950 1955 2366 2847 3237 3680 3900 4326 4640 4836 5239 5980 6070 7254 9039 30 000 ND ND ND ND ND
550 610 650 550 550 400 450 600 600 550 600 650 574 600 600 600 600 650 600 600 600 607 502 520 13 813
0 4 0 4 1 1 1 0 4 2 4 3 0 4 3 1 1 5 1 0 1 5 1 3 49
0 0.65 0 0.73 0.18 0.25 0.22 0 0.66 0.36 0.66 0.46 0 0.66 0.5 0.16 0.16 0.77 0.16 0 0.16 0.82 0.2 0.57 0.35 ŽES 0.06.
1 1 1 0 0 0 3 0 0 0 0 1 0 2 0 0 0 1 0 1 1 1 2 0 15
0.18 0.16 0.15 0 0 0 0.66 0 0 0 0 0.15 0 0.33 0 0 0 0.15 0 0.16 0.16 0.16 0.39 0 0.11 ŽES 0.03.
1 0 2 2 2 2 3 0 0 3 0 1 0 0 1 0 0 0 2 0 1 1 0 1 22
0.18 0 0.3 0.36 0.36 0.5 0.66 0 0 0.54 0 0.15 0 0 0.16 0 0 0 0.33 0 0.16 0.16 0 0.19 0.15 ŽES 0.04.
5 3 6 3 3 7 0 4 5 4 5 0 6 4 1 1 6 3 1 3 7 3 4 86
0.81 0.46 0.11 0.54 0.75 1.54 0 0.66 0.9 0.66 0.76 0 0.99 0.66 0.16 0.16 0.92 0.49 0.16 0.48 1.14 0.6 0.76 0.622 ŽES 0.08. a
Group B a b c d e f g Total
M F F F F F F
11 10 11 10 9 10 9
- 70 - 70 - 70 95 128 ND ND
600 600 600 264 580 600 600 3844
2 2 3 1 2 1 1 12
0.33 0.33 0.5 0.37 0.34 0.16 0.16 0.31 ŽES 0.04.
0 0 1 1 0 1 0 3
0 0 0.16 0.37 0 0.16 0 0.08 ŽES 0.05.
1 1 0 0 1 0 1 4
0.16 0.16 0 0 0.17 0 0.16 0.1 ŽES 0.03.
3 3 4 2 3 2 2 19
0.5 0.5 0.66 0.75 0.51 0.33 0.33 0.49 ŽES 0.06.
Group C I II III IV V VI VII VIII IX X XI Total
M F M M M M F F F F F
11 10 10 10 9 10 11 9 12 10 11
- 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70 - 70
650 550 650 600 600 570 550 448 530 553 600 6301
3 2 0 0 2 1 1 1 1 4 0 15
0.46 0.36 0 0 0.33 0.17 0.18 0.16 0.18 0.72 0 0.22 ŽES 0.07.
0 0 0 1 0 0 1 0 0 1 0 3
0 0 0 0 0 1 0.16 0 0 0 0 3 0.18 0 0 0 0 0 0.18 0 0 0 0.045 ŽES 0.02. 4
0 3 0 2 0.15 1 0 1 0 2 0.52 4 0 2 0 1 0 1 0 5 0 0 0.061 ŽES 0.05. 22
0.46 0.36 0.15 0.16 0.33 0.7 0.36 0.16 0.18 0.9 0 0.33 ŽES 0.08.
Group A, contaminated group; Group B, evacuated group; Group C, Russian control group; Ace q DM, acentric fagmentsq double minute; Dic q CR, dicentricsq centrics rings; Trl, translocations; ND, not determined. a Two-tailed Student’s t-test, statistically significant Ž p - 0.05. compared to control group.
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The two-tailed Student’s t probability test for independent samples was performed; both p - 0.05 and confidence limits at 95% were evaluated.
3. Results Results obtained by the conventional cytogenetic and the WBC measures for each subject are given in Table 1. As a general trend, data show that both group A and group B exhibit higher frequencies of chromosomal aberrations than the control group C. The aberrations, when present, were homogeneously distributed among the metaphases for each individual and only one aberration per cell was found. Statistical analyses were performed on both specific and total chromosome aberrations Žacentrics, dicentrics and translocations.. A significant difference has been revealed only for the mean frequency of the total chromosome aberrations between the contaminated group ŽA. and the Russian control group ŽC. Ž p 0.05; confidence limits at 95%, 0.47–0.77.. No statistically significant differences were observed for all the other comparisons. It is also evident that there is no correlation between chromosome aberrations of each subject of group A and their values of internal contamination. This is not surprising, considering that 137Cs mean life in human body is 20–110 days Žin children. w24,25x and it is not possible to evaluate the pattern of the internal contamination value for the last 10 years because of time elapsed after the accident, changes in diet habits and effects of countermeasures.
4. Discussion Chromosome aberrations can be regarded as a very important and useful indicator of radiation exposure. In this study, a cytogenetic analysis was carried out on a group of children differently exposed to the fallout of the Chernobyl accident. One group of exposed children Žgroup A. were living in a presumably still radioactive environment, while the other Žgroup B. had been supposedly exposed only to an initial acute dose. The cytogenetic analysis shows a statistically significant increase of
aberrations in children belonging to the first group with respect to the controls. This confirms that chromosome damage can persist in individuals exposed to relatively low chronic doses, in agreement with other authors w26,27,16,20x. These data reinforce those obtained in a previous work w20x and suggest that the increase of chromosomal aberrations in subjects living in contaminated areas could actually be due to the exposure to low but chronic doses of ionizing radiation possibly related to still existing problems in diet and lifestyle. Actually, the Cs WBC measures, too, appear to indicate a persisting contamination in group A. Elevated, but not statistically different frequencies of aberrations, are also detected in children of group B, which accordingly show much lower WBC values than children of group A. It can be supposed that, due to the long time elapsed since the initial exposure, a good part of the chromosome damage has been lost. As it is well known that in case of past exposure it is advised to analyse stable rather than unstable aberrations, which decay with time, a pilot study on these subjects is in progress using the chromosome painting technique Ždata not shown.. In the previous study, 10 healthy Italian children characterized by the same age but different ethnic, lifestyle and dietary background from Russian children were used as controls. Interestingly, a higher background of chromosomal aberrations was found in the Russian control group with respect with those Italian controls. Furthermore, when the data relative to the exposed groups of the present study were compared with the frequency of aberrations detected in the Italian children, the statistical analysis revealed a significance of p - 0.01 not only for group A but also for group B. On the basis of these data we put forth two possible hypotheses about differences arising from the comparison with group B and the two, Italian and Russian, control groups. Ža. Both groups B and C are exposed to some environmental < confounding factor 4 Ži.e. air pollution in the area of origin., so that they show a higher but similar background of chromosome aberrations and differences between them are not apparent. In this case, the Russian control group is the only one acceptable to be used as control. Žb. There is a bias due to a wrong selection of subjects Ži.e. they were not really excluded from the fall-out., therefore it is advisable,
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when subjects are not directly selected by the investigators, to have as much information as possible about them using personal health questionnaires. In conclusion, the present work confirms that for these groups of children, even if the frequency of aberrations is very low and no pathologies have been observed, the cytogenetic picture is not back to normal. This abnormal situation is clearly due to the effects of the Chernobyl accident. The elevated abnormal frequency of aberrations observed in unexposed Russian subjects used as control may also suggest a further investigation.
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Acknowledgements The authors thank Dr. F. Pacchierotti and Dr. M. Spano` for their critical suggestions.
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