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Chromosome analysis of peripheral lymphocytes from persons exposed to radioactive fallout in Norway from the Chernobyl accident
Environmental Mutagenesis
ELSEVIER
Mutation Research 361 (1996) 73-79
Chromosome analysis of peripheral lymphocytes from persons exposed to radioactive fallout in Norway from the Chernobyl accident A. Brcgger a, *, J.B. Reitan b,c, p. Strand c, I. Amundsen c a Department of Genetics, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway b Department of Oncology, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway c Norwegian Radiation Protection Authority, Oster~s, Norway
Received 8 February 1996; revised 14 May 1996; accepted 15 May 1996
Abstract
Chromosome analysis of peripheral lymphocytes from two Norwegian populations (44 reindeer herding South samis from RCros and Snfisa, 12 sheep farmers from Valdres) exposed to fallout from the Chernobyl accident were made. The doses from caesium through the years 1987-1991 were calculated based on whole-body measurements of 134Csand 137Csgiving a total cumulative mean internal dose of 5.54 mSv for the total group of 56 persons. Chromosome aberrations were within the normal range when compared with historical controls with the exception of dicentrics (0.3% per cell, which is a 10-fold increase) and rings (0.07% per cell). A dose-dependent increase in dicentrics and rings based on caesium exposure was not observed. Keywords: Chromosomeaberration; Chernobyl fallout; Radiation burden; Norwegian population
1. Introduction
After the Chernobyl accident in 1986, fairly high levels of radioactive contamination occurred in parts of Norway (Backe et al., 1987), in spots up to 600 k B q / m 2. This is higher than in most European countries outside the former Soviet Union. The doses to the Norwegian population have nevertheless been small, except for some rural populations living of
* Corresponding author. Department of Genetics, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, N0310 Oslo, Norway.
reindeer breeding and mountain sheep production (Strand et al., 1989; Strand et al., 1992a). Shortly after the accident, widespread public anxiety developed (Weismth, 1990), apparently dominant in the regions with highest pollution. Possible genotoxic effects represented one aspect of the public concern. However, there are few observations on populations, except for some occupational cohorts, continously exposed for years to artificial nuclides in the mSv range. Such studies are needed for an appropriate risk evaluation of the Chernobyl fallout. The Norwegian Radiation Protection Authority has carried out whole-body measurements of radioactive caesium in selected populations since the bomb tests in the 1960s (Strand et al., 1989, 1992a;
0165-1161/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0165-1161(96)00024-6
74
A. Brogger et al. / Mutation Research 361 (1996) 73 79
Westerlund et al., 1987), and it was regarded feasible to correlate genotoxic parameters with individual dose determinations. After contact with organizations of the affected populations, this study was therefore started.
2. Material and methods Wholebody caesium measurements of individuals were carried out according to previous programs in three different municipalities with fairly high levels of contamination. The individuals were measured using an 3" NaI detector connected to a Canberra Series $30 multichannel analyzer (Canberra, Connecticut, USA) as previously described (Strand et al., 1990). The doses per year were calculated based on body content of ~34Cs and 137Cs, using standard dose conversion factors recommended by ICRP (1979). Most persons measured in 1991 had also been measured previous years since 1987, making it possible both to analyse effects of cumulated doses and doses last year. Some missing values for persons not measured annually since 1987 had to be calculated by inter- and extrapolation. No correction for small residual amounts of 137Cs originating from the athmospberic atomic bomb tests were attempted.
Most of the persons measured in 1991 also consented in blood sampling, and the number of persons in this material therefore comprise 9 persons (5 males, 4 females) from Snfisa, 47 (27 males, 20 females) from RCros (reindeer herding South sami population) and 18 (17 males and I female) from Oystre Slidre, Valdres (sheep farming Norwegian Caucasian population), in total 74 individuals. The mean age was 44.7 years (SD 14.0 and range 13-80). Blood samples were transported as fast as possible to the Department of Genetics. The transport time from Sn3.sa was about 5 h, from ROros 2 h and from Oystre Slidre by car 3.5 h. The blood donors filled out a questionnaire giving information on smoking habits, use of drugs, exposure to medical X-rays, and any recent viral infection. Blood cultures were established at the time of arrival of the sample. Two parallel cultures were prepared for harvest at 48, 50 and 52 h culture time. Any control of cell cycling with BrdU incorporation was not done. The yield of mitotic cells is sometimes higher at 50 or 52 h than at 48. To obtain a sufficient number of metaphases the results scored have been pooled from the three culture times. Culture conditions, chromosome preparation, Giemsa staining method and scoring of aberrations were according to general standards and have been described in detail
Table 1 Internal doses in mSv pr. year in 1991 and for the years 1986-1991 in selected groups of Norwegians. Municipality (weighted mean) ~'
Study group (mean) b
Study group (range) max.
min.
1991
Snhsa + R0ros Oystre Slidre
0.09 c 0.13 ~
0.71 0.54
2.17 2.04
0.11 0.01
1986-1991
Sn~sa + R0ros Oystre Slidre f
0.10 d 0.16 d
1.08 0.83
12.98 4.14
0.09 c 0.01
Values for the study group of 74 persons based on individual measured body content of 134Cs and 137Cs, and for the municipalities calculated total mean based on food contamination in the same municipality. a Calculated weighted mean; see Strand et al., 1992a. b Mean of all measurements Data for 1989 a Data for 1986-1989 Highest recorded individual value in Snfisa in 1988 f No wholebody measurements performed in 1990
A. BrOgger et al. / Mutation Research 361 (1996) 73-79
previously (Brcgger et al., 1984). Counting of the chromosomes in each metaphase was for most of the material made with the computer programme KARY (Knut Eide, Pro* Data, Oslo, Norway). Scoring of chromosome aberrations in each case was made without knowledge of the exposure data. Statistical treatment of the data was made with the Statview programme (BrainPower, Calabasas, CA) on Macintosh, except for a part of the linear regression using Statgraphics 5.0 (STSC, Rockville, MD).
3. Results
The doses based on body content of 134Cs and 137Cs are shown in Table 1 together with the estimated mean doses to inhabitants of the municipalities in question. Successful cultures with at least 50 suitable metaphases from each subject were obtained from 44 persons (26 males and 18 females) of the R0ros/Snhsa South sami population and from 12 persons (11 males and 1 female) of the Caucasian Valdres population; altogether 56 persons. It was decided to analyse 500 metaphases from each person, but it was not possible to obtain this from each subject (the mean number of cells were 400 cells). The mean internal doses in the 56 persons with successful chromosome analysis are shown in Table 2. The result of the chromosome analysis is given in Table 3. Essentially, no difference was found be-
Table 2 Mean internal doses in the total material of 56 persons with successful chromosome analysis Year
Internal dose (mean mSV)
SD
Min.
Max.
1987 1988 1989 1990 1991
0.81 1.79 1.27 0.98 0.69
0.80 2.27 1.02 0.67 0.47
0.13 0.16 0.09 0.t 1 0.01
3.35 12.98 4.7 3.1 2.04
4.48
0.57
21.63
Total cumulative Dose 5.54 Mean Annual dose 1.11
75
Table 3 Chromosome aberrations observed in the two groups of persons and in the total material
Persons examined Cells examined
ROros/Snhsa
Oystre slidre
Total material
44 17 682
12 4747
56 22 429
SD
SD
SD
ctg csg ctb csb cte ace dic ring
0.5 0.2 0.6 0.02 0.04 0.5 0.3 0.07
0.5 0.3 0.5 0.07 0.1 0.6 0.4 0.1
0.8 0.5 1.2 0.02 0.02 0.7 0.2 0.05
0.7 0.4 0.6 0.06 0.06 0.7 0.3 0.09
0.6 0.2 0.7 0.02 0.04 0.5 0.3 0.07
0.5 0.3 0.6 0.07 0.09 0.6 0.4 0.1
Total
2.1
1.2
3.6
1.4
2.4
1.4
The mean value with standard deviation of the mean (SD) is given for the total amount of each type of aberration expressed as per cent aberrations per cell. Aberration types are: ctg, chromatid gap; csg, chromosome gap; ctb, chromatid break; csb, chromosome break; cte, chromatid exchange; ace, acentric fragment (in addition to fragments involved in the formation of dicentrics); dic, dicentric chromosome; ring, ring chromosome.
tween results from males compared with females. The results are therefore presented for both sexes together. The chromosome aberration data are within the normal range in a human population with the exception of the frequency of dicentrics, which is about 10 times as high as our own historical controls (6 dicentrics among 19841 cells, i.e., 0.3 X 10 -3 (Br0gger, unpublished). The occurrence of dicentrics
Table 4 Mean total internal exposures and chromosome aberrations in smokers and nonsmokers
Smokers (n = 11) Nonsmokers (n = 43) Diagnostic X-ray (n = 45) No diagnostic X-ray (n = 9)
Tot. exp. (mSV)
Tot. CA (%)
Dic + rings (%)
5.91 5.38 5.33 5.90
2.45 2.42 2.28 2.88
0.35 0.31 0.29 a 0.45 a
Not significantly different. Chi-square: 0.034:0.80 < p < 0.90.'
76
A. BrOgger et al. / Mutation Research 36l (1996) 73-79
÷
1.5
•
00
.5 0000
IO0
•
•
0 0 cumulative dose mSv
Fig. I. Linear regression of the sum of dicentrics and rings in per cent per cell for each of the 56 persons and their total cumulative internal exposure dose from caesium in mSv (y = -0.016 x + 0.411). There are overlapping points for cases 28 and 44 (dic + ring 0.0; dose 1.47,
1,49), cases 71 and 73 (0.2; 5.3, 5.33), and cases 35 and 46 (0.6; 4.61, 4.66).
in control populations has varied from 0.1 X 10 -3 to 2.1 X 10 -3 (review by Lloyd (1984). The increased frequency of dicentrics can not readily be explained by any confounding factors, such as smoking (Table 4), use of drugs, medical irradiation or viral infections. Regression analyses showed a slight positive correlation between gaps and total exposure as well as exposure during the last year, but not with any other chromosomal parameter, such as dicentrics and rings (Fig. 1), where the correlation was negative.
4. Discussion
The total dose from radioactivity is the sum of the dose from internal natural radioactive substances and nuclear pollution, and the external exposure from the same nuclides outside the body. The doses reported here do not represent the total dose, only the dose from internal 134Cs "~ 13VCs. Natural internal nuclides as 4°K and 21°po are excluded or not measured, and dose contributions from, e.g., 9°Sr have not been
estimated. The problem of intake of 21°po through the lichen-reindeer pathway have been discussed in an earlier report (Evensen et al., 1990). 21°po would probably increase the equivalent dose estimates in the counties of SnSsa and R0ros, but not to the same extent in Oystre Slidre. However, if the doses should be seriously underestimated in the Sami areas, this is not reflected in higher frequencies in chromosome aberrations. Quite contrary, the Oystre Slidre population shows slightly higher levels for total aberrations (3.6 + 1.4 vs. 2.1 _+ 1.2%). Moreover, wholebody counting gives a point estimation only of the body radiocaesium content during the year. The changes during the year in the populations studied here may be greater than the generally low variation in mean doses in community populations from month to month. This is because they represent reindeer breeders, mainly of sami origin, (Sn~tsa and R0ros) and mountain farmers (¢0ystre Slidre) with not completely ordinary diet habits (Strand et al., 1992a,b). However, we assume some correlation between the intake of Cs, Sr and other artificial radionuclides, although the correlation may
A. BrOgger et al. /Mutation Research 361 (1996) 73-79
be weak (Evensen et al., 1990; Strand et al., t992a). 137Cs originating from the bomb test fallout could be determined based on the known initial ratio of ~34Cs to ~37Cs. It was, however, not subtracted from the dose estimates, because possible effects were thought to be a result of both cesium origins. However, this also implies that the total cumulative dose before the Chernobyl accident is not taken into consideration. A close correlation was found between 1991 dose and cumulated 1987-1991 dose in the persons actually measured each year. The results in this study is presented by use of cumulative dose 1987-1991, although dose-effect regressions using the actual measurements 1991 would have shown generally the same pattern. Scoring of dicentric and ring chromosome aberrations is often regarded as the gold standard of biological dosimetry in radiation accidents, and is considered fairly reliable down to a detection limit of 50-100 mSv wholebody acute external dose (Lloyd, 1984, Littlefield and Lushbaugh, 1990). However, in persons irradiated at the plant during the Chernobyl accident, frequency of aberrations apparently correlated poorly with dose in the 100-1000 mSv range (Pilinskaya et al., 1993). The irradiation was probably inhomogeneous and not only of external origin, and the kinetics of induction and decay of aberrations induced by low dose-rate internal contamination is fairly unknown. There are numerous observations that priming with a rather small radiation dose (50-100 mGy) will reduce the chromosome abberration yield after a following large challenging dose, often referred to as an adaptive response (Olivieri et al., 1984, Shadley, 1994). This is generally interpreted as induction of repair mechanism, probably induced enzymes, but the dose-response relationship varies from study to study. Adaptive response has been descibed in Drosophila oocytes down to 0.2 mGy (SchiippiBiichi, 1994) but is generally descibed after doses in the 25-100 mGy range. In human lymphocytes there seem to be large inter- and intra-personal variability in the response (Shadley, 1994). A group of 26 Byelorussian children visiting Finland in 1990 were examined by Salomaa et al. (1992). They came from three polluted areas, but their internal cesium burden as evaluated by whole body measurements was not higher than in a matched
77
control group of 12 Finnish children. The total frequency of radiation-related chromosome type aberrations were significantly higher in the Byelorussian children, although the 4-fold increase in dicentrics was not statistically significant. Padovani et al. (1993) examined 41 children from Belarus, Ukraine and Russia on holiday in Italy in 1991-1992. Whole-body measurements revealed varying amounts of 137Cs internal contamination. Radiation-induced chromosome damage was found, although at a very low frequency. Sevan'kaev et al. (1995) have studied a large group (1855 persons including controls) who have lived continuously in the contaminated areas of Belarus and Russia, the majority being 10 cohorts of children. The mean yields of chromosome aberrations were above those in the controls, although statistically significant only in children evacuated from the Chernobyl area and in one community with contamination about 1000 k B q / m 2. The average dose over 1987-1991 estimated from chromosome damage or from the radioactive contamination was in both cases approx. 100 mSv. An elevated frequency of chromosome aberrations has also been found in most of the subjects in a group of 35 adults in the Gomel area in Belarus (Verschaeve et al., 1993) and in five of 15 exposed adults from Kiev and Minsk (Scheid et al., 1993). In another study from Belarus, similar findings are reported, with about a factor 3 higher frequency of dicentrics and tings in lymphocytes from persons living in contaminated areas compared to control areas (Lazjuk et al., 1995). Several other cytogenetic studies have been performed on persons exposed to fallout from the Chernobyl accident. One year after the accident 16 volunteers were examined in the Salzburg area (PohlRiiling et al., 1991). The 137Cs content of the individuals was estimated by whole-body counting. The additional external plus internal radiation doses in the year 1987 ranged between 15 and 68% of the pre-accident, normal environmental burden in the area. The aberration frequencies showed a sharp increase of about a factor of 6 from the pre-Chernobyl dose rate (0.9 m G y / y e a r ) to the post-Chernobyl dose-rate (about 2 mGy/year). Braselmann et al. (1992) studied 30 persons sampled from March to September 1989 in the Berchtesgaden area. This
78
A. BrOgger et al./Mutation Research 361 (1996) 73-79
Bavarian area received the highest amount of radioactive cesium ( > 42 k B q / m 2) in Germany. Any elevation in the frequency of chromosome aberrations compared to a large pre-Chernobyl control group was not seen. Similar negative studies were obtained in another group of 29 subjects from the Berchtesgaden area (Stephan and Oestreicber, 1993). The chromosome aberration frequencies in our study are within the normal range with the exception of the amount of dicentrics and rings. The amount of dicentrics appears to be almost ten times as large as expected. Since dicentrics and tings are aberrations typically induced by ionizing radiation, and since the population has received an increased exposure to radioactivity, we suggest that the increased chromosome damage is due to the Chernobyl fallout. We are aware of the lack of a dose response related to the internal radiation measured. Several factors may explain this lack of correlation. We have not measured the individual external radiation and any possible radiation from alfa- or beta-emitters. Different, individual radiosensitivity may also disturb a dose response in a small sample of persons. The dose-effect relationship of radiation induced chromosome abberrations below 50-100 mSv, and of the risk of cancer induction, is not fully known. The health physics linear extrapolation down to zero dose may be appropriate for radiation protection purposes, but is not adequately substantiated with biological observations. Oftedal (1991) suggested the existence of a threshold in the very low dose range where the cell/organism does not sense the need for repair mobilization. According to this view, very low dose events will probably not be repaired (lack of repair induction), medium doses will be repaired, and high-dose damage exceeding repair capacity will not be repaired either. This should imply a biphasic dose-effect relationship with a top in the very low dose region. Such biphasic relationships have been found for many biological endpoints (Burlakova, 1995). In this study, 10 times our own control values for dicentric and ring chromosome abberations were found in individuals exposed to low but significant doses from internal radionuclides, with a tendency to a negative dose-effect correlation. Although rather speculative, this finding may represent fixation of chromosome damage not reaching the level of inducible repair.
Acknowledgements We are grateful to P~_I MOiler, MD, PhD, for his contribution to the computer-assisted chromosome counting (KARY programme) and helpful discussions, and to Jon Dahlen, Anne Kari Gangna~s Hammer, Berit HammerO and Mette Ekn~es Lier for expert technical assistance.
References Backe, S., H. Bjerke, A.L. Rudjord and F. Ugletveit (1987) The fallout of caesium in Norway alter the Chernobyl accident. Radiat. Protein Dosim., 18, 105-107. Braselmann, H., E. Schmid and M. Bauchinger (1992) Chromosome analysis in a population living in an area of Germany with the highest fallout deposition from the Chernobyl accident, Mutation Res., 283, 221-225. BrC.gger, A., R.B. Nicolaysen, R. Nygaard, H. Waksvik, P.J. Moe and J. Cohn (1984) Structural chromosome aberrations in lymphocytes from children with one to five years of total remission after chemotherapy of acute lymphoblastic leukaemia, Eur. Paediatr. Haematol. Oncol., 1, 39-46. Burlakova, E.B. (1995) Low intensity radiation: Radiobiological aspects, Radiat. Protein Dosim., 62, 13-18. Evensen, J.F., J.B. Reitan, E.A. Westerlund and A. BrC,gger (1990) Cesium-137 body burden and chromosome aberrations in Norwegian lapps 1965-1978, in: T. Brustad, F. Langmark and J.B. Reitan (Eds), Radiation and cancer risk, Hemisphere. New York, pp. 21-30. ICRP (1979) Limits for intakes of radionuclides by workers, International Commission on Radiological Protection, Publication 30. Pergamon Press, Oxford. Lazjuk, G.I., I.A. Kirillova, D.L. Nikolaev, I.V. Novikova, Z.N. Fomina and R.D. Khmel (1995) Frequency changes of inherited anomalies in The Republic of Belarus after the Chernobyl accident, Radiat. Protein Dosim., 62, 71-74. Littlefield, L.G. and C.C. Lushbaugh (1990) Cytogenetic dosimetry for radiation accidents - 'The good, the bad and the ugly'. In: R.C. Ricks and S.A. Fry (Eds.): The medical basis for radiation accident preparedness II. Elsevier, Amsterdam, pp. 461-479. Lloyd, D.C. (1984) An overview of radiation dosimetry by conventional methods, in W.G. Eisert and M.L. Mendelsohn (Eds.), Biological dosimetry, Springer, Berlin, pp. 3-14. Oftedal. P. (1991) Biological low-dose radiation effects, Mutation Res., 258, 191-205. Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science, 223, 594-597. Padovani, L., D. Caporossi, B. Tedeschi, P. Vernole, B. Nicoletti and F. Mauro (1993) Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident, Mutation Res., 319, 55-60.
A. BrCgger et al. / Mutation Research 361 (1996) 73- 79 Pilinskaya, M.A., A.M. Shemetun, S.S. Dybskii, D.V. Red'ko and M.N. Eremeeva (1993) Cytogenetic effects in blood lymphocytes as an indicator of Chernobyl accident impact on human health, Radiat. Biol. Ecol. (Moscow), 33, 99-103. Pohl-Riiling, J., O. Haas, A. BrCgger, G. Obe, H. Lettner, F. Daschil, C. Atzmiiller, C., D. Lloyd, R. Kubiak and A.T. Natarajan (1991) The effect on lymphocyte chromosomes of additional radiation burden due to fallout in Salzburg (Austria) from the Chernobyl accident, Mutation Res., 262, 209-217. Salomaa, S., A. Koivistoinen, W. Paile, T. Rahola and Ryttimaa, T. (1992) Chromosomal aberration analyses of Byelorussian children exposed to Chernobyl fallout, in Proceedings of International Radiation Protection Association (IRPA 8), IRPA, Montreal, pp. 801-804. Sch~ippi-Bfichi, C. (1994) On the genetic background of the adaptive response to X-rays in Drosophila melanogaster, Int. J. Radiat. Biol., 65, 427-435. Scheid, W., J. Weber, S. Petrenko and H. Traut (1993) Chromosome aberrations in human lymphocytes apparently induced by Chernobyl fallout, Health Phys., 64, 531-534. Sevan'kaev, A.V., D.C. Lloyd, O.I. Potetnya, A.A. Zhloba, V.V. Moiseenko and A.A. Edwards (1995) Chromosomal aberrations in lymphocytes of residents of areas contaminated by radioactive discharges from the Chemobyl accident, Radiat. Protein Dosim., (in press). Shadley, J.D. (1994) Chromosomal adaptive response in human lymphocytes, Rad. Res., 138, $9-S12. Stephan, G. and U. Oestreicher (1993) Chromosome investigation of individuals living in areas of Southern Germany contami-
79
nated by fallout from the Chernobyl reactor accident, Mutation Res., 319, 189-196. Strand, P., E. B~e, L. Berteig, T. Berthelsen, T. Strand, K. Trygg and O. Harbitz (1989) Whole-body counting and dietary surveys in Norway during the first year after the Chernobyl accident, Radiat. Protein Dosim., 27, 163-171. Strand, P., T. Seln~es and A. Andersson (1990) Wholebody measurements in South Norway after the Chernobyl accident 1987-1989, (In Norwegian with English summary), SIS-report 1990:4, SIS, Oster~s, Norway. Strand, P., T.D. Seln~es and J,B. Reitan (1992a) Area and time distribution of external and internal doses from the Chernobyl fallout: The lack of correlation in Norway, Health Phys., 62, 512-518. Strand, P., T.D. Seln~es, E. Bcie A. Anderson-S0rlie (1992b) Internal doses to different parts of the Norwegian population after the Chernobyl accident and the effect of dietary advice, Health Phys., 64, 385-392. Verschaeve, L., E.V. Domracheva, S.A. Kuznetsov and V,V. Nechai (1993) Chromosome aberrations in inhabitants of Byelorussia: consequence of the Chernobyl accident, Mutation Res., 287, 253-259. Weis~eth, L. (1990) Reactions in Norway to fallout from the Chernobyl disaster, in: T. Brustad, F. Langmark and J.B. Reitan (Eds.), Radiation and cancer risk, Hemisphere, New York, pp 149-155. Westerlund, E.A., T. Berthelsen and L. Berteig (1987) Cesium-137 body burdens in Norwegian Lapps, Health Phys., 52, 171-177.
ELSEVIER
Mutation Research 361 (1996) 73-79
Chromosome analysis of peripheral lymphocytes from persons exposed to radioactive fallout in Norway from the Chernobyl accident A. Brcgger a, *, J.B. Reitan b,c, p. Strand c, I. Amundsen c a Department of Genetics, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway b Department of Oncology, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway c Norwegian Radiation Protection Authority, Oster~s, Norway
Received 8 February 1996; revised 14 May 1996; accepted 15 May 1996
Abstract
Chromosome analysis of peripheral lymphocytes from two Norwegian populations (44 reindeer herding South samis from RCros and Snfisa, 12 sheep farmers from Valdres) exposed to fallout from the Chernobyl accident were made. The doses from caesium through the years 1987-1991 were calculated based on whole-body measurements of 134Csand 137Csgiving a total cumulative mean internal dose of 5.54 mSv for the total group of 56 persons. Chromosome aberrations were within the normal range when compared with historical controls with the exception of dicentrics (0.3% per cell, which is a 10-fold increase) and rings (0.07% per cell). A dose-dependent increase in dicentrics and rings based on caesium exposure was not observed. Keywords: Chromosomeaberration; Chernobyl fallout; Radiation burden; Norwegian population
1. Introduction
After the Chernobyl accident in 1986, fairly high levels of radioactive contamination occurred in parts of Norway (Backe et al., 1987), in spots up to 600 k B q / m 2. This is higher than in most European countries outside the former Soviet Union. The doses to the Norwegian population have nevertheless been small, except for some rural populations living of
* Corresponding author. Department of Genetics, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, N0310 Oslo, Norway.
reindeer breeding and mountain sheep production (Strand et al., 1989; Strand et al., 1992a). Shortly after the accident, widespread public anxiety developed (Weismth, 1990), apparently dominant in the regions with highest pollution. Possible genotoxic effects represented one aspect of the public concern. However, there are few observations on populations, except for some occupational cohorts, continously exposed for years to artificial nuclides in the mSv range. Such studies are needed for an appropriate risk evaluation of the Chernobyl fallout. The Norwegian Radiation Protection Authority has carried out whole-body measurements of radioactive caesium in selected populations since the bomb tests in the 1960s (Strand et al., 1989, 1992a;
0165-1161/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S0165-1161(96)00024-6
74
A. Brogger et al. / Mutation Research 361 (1996) 73 79
Westerlund et al., 1987), and it was regarded feasible to correlate genotoxic parameters with individual dose determinations. After contact with organizations of the affected populations, this study was therefore started.
2. Material and methods Wholebody caesium measurements of individuals were carried out according to previous programs in three different municipalities with fairly high levels of contamination. The individuals were measured using an 3" NaI detector connected to a Canberra Series $30 multichannel analyzer (Canberra, Connecticut, USA) as previously described (Strand et al., 1990). The doses per year were calculated based on body content of ~34Cs and 137Cs, using standard dose conversion factors recommended by ICRP (1979). Most persons measured in 1991 had also been measured previous years since 1987, making it possible both to analyse effects of cumulated doses and doses last year. Some missing values for persons not measured annually since 1987 had to be calculated by inter- and extrapolation. No correction for small residual amounts of 137Cs originating from the athmospberic atomic bomb tests were attempted.
Most of the persons measured in 1991 also consented in blood sampling, and the number of persons in this material therefore comprise 9 persons (5 males, 4 females) from Snfisa, 47 (27 males, 20 females) from RCros (reindeer herding South sami population) and 18 (17 males and I female) from Oystre Slidre, Valdres (sheep farming Norwegian Caucasian population), in total 74 individuals. The mean age was 44.7 years (SD 14.0 and range 13-80). Blood samples were transported as fast as possible to the Department of Genetics. The transport time from Sn3.sa was about 5 h, from ROros 2 h and from Oystre Slidre by car 3.5 h. The blood donors filled out a questionnaire giving information on smoking habits, use of drugs, exposure to medical X-rays, and any recent viral infection. Blood cultures were established at the time of arrival of the sample. Two parallel cultures were prepared for harvest at 48, 50 and 52 h culture time. Any control of cell cycling with BrdU incorporation was not done. The yield of mitotic cells is sometimes higher at 50 or 52 h than at 48. To obtain a sufficient number of metaphases the results scored have been pooled from the three culture times. Culture conditions, chromosome preparation, Giemsa staining method and scoring of aberrations were according to general standards and have been described in detail
Table 1 Internal doses in mSv pr. year in 1991 and for the years 1986-1991 in selected groups of Norwegians. Municipality (weighted mean) ~'
Study group (mean) b
Study group (range) max.
min.
1991
Snhsa + R0ros Oystre Slidre
0.09 c 0.13 ~
0.71 0.54
2.17 2.04
0.11 0.01
1986-1991
Sn~sa + R0ros Oystre Slidre f
0.10 d 0.16 d
1.08 0.83
12.98 4.14
0.09 c 0.01
Values for the study group of 74 persons based on individual measured body content of 134Cs and 137Cs, and for the municipalities calculated total mean based on food contamination in the same municipality. a Calculated weighted mean; see Strand et al., 1992a. b Mean of all measurements Data for 1989 a Data for 1986-1989 Highest recorded individual value in Snfisa in 1988 f No wholebody measurements performed in 1990
A. BrOgger et al. / Mutation Research 361 (1996) 73-79
previously (Brcgger et al., 1984). Counting of the chromosomes in each metaphase was for most of the material made with the computer programme KARY (Knut Eide, Pro* Data, Oslo, Norway). Scoring of chromosome aberrations in each case was made without knowledge of the exposure data. Statistical treatment of the data was made with the Statview programme (BrainPower, Calabasas, CA) on Macintosh, except for a part of the linear regression using Statgraphics 5.0 (STSC, Rockville, MD).
3. Results
The doses based on body content of 134Cs and 137Cs are shown in Table 1 together with the estimated mean doses to inhabitants of the municipalities in question. Successful cultures with at least 50 suitable metaphases from each subject were obtained from 44 persons (26 males and 18 females) of the R0ros/Snhsa South sami population and from 12 persons (11 males and 1 female) of the Caucasian Valdres population; altogether 56 persons. It was decided to analyse 500 metaphases from each person, but it was not possible to obtain this from each subject (the mean number of cells were 400 cells). The mean internal doses in the 56 persons with successful chromosome analysis are shown in Table 2. The result of the chromosome analysis is given in Table 3. Essentially, no difference was found be-
Table 2 Mean internal doses in the total material of 56 persons with successful chromosome analysis Year
Internal dose (mean mSV)
SD
Min.
Max.
1987 1988 1989 1990 1991
0.81 1.79 1.27 0.98 0.69
0.80 2.27 1.02 0.67 0.47
0.13 0.16 0.09 0.t 1 0.01
3.35 12.98 4.7 3.1 2.04
4.48
0.57
21.63
Total cumulative Dose 5.54 Mean Annual dose 1.11
75
Table 3 Chromosome aberrations observed in the two groups of persons and in the total material
Persons examined Cells examined
ROros/Snhsa
Oystre slidre
Total material
44 17 682
12 4747
56 22 429
SD
SD
SD
ctg csg ctb csb cte ace dic ring
0.5 0.2 0.6 0.02 0.04 0.5 0.3 0.07
0.5 0.3 0.5 0.07 0.1 0.6 0.4 0.1
0.8 0.5 1.2 0.02 0.02 0.7 0.2 0.05
0.7 0.4 0.6 0.06 0.06 0.7 0.3 0.09
0.6 0.2 0.7 0.02 0.04 0.5 0.3 0.07
0.5 0.3 0.6 0.07 0.09 0.6 0.4 0.1
Total
2.1
1.2
3.6
1.4
2.4
1.4
The mean value with standard deviation of the mean (SD) is given for the total amount of each type of aberration expressed as per cent aberrations per cell. Aberration types are: ctg, chromatid gap; csg, chromosome gap; ctb, chromatid break; csb, chromosome break; cte, chromatid exchange; ace, acentric fragment (in addition to fragments involved in the formation of dicentrics); dic, dicentric chromosome; ring, ring chromosome.
tween results from males compared with females. The results are therefore presented for both sexes together. The chromosome aberration data are within the normal range in a human population with the exception of the frequency of dicentrics, which is about 10 times as high as our own historical controls (6 dicentrics among 19841 cells, i.e., 0.3 X 10 -3 (Br0gger, unpublished). The occurrence of dicentrics
Table 4 Mean total internal exposures and chromosome aberrations in smokers and nonsmokers
Smokers (n = 11) Nonsmokers (n = 43) Diagnostic X-ray (n = 45) No diagnostic X-ray (n = 9)
Tot. exp. (mSV)
Tot. CA (%)
Dic + rings (%)
5.91 5.38 5.33 5.90
2.45 2.42 2.28 2.88
0.35 0.31 0.29 a 0.45 a
Not significantly different. Chi-square: 0.034:0.80 < p < 0.90.'
76
A. BrOgger et al. / Mutation Research 36l (1996) 73-79
÷
1.5
•
00
.5 0000
IO0
•
•
0 0 cumulative dose mSv
Fig. I. Linear regression of the sum of dicentrics and rings in per cent per cell for each of the 56 persons and their total cumulative internal exposure dose from caesium in mSv (y = -0.016 x + 0.411). There are overlapping points for cases 28 and 44 (dic + ring 0.0; dose 1.47,
1,49), cases 71 and 73 (0.2; 5.3, 5.33), and cases 35 and 46 (0.6; 4.61, 4.66).
in control populations has varied from 0.1 X 10 -3 to 2.1 X 10 -3 (review by Lloyd (1984). The increased frequency of dicentrics can not readily be explained by any confounding factors, such as smoking (Table 4), use of drugs, medical irradiation or viral infections. Regression analyses showed a slight positive correlation between gaps and total exposure as well as exposure during the last year, but not with any other chromosomal parameter, such as dicentrics and rings (Fig. 1), where the correlation was negative.
4. Discussion
The total dose from radioactivity is the sum of the dose from internal natural radioactive substances and nuclear pollution, and the external exposure from the same nuclides outside the body. The doses reported here do not represent the total dose, only the dose from internal 134Cs "~ 13VCs. Natural internal nuclides as 4°K and 21°po are excluded or not measured, and dose contributions from, e.g., 9°Sr have not been
estimated. The problem of intake of 21°po through the lichen-reindeer pathway have been discussed in an earlier report (Evensen et al., 1990). 21°po would probably increase the equivalent dose estimates in the counties of SnSsa and R0ros, but not to the same extent in Oystre Slidre. However, if the doses should be seriously underestimated in the Sami areas, this is not reflected in higher frequencies in chromosome aberrations. Quite contrary, the Oystre Slidre population shows slightly higher levels for total aberrations (3.6 + 1.4 vs. 2.1 _+ 1.2%). Moreover, wholebody counting gives a point estimation only of the body radiocaesium content during the year. The changes during the year in the populations studied here may be greater than the generally low variation in mean doses in community populations from month to month. This is because they represent reindeer breeders, mainly of sami origin, (Sn~tsa and R0ros) and mountain farmers (¢0ystre Slidre) with not completely ordinary diet habits (Strand et al., 1992a,b). However, we assume some correlation between the intake of Cs, Sr and other artificial radionuclides, although the correlation may
A. BrOgger et al. /Mutation Research 361 (1996) 73-79
be weak (Evensen et al., 1990; Strand et al., t992a). 137Cs originating from the bomb test fallout could be determined based on the known initial ratio of ~34Cs to ~37Cs. It was, however, not subtracted from the dose estimates, because possible effects were thought to be a result of both cesium origins. However, this also implies that the total cumulative dose before the Chernobyl accident is not taken into consideration. A close correlation was found between 1991 dose and cumulated 1987-1991 dose in the persons actually measured each year. The results in this study is presented by use of cumulative dose 1987-1991, although dose-effect regressions using the actual measurements 1991 would have shown generally the same pattern. Scoring of dicentric and ring chromosome aberrations is often regarded as the gold standard of biological dosimetry in radiation accidents, and is considered fairly reliable down to a detection limit of 50-100 mSv wholebody acute external dose (Lloyd, 1984, Littlefield and Lushbaugh, 1990). However, in persons irradiated at the plant during the Chernobyl accident, frequency of aberrations apparently correlated poorly with dose in the 100-1000 mSv range (Pilinskaya et al., 1993). The irradiation was probably inhomogeneous and not only of external origin, and the kinetics of induction and decay of aberrations induced by low dose-rate internal contamination is fairly unknown. There are numerous observations that priming with a rather small radiation dose (50-100 mGy) will reduce the chromosome abberration yield after a following large challenging dose, often referred to as an adaptive response (Olivieri et al., 1984, Shadley, 1994). This is generally interpreted as induction of repair mechanism, probably induced enzymes, but the dose-response relationship varies from study to study. Adaptive response has been descibed in Drosophila oocytes down to 0.2 mGy (SchiippiBiichi, 1994) but is generally descibed after doses in the 25-100 mGy range. In human lymphocytes there seem to be large inter- and intra-personal variability in the response (Shadley, 1994). A group of 26 Byelorussian children visiting Finland in 1990 were examined by Salomaa et al. (1992). They came from three polluted areas, but their internal cesium burden as evaluated by whole body measurements was not higher than in a matched
77
control group of 12 Finnish children. The total frequency of radiation-related chromosome type aberrations were significantly higher in the Byelorussian children, although the 4-fold increase in dicentrics was not statistically significant. Padovani et al. (1993) examined 41 children from Belarus, Ukraine and Russia on holiday in Italy in 1991-1992. Whole-body measurements revealed varying amounts of 137Cs internal contamination. Radiation-induced chromosome damage was found, although at a very low frequency. Sevan'kaev et al. (1995) have studied a large group (1855 persons including controls) who have lived continuously in the contaminated areas of Belarus and Russia, the majority being 10 cohorts of children. The mean yields of chromosome aberrations were above those in the controls, although statistically significant only in children evacuated from the Chernobyl area and in one community with contamination about 1000 k B q / m 2. The average dose over 1987-1991 estimated from chromosome damage or from the radioactive contamination was in both cases approx. 100 mSv. An elevated frequency of chromosome aberrations has also been found in most of the subjects in a group of 35 adults in the Gomel area in Belarus (Verschaeve et al., 1993) and in five of 15 exposed adults from Kiev and Minsk (Scheid et al., 1993). In another study from Belarus, similar findings are reported, with about a factor 3 higher frequency of dicentrics and tings in lymphocytes from persons living in contaminated areas compared to control areas (Lazjuk et al., 1995). Several other cytogenetic studies have been performed on persons exposed to fallout from the Chernobyl accident. One year after the accident 16 volunteers were examined in the Salzburg area (PohlRiiling et al., 1991). The 137Cs content of the individuals was estimated by whole-body counting. The additional external plus internal radiation doses in the year 1987 ranged between 15 and 68% of the pre-accident, normal environmental burden in the area. The aberration frequencies showed a sharp increase of about a factor of 6 from the pre-Chernobyl dose rate (0.9 m G y / y e a r ) to the post-Chernobyl dose-rate (about 2 mGy/year). Braselmann et al. (1992) studied 30 persons sampled from March to September 1989 in the Berchtesgaden area. This
78
A. BrOgger et al./Mutation Research 361 (1996) 73-79
Bavarian area received the highest amount of radioactive cesium ( > 42 k B q / m 2) in Germany. Any elevation in the frequency of chromosome aberrations compared to a large pre-Chernobyl control group was not seen. Similar negative studies were obtained in another group of 29 subjects from the Berchtesgaden area (Stephan and Oestreicber, 1993). The chromosome aberration frequencies in our study are within the normal range with the exception of the amount of dicentrics and rings. The amount of dicentrics appears to be almost ten times as large as expected. Since dicentrics and tings are aberrations typically induced by ionizing radiation, and since the population has received an increased exposure to radioactivity, we suggest that the increased chromosome damage is due to the Chernobyl fallout. We are aware of the lack of a dose response related to the internal radiation measured. Several factors may explain this lack of correlation. We have not measured the individual external radiation and any possible radiation from alfa- or beta-emitters. Different, individual radiosensitivity may also disturb a dose response in a small sample of persons. The dose-effect relationship of radiation induced chromosome abberrations below 50-100 mSv, and of the risk of cancer induction, is not fully known. The health physics linear extrapolation down to zero dose may be appropriate for radiation protection purposes, but is not adequately substantiated with biological observations. Oftedal (1991) suggested the existence of a threshold in the very low dose range where the cell/organism does not sense the need for repair mobilization. According to this view, very low dose events will probably not be repaired (lack of repair induction), medium doses will be repaired, and high-dose damage exceeding repair capacity will not be repaired either. This should imply a biphasic dose-effect relationship with a top in the very low dose region. Such biphasic relationships have been found for many biological endpoints (Burlakova, 1995). In this study, 10 times our own control values for dicentric and ring chromosome abberations were found in individuals exposed to low but significant doses from internal radionuclides, with a tendency to a negative dose-effect correlation. Although rather speculative, this finding may represent fixation of chromosome damage not reaching the level of inducible repair.
Acknowledgements We are grateful to P~_I MOiler, MD, PhD, for his contribution to the computer-assisted chromosome counting (KARY programme) and helpful discussions, and to Jon Dahlen, Anne Kari Gangna~s Hammer, Berit HammerO and Mette Ekn~es Lier for expert technical assistance.
References Backe, S., H. Bjerke, A.L. Rudjord and F. Ugletveit (1987) The fallout of caesium in Norway alter the Chernobyl accident. Radiat. Protein Dosim., 18, 105-107. Braselmann, H., E. Schmid and M. Bauchinger (1992) Chromosome analysis in a population living in an area of Germany with the highest fallout deposition from the Chernobyl accident, Mutation Res., 283, 221-225. BrC.gger, A., R.B. Nicolaysen, R. Nygaard, H. Waksvik, P.J. Moe and J. Cohn (1984) Structural chromosome aberrations in lymphocytes from children with one to five years of total remission after chemotherapy of acute lymphoblastic leukaemia, Eur. Paediatr. Haematol. Oncol., 1, 39-46. Burlakova, E.B. (1995) Low intensity radiation: Radiobiological aspects, Radiat. Protein Dosim., 62, 13-18. Evensen, J.F., J.B. Reitan, E.A. Westerlund and A. BrC,gger (1990) Cesium-137 body burden and chromosome aberrations in Norwegian lapps 1965-1978, in: T. Brustad, F. Langmark and J.B. Reitan (Eds), Radiation and cancer risk, Hemisphere. New York, pp. 21-30. ICRP (1979) Limits for intakes of radionuclides by workers, International Commission on Radiological Protection, Publication 30. Pergamon Press, Oxford. Lazjuk, G.I., I.A. Kirillova, D.L. Nikolaev, I.V. Novikova, Z.N. Fomina and R.D. Khmel (1995) Frequency changes of inherited anomalies in The Republic of Belarus after the Chernobyl accident, Radiat. Protein Dosim., 62, 71-74. Littlefield, L.G. and C.C. Lushbaugh (1990) Cytogenetic dosimetry for radiation accidents - 'The good, the bad and the ugly'. In: R.C. Ricks and S.A. Fry (Eds.): The medical basis for radiation accident preparedness II. Elsevier, Amsterdam, pp. 461-479. Lloyd, D.C. (1984) An overview of radiation dosimetry by conventional methods, in W.G. Eisert and M.L. Mendelsohn (Eds.), Biological dosimetry, Springer, Berlin, pp. 3-14. Oftedal. P. (1991) Biological low-dose radiation effects, Mutation Res., 258, 191-205. Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science, 223, 594-597. Padovani, L., D. Caporossi, B. Tedeschi, P. Vernole, B. Nicoletti and F. Mauro (1993) Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident, Mutation Res., 319, 55-60.
A. BrCgger et al. / Mutation Research 361 (1996) 73- 79 Pilinskaya, M.A., A.M. Shemetun, S.S. Dybskii, D.V. Red'ko and M.N. Eremeeva (1993) Cytogenetic effects in blood lymphocytes as an indicator of Chernobyl accident impact on human health, Radiat. Biol. Ecol. (Moscow), 33, 99-103. Pohl-Riiling, J., O. Haas, A. BrCgger, G. Obe, H. Lettner, F. Daschil, C. Atzmiiller, C., D. Lloyd, R. Kubiak and A.T. Natarajan (1991) The effect on lymphocyte chromosomes of additional radiation burden due to fallout in Salzburg (Austria) from the Chernobyl accident, Mutation Res., 262, 209-217. Salomaa, S., A. Koivistoinen, W. Paile, T. Rahola and Ryttimaa, T. (1992) Chromosomal aberration analyses of Byelorussian children exposed to Chernobyl fallout, in Proceedings of International Radiation Protection Association (IRPA 8), IRPA, Montreal, pp. 801-804. Sch~ippi-Bfichi, C. (1994) On the genetic background of the adaptive response to X-rays in Drosophila melanogaster, Int. J. Radiat. Biol., 65, 427-435. Scheid, W., J. Weber, S. Petrenko and H. Traut (1993) Chromosome aberrations in human lymphocytes apparently induced by Chernobyl fallout, Health Phys., 64, 531-534. Sevan'kaev, A.V., D.C. Lloyd, O.I. Potetnya, A.A. Zhloba, V.V. Moiseenko and A.A. Edwards (1995) Chromosomal aberrations in lymphocytes of residents of areas contaminated by radioactive discharges from the Chemobyl accident, Radiat. Protein Dosim., (in press). Shadley, J.D. (1994) Chromosomal adaptive response in human lymphocytes, Rad. Res., 138, $9-S12. Stephan, G. and U. Oestreicher (1993) Chromosome investigation of individuals living in areas of Southern Germany contami-
79
nated by fallout from the Chernobyl reactor accident, Mutation Res., 319, 189-196. Strand, P., E. B~e, L. Berteig, T. Berthelsen, T. Strand, K. Trygg and O. Harbitz (1989) Whole-body counting and dietary surveys in Norway during the first year after the Chernobyl accident, Radiat. Protein Dosim., 27, 163-171. Strand, P., T. Seln~es and A. Andersson (1990) Wholebody measurements in South Norway after the Chernobyl accident 1987-1989, (In Norwegian with English summary), SIS-report 1990:4, SIS, Oster~s, Norway. Strand, P., T.D. Seln~es and J,B. Reitan (1992a) Area and time distribution of external and internal doses from the Chernobyl fallout: The lack of correlation in Norway, Health Phys., 62, 512-518. Strand, P., T.D. Seln~es, E. Bcie A. Anderson-S0rlie (1992b) Internal doses to different parts of the Norwegian population after the Chernobyl accident and the effect of dietary advice, Health Phys., 64, 385-392. Verschaeve, L., E.V. Domracheva, S.A. Kuznetsov and V,V. Nechai (1993) Chromosome aberrations in inhabitants of Byelorussia: consequence of the Chernobyl accident, Mutation Res., 287, 253-259. Weis~eth, L. (1990) Reactions in Norway to fallout from the Chernobyl disaster, in: T. Brustad, F. Langmark and J.B. Reitan (Eds.), Radiation and cancer risk, Hemisphere, New York, pp 149-155. Westerlund, E.A., T. Berthelsen and L. Berteig (1987) Cesium-137 body burdens in Norwegian Lapps, Health Phys., 52, 171-177.