- Email: [email protected]
Long-term genetic effects of radiation exposure
Mutation Research 544 (2003) 433–439
Long-term genetic effects of radiation exposure Yuri E. Dubrova∗ Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK Accepted 30 May 2003
Abstract To date, there has been little experimental knowledge on the genetic risks of human exposure to ionising radiation for humans. Recent data suggest that hypervariable tandem repeat minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced germline mutation in humans. Here, I review the results of studies on minisatellite mutation rates in human populations exposed to radioactive fallout after the Chernobyl accident and nuclear weapon tests in Kazakhstan. © 2003 Elsevier B.V. All rights reserved. Keywords: Ionising radiation; Genetic risk; Germline mutation; Chernobyl accident; Nuclear weapon tests
1. Introduction The effort to predict the genetic consequences for humans of exposure to ionising radiation has certainly been one of the most important issues of human genetics in the past 50 years [1]. Given that the majority of chromosome aberrations and many gene mutations lead to inherited diseases, the analysis of radiation-induced changes in germline mutation rates could provide important data on the genetic risk of human exposure to ionising radiation. However, despite numerous experimental studies, experimental evidence for radiation-induced germline mutation in humans still remains highly controversial and all current estimates of genetic hazard for humans rely heavily on extrapolation from the mouse data [1,2]. It should be stressed that the lack of reliable approaches for monitoring germline mutation currently presents the main obstacle on our way to evaluate the genetic risk of human exposure to ionising radiation. It is ∗ Tel.: +44-116-252-5654; fax: +44-115-252-3378. E-mail address: [email protected] (Y.E. Dubrova).
however hoped that the recent spectacular developments in the Human Genome Project [3] could provide such approaches. Here, I present the summary of recent publications analysing mutation induction at the DNA level.
2. Accidentally exposed families from the former USSR Currently, the genetic risk of ionising radiation can potentially be analysed by studying three large cohorts of families, containing occupationally or accidentally exposed parents, as well as parents who received radiotherapy [1]. In most cases, the doses of occupational exposure are too small to induce substantial changes in germline mutation, and the analysis of this group would therefore require an exceptionally large study cohort. On the other hand, the radiotherapy patients is characterised by relatively high doses of parental exposure. However, the mutational analysis of this group is very complicated, given the fact that radiotherapy is normally administered with chemotherapy, which
1383-5742/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2003.05.003
434
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
Table 1 Summary of reported associations between reproductive outcomes and the Chernobyl accident Increased
No change
Country
References
Country
References
Miscarriages
Finland
[5]
Sweden Austria
[6] [7]
Perinatal death
Germany
[8]
Belarus, Hungary, Norway
[9]
Malformations: In embryos At birth
Belarus Belarus
[10] [10]
No data Nine countries of Europe
[11]
Down’s syndrome
Belarus West Berlin Scotland
[12] [14] [16]
Hungary Finland
[13] [15]
Neural tube defects
Turkey
[17]
Nine countries of Europe
[18]
itself may also be mutagenic. Accidentally exposed families consist of parents which have received different doses of chronic and acute ionising radiation mainly following the accidents on the nuclear facilities. For some of these families, the level of exposure from external and internal sources is relatively high and their analysis is therefore essential for estimating the genetic hazards of radiation in humans. The largest cohorts of accidentally exposed families are currently available on the territory of the former USSR, which include parents irradiated after main technological accidents on 1949–1956 at southern Urals and 1986 at Chernobyl, as well as following nuclear weapon tests in Kazakhstan. The number of exposed families and doses of ionising radiation are unprecedented, therefore providing a unique opportunity for the mutational analysis. Thus, the Chernobyl accident resulted in a largest accidental release of radioactive materials [4]. Many regions within the European part of the former USSR were heavily contaminated by radioactive fallout. The highest doses of exposure to ionising radiation following the Chernobyl accident were received by the workers involved in the accident, either during the emergency period or during the clean-up phase and by the inhabitants of heavily contaminated areas of Belarus, Ukraine and Russia [4]. Table 1 presents a summary of the data collected over recent years by analysing reproductive outcomes in the families affected by the Chernobyl accident. The results of these studies are far from being consistent,
thus clearly illustrating that the analysis of the incidence of mortality and the occurrence of genetic diseases among the offspring of irradiated parents cannot be regarded as a sensitive approach for monitoring radiation-induced germline mutation in humans. That is why we and others proposed that hypervariable tandem repeat DNA loci may provide a new experimental approach to the evaluation of germline mutation induction [19–21].
3. Tandem repeat minisatellite loci Minisatellites are the most unstable loci in human genome. They consist of 10–60 bp long repeats, which show considerable sequence variation along the array [22–25]. Mutation at these loci is almost completely restricted to the germline, with very rare and simple mutational events occurring in the somatic cells [26]. Germline mutation at human minisatellites is attributed to complex gene-conversion like events altering repeat unit copy number [22–26]. Previous studies have shown very high spontaneous germline mutation rates for some minisatellite loci, ranging from 0.5 up to 13% per gamete [25,27], which potentially make these loci useful markers for monitoring germline mutation in humans. Minisatellite mutation rates in the germline of parents are evaluated by profiling their offspring. DNA samples are most often extracted from peripheral blood lymphocytes of the parents and their offspring.
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
DNA samples are digested to completion with enzymes that do not cut within the minisatellite repeats, separated on agarose gel and minisatellite loci are then detected by hybridisation with locus-specific probes. Mutants are identified as novel DNA fragments present in the offspring that cannot be ascribed to either parent. Given the very high number of differently-sized alleles seen at hypervariable minisatellite loci, establishing the parental origin of mutants (paternal or maternal mutations) is practically always possible [27]. Mutation rate in the germline of parents is estimated by dividing the total number of mutations scored in the offspring by the total number of minisatellite bands.
4. Minisatellite mutation rates in the post-Chernobyl families To date, the frequency of minisatellite mutation has been estimated in human families inhabiting the heavily polluted rural areas of Belarus and Ukraine [21,28,29], as well as in the offspring of Chernobyl clean-up workers [30,31]. The frequency of minisatellite mutation was estimated in human families inhabiting the heavily polluted rural areas of the Mogilev district of Belarus following the Chernobyl accident and compared it to mutation frequency in a control data set from the United Kingdom [21,28]. To verify the results of this study, we extended this analysis to the group of exposed families inhabiting rural areas of the Kiev and Zhitomir regions of Ukraine [29]. In contrast to our previous study, the control group was composed of children born in the Kiev and Zhitomir regions of Ukraine and conceived before the Chernobyl accident. The exposed group contained children conceived after the Chernobyl accident. The control and exposed groups from Ukraine were matched by many confounding factors, including ethnicity and occupation, which provided a much better control population than used for the previous study of the Belarus families. Importantly, the Ukrainian families did not significantly differ by parental age and occupation from the Belarus families. In both studies, minisatellite germline mutations were scored using eight hypervariable minisatellite probes B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31 and MS32, chosen for their high spontaneous
435
mutation rate [23–25,27]. A statistically significant increase in the paternal mutation rate was found in the exposed families from Ukraine and Belarus, whereas maternal mutation rate in this cohort was not elevated (Fig. 1a). Our data therefore show that the elevated mutation rate in the germline of exposed fathers is most likely radiation-induced and raise the issue of human exposure after the Chernobyl accident. The Chernobyl accident resulted in a release of a wide spectrum of short-lived isotopes. During the first days after the Chernobyl accident, their contribution to the absorbed dose in air was extremely high [4,32], which, given the short half-life of unstable radionuclides, can no longer be evaluated by means of physical dosimetry. However, retrospective dosimetry provides an alternative approach for the evaluation of the doses for the populations of contaminated territories. The analysis of stable and unstable chromosome aberrations has provided an estimate of the mean doses of exposure for the residents of heavily contaminated areas of Ukraine and Belarus ranging up to 0.5 Gy [33–35]. Similar data were obtained by measuring the thermoluminescence of the bricks collected within contaminated Gomel-Bryansk area [36]. These estimates are markedly higher than those obtained by physical dosimetry [4,37] and reflect the initial external and internal exposure to the short-lived radionuclides. Assuming that the doubling dose for germline mutation in humans is 1 Gy [1,2], an exposure to 0.5 Gy can lead to a 1.6-fold increase in minisatellite mutation rate as found in the families from Ukraine and Belarus. These data therefore provide strong evidence that the elevated minisatellite mutation rates in the Ukrainian and Belarus families is attributed to post-Chernobyl radioactive exposure. Another two studies of germline minisatellite mutation rate were carried out on the offspring of Chernobyl clean-up workers [30,31]. The doses for this group of workers are thought to be extremely heterogeneous, and most of the participants involved in the decontamination work around the Chernobyl nuclear power plant, sarcophagus construction and other clean-up operations received doses less than 0.25 Gy [38]. Importantly, the group of Chernobyl clean-up workers was exposed to repeated small daily doses of ionising radiation. Previous studies in male mice have clearly shown that the yield of germline mutations after such an acute external fractionated exposure was less than
436
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
Paternal mutation rate, 95% CI
0.10
0.08
0.06
0.04 Control Exposed 0.02 Ukraine
UK
(a)
Ukraine
Belarus
Group 0.08
Mutation rate, 95% CI
0.07 0.06 0.05 n=44
n=36 n=46
n=45
0.04 n=37 n=24
0.03 0.02 0.01 1920-35
(b)
36-40
41-50
50-60
61-65
66-74
Parental year of birth
Fig. 1. Minisatellite germline mutation rates in human families exposed to ionising radiation (data from [21,28,29,42]). (a) Paternal mutation rates in the control and post-Chernobyl exposed groups from Ukraine, Belarus and the United Kingdom (data from [21,28,29]). The probability of difference (Fisher’s exact test, two-tailed) from the Ukrainian control group, P = 0.0299 and 0.0121 for the Ukrainian and Belarus exposed families, respectively. (b) Mutation rates in the cohort of parents exposed to nuclear weapon tests in Kazakhstan. The number of offspring analysed in each cohort of irradiated parents is shown. The dashed line represents mutation rate for the control group. 95% confidence intervals (CI) for mutation rate estimated from the Poisson distribution are shown in both graphs.
when the same dose was given in a single exposure [39]. This, together with the relatively low-dose exposure to the Chernobyl clean-up workers (mean dose 0.11 cSv [31]), suggests that the expected increase in mutation rate in this group may be too small to detect. It should be noted that a seven-fold increase in mutation rate was recently reported in the cohort of
Chernobyl liquidators [40], thus representing the most dramatic evidence for radiation-induced increase in human germline mutation rate. The authors used short random-sequence PCR primers to amplify DNA segments from the human genome (RAPD-PCR), a technique has not been validated for monitoring germline mutation in humans. The results of this study are at
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
odds with all known estimates of spontaneous mutation rate in humans, hence making this claim highly implausible [41]. It therefore appears that the novel DNA bands interpreted by the authors of this publication as the ‘mutants’ may well be attributed to non-paternity or some other factors [41].
5. Nuclear weapon tests and minisatellite mutation rates Another evidence for radiation-induced increase in minisatellite mutation rate was obtained on the offspring of irradiated parents living in the vicinity of the Semipalatinsk nuclear test site in Kazakhstan [42]. The Semipalatinsk nuclear test site has been the site for 470 nuclear tests performed by the Soviet Union during the period 1949–1989, including atmospheric and surface explosions (1949–1963), and underground tests (1963–1989) [43]. The surrounding population was mainly exposed to the fresh radioactive fallout from four surface explosions conducted between 1949 and 1956, and currently the radioactive contamination outside the test zone is low [43,44]. In our study, minisatellite mutation rates in the germline of irradiated parents living around the Semipalatinsk nuclear test site were evaluated by profiling their offspring. These data were compared with the estimates of mutation rates in the germline of non-irradiated parents from the rural area of the former Taldy Kurgan district of Kazakhstan. Both groups of parents were matched by the year of birth, parental age, occupation and smoking. Minisatellite mutation rates in these families were established using the same minisatellite probes used in our previous studies on the Chernobyl families. Similar to the Chernobyl data, an elevated minisatellite mutation rates were also found in the exposed cohort from Kazakhstan [42]. Within the exposed group, mutation rates did not show homogeneity across the cohorts of exposed parents grouped according to year of birth (Fig. 1b). Thus, for all parents born between 1926 and 1960, the germline mutation rate remained stable, and significantly exceeded that for the control group, whereas in the later cohorts it showed a negative correlation with the parental year of birth. This heterogeneity in the mutation rate could be explained by the differential exposure to ionising radiation within the
437
Semipalatinsk group. All parents born between 1926 and 1960 were exposed to relatively high levels of ionising radiation immediately after four surface explosions carried out in 1949, 1951, 1953 and 1956 that resulted in the highest contamination of the surrounding territory [43,44]. This implies that, before conception, the parents from this group were exposed to at least one surface explosion, which resulted in the accumulation of radiation-induced damage in their germline that subsequently affected the stability of minisatellite loci. In contrast, the later cohorts were exposed to the decreased doses of ionising radiation following the decay of radioisotopes in the late 1950s after the cessation of surface and atmospheric nuclear tests. This correlation implies the presence of a dose-response for minisatellite mutation induction and suggests that an elevated mutation rate in the affected families is indeed radiation-induced. Most importantly, our data show that the Moscow treaty banning nuclear weapon tests in the atmosphere (August 1963) has been effective in reducing genetic risk to the affected population.
6. Conclusions The results of our studies have shown that hypervariable tandem repeat minisatellite loci provide a useful and convenient tool for monitoring radiation-induced mutation in humans. Using this technique, we have obtained the first experimental evidence for elevated mutation rates in the germline of parents exposed to ionising radiation. However, taking into account the negative results of other studies [20,30,31], it should be stressed that the evidence for mutation induction at minisatellite loci in humans still remains controversial. Given that all above-mentioned data in humans were derived from relatively small numbers of families, additional surveys are clearly needed to evaluate in detail the mechanisms of mutation induction at human minisatellite loci.
Acknowledgements The author acknowledges the members of his group and numerous colleagues who have made important contributions to this work. This work was supported by the grants from the Wellcome Trust and
438
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
by the INCO-Copernicus grant from the European Commission. References [1] UNSCEAR, Hereditary Effects of Radiation, United Nations, New York, 2001. [2] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic risks. XI. The doubling dose estimates from the mid-1950s to present and the conceptual change to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations, Mutat. Res. 453 (2000) 107–127. [3] T.G. Wolfsberg, K.A. Wetterstrand, M.S. Guyer, F.S. Collins, A.D. Baxevanis, A user’s guide to the human genome, Nat. Genet. 32 (Suppl.) (2002) 1–79. [4] UNCEAR, Sources and Effects of Ionizing radiation, vol. II, Annex J. Exposures and Effects of the Chernobyl Accident, United Nations, New York, 2000. [5] A. Auvinen, M. Vahteristo, H. Arvela, M. Suomela, T. Rahola, M. Hakama, T. Rytomaa, Chernobyl fallout and outcome of pregnancy in Finland, Environ. Health Perspect. 109 (2001) 179–185. [6] A. Ericson, B. Kallen, Pregnancy outcome in Sweden after the Chernobyl accident, Environ. Res. 67 (1994) 149–159. [7] M.C.H. Haeusler, A. Berghold, W. Schoell, P. Hofer, M. Schaffer, The influence of the post-Chernobyl fallout on birth defects and abortion rates in Austria, Am. J. Obstet. Gynecol. 167 (1992) 1025–1031. [8] G. Luning, J. Scheer, M. Schmidt, H. Ziggel, Early infant mortality in west Germany before and after Chernobyl, Lancet 2 (1989) 1081–1083. [9] J. Little, The Chernobyl accident, congenital anomalies and other reproductive outcomes, Paediatr. Perinat. Epidemiol. 7 (1993) 121–151. [10] G.I. Lazjuk, I.A. Kirillova, D.L. Nikolaev, I.V. Novikova, Z.N. Fomina, R.D. Khmel, Frequency changes of inherited anomalies in the Republic of Belarus after the Chernobyl accident, Radiat. Protec. Dosim. 62 (1995) 71–74. [11] H. Dolk, R. Nichols, EUROCAT Working Group: evaluation of the impact of Chernobyl on the prevalence of congenital anomalies in 16 regions of Europe, Int. J. Epidemiol. 28 (1999) 941–948. [12] G.I. Laziuk, I.O. Zatsepin, P. Verger, V. Gagniere, E. Robert, Zh.P. Kravchuk, R.D. Khmel, Down syndrome and ionizing radiation: causal effect or coincidence, Radiats. Biol. Radioecol. 42 (2002) 678–683. [13] A. Czeizel, Hungarian surveillance of germinal mutations: lack of detectable increase in indicator conditions caused by germinal mutations following the Chernobyl accident, Hum. Genet. 82 (1989) 359–366. [14] K. Sperling, J. Pelz, R.D. Wegner, A. Dorries, A. Gruters, M. Mikkelsen, Significant increase in trisomy 21 in Berlin nine months after the Chernobyl reactor accident: temporal correlation or causal relation? Br. Med. J. 309 (1994) 158– 162.
[15] T. Harjulehto-Mervaala, R. Salonen, T. Aro, L. Saxen, The accident at Chernobyl and trisomy 21 in Finland, Mutat. Res. 275 (1992) 81–86. [16] C.N. Ramsay, P.M. Ellis, H. Zealley, Down’s syndrome in the Lothian region of Scotland—1978 to 1989, Biomed. Pharmacother. 45 (1991) 267–272. [17] H. Mocan, H. Bozkaya, M.H. Mocan, E.M. Furtun, Changing incidence of anencephaly in the eastern Black Sea region of Turkey and Chernobyl, Paediatr. Perinat. Epidemiol. 4 (1990) 264–268. [18] The EUROCAT Working Group: preliminary evaluation of the impact of the Chernobyl radiological contamination on the frequency of central nervous system malformations in 18 regions of Europe, Paediatr. Perinat. Epidemiol. 2 (1988) 253–264. [19] Y.E. Dubrova, A.J. Jeffreys, A.M. Malashenko, Mouse minisatellite mutations induced by ionizing radiation, Nat. Genet. 5 (1993) 92–94. [20] M. Kodaira, C. Satoh, K. Hiyama, K. Toyama, Lack of effects of atomic-bomb radiation on genetic instability of tandem-repetitive elements in human germ-cells, Am. J. Hum. Genet. 57 (1995) 1275–1283. [21] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 (1996) 683–686. [22] A.J. Jeffreys, A. MacLeod, K. Tamaki, D.L. Neil, D.G. Monckton, Minisatellite repeat coding as a digital approach to DNA typing, Nature 354 (1991) 204–209. [23] J. Buard, A. Bourdet, J. Yardley, Y.E. Dubrova, A.J. Jeffreys, Influence of array size and homogeneity on minisatellite mutation, EMBO J. 17 (1998) 3495–3502. [24] K. Tamaki, C.A. May, Y.E. Dubrova, A.J. Jeffreys, Extremely complex repeat shuffling during germline mutation at human minisatellite B6.7, Hum. Mol. Genet. 8 (1999) 879–888. [25] G. Vergnaud, F. Denoeud, Minisatellites: mutability and genome architecture, Genome Res. 10 (2000) 899–907. [26] A.J. Jeffreys, K. Tamaki, A. MacLeod, D.G. Monckton, D.L. Neil, A.J.L. Armour, Complex gene conversion events in germline mutation at human minisatellites, Nat. Genet. 6 (1994) 136–145. [27] A.J. Jeffreys, N.J. Royle, V. Wilson, Z. Wong, Spontaneous mutation rate to new length alleles at tandem-repeat hypervariable loci in human DNA, Nature 332 (1988) 278– 281. [28] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, G. Vergnaud, F. Giraudeau, J. Buard, A.J. Jeffreys, Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident, Mutat. Res. 381 (1997) 267–278. [29] Y.E. Dubrova, G. Grant, A.A. Chumak, V.A. Stezhka, A.N. Karakasian, Elevated minisatellite mutation rate in the post-Chernobyl families from Ukraine, Am. J. Hum. Genet. 71 (2002) 801–809. [30] L.A. Livshits, S.G. Malyarchuk, E.M. Lukyanova, Y.G. Antipkin, L.P. Arabskaya, S.A. Kravchenko, G.H. Matsuka, E. Petit, F. Giraudeau, P. Gourmelon, G. Vergnaud, B. Le
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
[31]
[32]
[33]
[34]
[35]
[36]
Guen, Children of Chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles, Radiat. Res. 155 (2001) 74–80. A. Kiuru, A. Auvinen, M. Luokkamaki, K. Makkonen, T. Veidebaum, M. Tekkel, M. Rahu, T. Hakulinen, K. Servomaa, T. Rytomaa, R. Mustonen, Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers, Radiat. Res. 159 (2003) 651–655. M.I. Balonov, Overview of doses to the Soviet population from the Chernobyl accident and the protective actions applied, in: S.E. Mervin, M.I. Balonov (Eds.), The Chernobyl Papers, vol. 1, Research Enterprises, Richland, Washington, 1993, pp. 23–46. F. Darroudi, A.T. Natarajan, Biological dosimetric studies in the Chernobyl radiation accident, on populations living in the contaminated areas (Gomel regions) and in Estonian clean-up workers, using FISH technique, in: A. Karaoglou, G. Desmet, G.N. Kelly, H.G. Menzel (Eds.), The Radiological Consequences of the Chernobyl accident, European Commission, Luxembourg, 1996, pp. 1067– 1072. N.A. Maznik, V.A. Vinnikov, D.C. Lloyd, A.A. Edwards, Chromosomal dosimetry for some groups of evacuees from Prypiat and Ukrainian liquidators at Chernobyl, Radiat. Protec. Dosim. 74 (1997) 5–11. L.S. Mikhalevich, D.C. Lloyd, A.A. Edwards, G.A. Perepetskaya, N.A. Kartel, Dose estimates made by dicentric analysis for some Belarussian children irradiated by the Chernobyl accident, Radiat. Protec. Dosim. 87 (2000) 109– 114. H. Sato, T. Takatsuji, J. Takada, S. Endo, M. Hoshi, V.F. Sharifov, I.I. Veselkina, I.V. Pilenko, W.A. Kalimullin, V.B. Masyakin, I. Yoshikawa, T. Nagatomo, S. Okajima, Measuring the external exposure dose in the contaminated area near the Chernobyl nuclear power station using the thermoluminescence of quartz in bricks, Health Phys. 83 (2002) 227–236.
439
[37] I.A. Likhtarev, L.N. Kovgan, P. Jacob, L.R. Anspaugh, Chernobyl accident: retrospective and prospective estimates of external dose of the population of Ukraine, Health Phys. 82 (2002) 290–303. [38] V.A. Pitkevitch, V.K. Ivanov, A.F. Tsyb, M.A. Maksyoutov, V.A. Matiash, N.V. Shchukina, Exposure levels for persons involved in recovery operations after the Chernobyl accident: statistical analysis based on the data of the Russian National Medical and Dosimetric Registry (RNMDR), Radiat. Environ. Biophys. 36 (1997) 149–160. [39] M.F. Lyon, R.J.S. Phillips, H.J. Bailey, Mutagenic effects of repeated small doses to mouse spermatogonia. I. Specific-locus mutation rates, Mutat. Res. 15 (1972) 185–190. [40] H.S. Weinberg, A.B. Korol, V.M. Kirzhner, A. Avivi, T. Fahima, E. Nevo, S. Shapiro, G. Rennert, O. Piatak, E.I. Stepanova, E. Skvarskaja, Very high mutation rate in offspring of Chernobyl accident liquidators, Proc. Roy. Soc. B 268 (2001) 1001–1005. [41] A.J. Jeffreys, Y.E. Dubrova, Monitoring spontaneous and induced human mutation by RAPD PCR, Proc. Roy. Soc. B 268 (2001) 2493–2494. [42] Y.E. Dubrova, R.I. Bersimbaev, L.B. Djansugurova, M.K. Tankimanova, Z.Zh. Mamyrbaeva, R. Mustonen, C. Lindholm, M. Hultén, S. Salomaa, Nuclear weapons tests and human germline mutation rate, Science 295 (2002) 1037. [43] A.M. Matsuschenko, G.A. Tsyrkov, A.K. Chernyshov, Y.V. Dubasov, G.A. Krasilov, V.A. Logachev, S.G. Smagulov, Y.S. Tsaturov, S.A. Zelentsov, Chronological list of nuclear tests at the Semipalatinsk Test Site and their radiation effects, in: C.S. Shapiro, V.I. Kiselev, E.V. Zaitsev (Eds.), Nuclear Tests, Long-Term Consequences in the Semipalatinsk/Altai Region, Springer, Berlin, 1998, pp. 89–97. [44] B.I. Gusev, Zh.N. Abylkassimova, K.N. Apsalikov, The Semipalatinsk nuclear test site: a first assessment of the radiological situation and the test-related radiation doses in the surrounding territories, Radiat. Environ. Biophys. 36 (1997) 201–204.
Long-term genetic effects of radiation exposure Yuri E. Dubrova∗ Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK Accepted 30 May 2003
Abstract To date, there has been little experimental knowledge on the genetic risks of human exposure to ionising radiation for humans. Recent data suggest that hypervariable tandem repeat minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced germline mutation in humans. Here, I review the results of studies on minisatellite mutation rates in human populations exposed to radioactive fallout after the Chernobyl accident and nuclear weapon tests in Kazakhstan. © 2003 Elsevier B.V. All rights reserved. Keywords: Ionising radiation; Genetic risk; Germline mutation; Chernobyl accident; Nuclear weapon tests
1. Introduction The effort to predict the genetic consequences for humans of exposure to ionising radiation has certainly been one of the most important issues of human genetics in the past 50 years [1]. Given that the majority of chromosome aberrations and many gene mutations lead to inherited diseases, the analysis of radiation-induced changes in germline mutation rates could provide important data on the genetic risk of human exposure to ionising radiation. However, despite numerous experimental studies, experimental evidence for radiation-induced germline mutation in humans still remains highly controversial and all current estimates of genetic hazard for humans rely heavily on extrapolation from the mouse data [1,2]. It should be stressed that the lack of reliable approaches for monitoring germline mutation currently presents the main obstacle on our way to evaluate the genetic risk of human exposure to ionising radiation. It is ∗ Tel.: +44-116-252-5654; fax: +44-115-252-3378. E-mail address: [email protected] (Y.E. Dubrova).
however hoped that the recent spectacular developments in the Human Genome Project [3] could provide such approaches. Here, I present the summary of recent publications analysing mutation induction at the DNA level.
2. Accidentally exposed families from the former USSR Currently, the genetic risk of ionising radiation can potentially be analysed by studying three large cohorts of families, containing occupationally or accidentally exposed parents, as well as parents who received radiotherapy [1]. In most cases, the doses of occupational exposure are too small to induce substantial changes in germline mutation, and the analysis of this group would therefore require an exceptionally large study cohort. On the other hand, the radiotherapy patients is characterised by relatively high doses of parental exposure. However, the mutational analysis of this group is very complicated, given the fact that radiotherapy is normally administered with chemotherapy, which
1383-5742/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2003.05.003
434
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
Table 1 Summary of reported associations between reproductive outcomes and the Chernobyl accident Increased
No change
Country
References
Country
References
Miscarriages
Finland
[5]
Sweden Austria
[6] [7]
Perinatal death
Germany
[8]
Belarus, Hungary, Norway
[9]
Malformations: In embryos At birth
Belarus Belarus
[10] [10]
No data Nine countries of Europe
[11]
Down’s syndrome
Belarus West Berlin Scotland
[12] [14] [16]
Hungary Finland
[13] [15]
Neural tube defects
Turkey
[17]
Nine countries of Europe
[18]
itself may also be mutagenic. Accidentally exposed families consist of parents which have received different doses of chronic and acute ionising radiation mainly following the accidents on the nuclear facilities. For some of these families, the level of exposure from external and internal sources is relatively high and their analysis is therefore essential for estimating the genetic hazards of radiation in humans. The largest cohorts of accidentally exposed families are currently available on the territory of the former USSR, which include parents irradiated after main technological accidents on 1949–1956 at southern Urals and 1986 at Chernobyl, as well as following nuclear weapon tests in Kazakhstan. The number of exposed families and doses of ionising radiation are unprecedented, therefore providing a unique opportunity for the mutational analysis. Thus, the Chernobyl accident resulted in a largest accidental release of radioactive materials [4]. Many regions within the European part of the former USSR were heavily contaminated by radioactive fallout. The highest doses of exposure to ionising radiation following the Chernobyl accident were received by the workers involved in the accident, either during the emergency period or during the clean-up phase and by the inhabitants of heavily contaminated areas of Belarus, Ukraine and Russia [4]. Table 1 presents a summary of the data collected over recent years by analysing reproductive outcomes in the families affected by the Chernobyl accident. The results of these studies are far from being consistent,
thus clearly illustrating that the analysis of the incidence of mortality and the occurrence of genetic diseases among the offspring of irradiated parents cannot be regarded as a sensitive approach for monitoring radiation-induced germline mutation in humans. That is why we and others proposed that hypervariable tandem repeat DNA loci may provide a new experimental approach to the evaluation of germline mutation induction [19–21].
3. Tandem repeat minisatellite loci Minisatellites are the most unstable loci in human genome. They consist of 10–60 bp long repeats, which show considerable sequence variation along the array [22–25]. Mutation at these loci is almost completely restricted to the germline, with very rare and simple mutational events occurring in the somatic cells [26]. Germline mutation at human minisatellites is attributed to complex gene-conversion like events altering repeat unit copy number [22–26]. Previous studies have shown very high spontaneous germline mutation rates for some minisatellite loci, ranging from 0.5 up to 13% per gamete [25,27], which potentially make these loci useful markers for monitoring germline mutation in humans. Minisatellite mutation rates in the germline of parents are evaluated by profiling their offspring. DNA samples are most often extracted from peripheral blood lymphocytes of the parents and their offspring.
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
DNA samples are digested to completion with enzymes that do not cut within the minisatellite repeats, separated on agarose gel and minisatellite loci are then detected by hybridisation with locus-specific probes. Mutants are identified as novel DNA fragments present in the offspring that cannot be ascribed to either parent. Given the very high number of differently-sized alleles seen at hypervariable minisatellite loci, establishing the parental origin of mutants (paternal or maternal mutations) is practically always possible [27]. Mutation rate in the germline of parents is estimated by dividing the total number of mutations scored in the offspring by the total number of minisatellite bands.
4. Minisatellite mutation rates in the post-Chernobyl families To date, the frequency of minisatellite mutation has been estimated in human families inhabiting the heavily polluted rural areas of Belarus and Ukraine [21,28,29], as well as in the offspring of Chernobyl clean-up workers [30,31]. The frequency of minisatellite mutation was estimated in human families inhabiting the heavily polluted rural areas of the Mogilev district of Belarus following the Chernobyl accident and compared it to mutation frequency in a control data set from the United Kingdom [21,28]. To verify the results of this study, we extended this analysis to the group of exposed families inhabiting rural areas of the Kiev and Zhitomir regions of Ukraine [29]. In contrast to our previous study, the control group was composed of children born in the Kiev and Zhitomir regions of Ukraine and conceived before the Chernobyl accident. The exposed group contained children conceived after the Chernobyl accident. The control and exposed groups from Ukraine were matched by many confounding factors, including ethnicity and occupation, which provided a much better control population than used for the previous study of the Belarus families. Importantly, the Ukrainian families did not significantly differ by parental age and occupation from the Belarus families. In both studies, minisatellite germline mutations were scored using eight hypervariable minisatellite probes B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31 and MS32, chosen for their high spontaneous
435
mutation rate [23–25,27]. A statistically significant increase in the paternal mutation rate was found in the exposed families from Ukraine and Belarus, whereas maternal mutation rate in this cohort was not elevated (Fig. 1a). Our data therefore show that the elevated mutation rate in the germline of exposed fathers is most likely radiation-induced and raise the issue of human exposure after the Chernobyl accident. The Chernobyl accident resulted in a release of a wide spectrum of short-lived isotopes. During the first days after the Chernobyl accident, their contribution to the absorbed dose in air was extremely high [4,32], which, given the short half-life of unstable radionuclides, can no longer be evaluated by means of physical dosimetry. However, retrospective dosimetry provides an alternative approach for the evaluation of the doses for the populations of contaminated territories. The analysis of stable and unstable chromosome aberrations has provided an estimate of the mean doses of exposure for the residents of heavily contaminated areas of Ukraine and Belarus ranging up to 0.5 Gy [33–35]. Similar data were obtained by measuring the thermoluminescence of the bricks collected within contaminated Gomel-Bryansk area [36]. These estimates are markedly higher than those obtained by physical dosimetry [4,37] and reflect the initial external and internal exposure to the short-lived radionuclides. Assuming that the doubling dose for germline mutation in humans is 1 Gy [1,2], an exposure to 0.5 Gy can lead to a 1.6-fold increase in minisatellite mutation rate as found in the families from Ukraine and Belarus. These data therefore provide strong evidence that the elevated minisatellite mutation rates in the Ukrainian and Belarus families is attributed to post-Chernobyl radioactive exposure. Another two studies of germline minisatellite mutation rate were carried out on the offspring of Chernobyl clean-up workers [30,31]. The doses for this group of workers are thought to be extremely heterogeneous, and most of the participants involved in the decontamination work around the Chernobyl nuclear power plant, sarcophagus construction and other clean-up operations received doses less than 0.25 Gy [38]. Importantly, the group of Chernobyl clean-up workers was exposed to repeated small daily doses of ionising radiation. Previous studies in male mice have clearly shown that the yield of germline mutations after such an acute external fractionated exposure was less than
436
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
Paternal mutation rate, 95% CI
0.10
0.08
0.06
0.04 Control Exposed 0.02 Ukraine
UK
(a)
Ukraine
Belarus
Group 0.08
Mutation rate, 95% CI
0.07 0.06 0.05 n=44
n=36 n=46
n=45
0.04 n=37 n=24
0.03 0.02 0.01 1920-35
(b)
36-40
41-50
50-60
61-65
66-74
Parental year of birth
Fig. 1. Minisatellite germline mutation rates in human families exposed to ionising radiation (data from [21,28,29,42]). (a) Paternal mutation rates in the control and post-Chernobyl exposed groups from Ukraine, Belarus and the United Kingdom (data from [21,28,29]). The probability of difference (Fisher’s exact test, two-tailed) from the Ukrainian control group, P = 0.0299 and 0.0121 for the Ukrainian and Belarus exposed families, respectively. (b) Mutation rates in the cohort of parents exposed to nuclear weapon tests in Kazakhstan. The number of offspring analysed in each cohort of irradiated parents is shown. The dashed line represents mutation rate for the control group. 95% confidence intervals (CI) for mutation rate estimated from the Poisson distribution are shown in both graphs.
when the same dose was given in a single exposure [39]. This, together with the relatively low-dose exposure to the Chernobyl clean-up workers (mean dose 0.11 cSv [31]), suggests that the expected increase in mutation rate in this group may be too small to detect. It should be noted that a seven-fold increase in mutation rate was recently reported in the cohort of
Chernobyl liquidators [40], thus representing the most dramatic evidence for radiation-induced increase in human germline mutation rate. The authors used short random-sequence PCR primers to amplify DNA segments from the human genome (RAPD-PCR), a technique has not been validated for monitoring germline mutation in humans. The results of this study are at
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
odds with all known estimates of spontaneous mutation rate in humans, hence making this claim highly implausible [41]. It therefore appears that the novel DNA bands interpreted by the authors of this publication as the ‘mutants’ may well be attributed to non-paternity or some other factors [41].
5. Nuclear weapon tests and minisatellite mutation rates Another evidence for radiation-induced increase in minisatellite mutation rate was obtained on the offspring of irradiated parents living in the vicinity of the Semipalatinsk nuclear test site in Kazakhstan [42]. The Semipalatinsk nuclear test site has been the site for 470 nuclear tests performed by the Soviet Union during the period 1949–1989, including atmospheric and surface explosions (1949–1963), and underground tests (1963–1989) [43]. The surrounding population was mainly exposed to the fresh radioactive fallout from four surface explosions conducted between 1949 and 1956, and currently the radioactive contamination outside the test zone is low [43,44]. In our study, minisatellite mutation rates in the germline of irradiated parents living around the Semipalatinsk nuclear test site were evaluated by profiling their offspring. These data were compared with the estimates of mutation rates in the germline of non-irradiated parents from the rural area of the former Taldy Kurgan district of Kazakhstan. Both groups of parents were matched by the year of birth, parental age, occupation and smoking. Minisatellite mutation rates in these families were established using the same minisatellite probes used in our previous studies on the Chernobyl families. Similar to the Chernobyl data, an elevated minisatellite mutation rates were also found in the exposed cohort from Kazakhstan [42]. Within the exposed group, mutation rates did not show homogeneity across the cohorts of exposed parents grouped according to year of birth (Fig. 1b). Thus, for all parents born between 1926 and 1960, the germline mutation rate remained stable, and significantly exceeded that for the control group, whereas in the later cohorts it showed a negative correlation with the parental year of birth. This heterogeneity in the mutation rate could be explained by the differential exposure to ionising radiation within the
437
Semipalatinsk group. All parents born between 1926 and 1960 were exposed to relatively high levels of ionising radiation immediately after four surface explosions carried out in 1949, 1951, 1953 and 1956 that resulted in the highest contamination of the surrounding territory [43,44]. This implies that, before conception, the parents from this group were exposed to at least one surface explosion, which resulted in the accumulation of radiation-induced damage in their germline that subsequently affected the stability of minisatellite loci. In contrast, the later cohorts were exposed to the decreased doses of ionising radiation following the decay of radioisotopes in the late 1950s after the cessation of surface and atmospheric nuclear tests. This correlation implies the presence of a dose-response for minisatellite mutation induction and suggests that an elevated mutation rate in the affected families is indeed radiation-induced. Most importantly, our data show that the Moscow treaty banning nuclear weapon tests in the atmosphere (August 1963) has been effective in reducing genetic risk to the affected population.
6. Conclusions The results of our studies have shown that hypervariable tandem repeat minisatellite loci provide a useful and convenient tool for monitoring radiation-induced mutation in humans. Using this technique, we have obtained the first experimental evidence for elevated mutation rates in the germline of parents exposed to ionising radiation. However, taking into account the negative results of other studies [20,30,31], it should be stressed that the evidence for mutation induction at minisatellite loci in humans still remains controversial. Given that all above-mentioned data in humans were derived from relatively small numbers of families, additional surveys are clearly needed to evaluate in detail the mechanisms of mutation induction at human minisatellite loci.
Acknowledgements The author acknowledges the members of his group and numerous colleagues who have made important contributions to this work. This work was supported by the grants from the Wellcome Trust and
438
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
by the INCO-Copernicus grant from the European Commission. References [1] UNSCEAR, Hereditary Effects of Radiation, United Nations, New York, 2001. [2] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic risks. XI. The doubling dose estimates from the mid-1950s to present and the conceptual change to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations, Mutat. Res. 453 (2000) 107–127. [3] T.G. Wolfsberg, K.A. Wetterstrand, M.S. Guyer, F.S. Collins, A.D. Baxevanis, A user’s guide to the human genome, Nat. Genet. 32 (Suppl.) (2002) 1–79. [4] UNCEAR, Sources and Effects of Ionizing radiation, vol. II, Annex J. Exposures and Effects of the Chernobyl Accident, United Nations, New York, 2000. [5] A. Auvinen, M. Vahteristo, H. Arvela, M. Suomela, T. Rahola, M. Hakama, T. Rytomaa, Chernobyl fallout and outcome of pregnancy in Finland, Environ. Health Perspect. 109 (2001) 179–185. [6] A. Ericson, B. Kallen, Pregnancy outcome in Sweden after the Chernobyl accident, Environ. Res. 67 (1994) 149–159. [7] M.C.H. Haeusler, A. Berghold, W. Schoell, P. Hofer, M. Schaffer, The influence of the post-Chernobyl fallout on birth defects and abortion rates in Austria, Am. J. Obstet. Gynecol. 167 (1992) 1025–1031. [8] G. Luning, J. Scheer, M. Schmidt, H. Ziggel, Early infant mortality in west Germany before and after Chernobyl, Lancet 2 (1989) 1081–1083. [9] J. Little, The Chernobyl accident, congenital anomalies and other reproductive outcomes, Paediatr. Perinat. Epidemiol. 7 (1993) 121–151. [10] G.I. Lazjuk, I.A. Kirillova, D.L. Nikolaev, I.V. Novikova, Z.N. Fomina, R.D. Khmel, Frequency changes of inherited anomalies in the Republic of Belarus after the Chernobyl accident, Radiat. Protec. Dosim. 62 (1995) 71–74. [11] H. Dolk, R. Nichols, EUROCAT Working Group: evaluation of the impact of Chernobyl on the prevalence of congenital anomalies in 16 regions of Europe, Int. J. Epidemiol. 28 (1999) 941–948. [12] G.I. Laziuk, I.O. Zatsepin, P. Verger, V. Gagniere, E. Robert, Zh.P. Kravchuk, R.D. Khmel, Down syndrome and ionizing radiation: causal effect or coincidence, Radiats. Biol. Radioecol. 42 (2002) 678–683. [13] A. Czeizel, Hungarian surveillance of germinal mutations: lack of detectable increase in indicator conditions caused by germinal mutations following the Chernobyl accident, Hum. Genet. 82 (1989) 359–366. [14] K. Sperling, J. Pelz, R.D. Wegner, A. Dorries, A. Gruters, M. Mikkelsen, Significant increase in trisomy 21 in Berlin nine months after the Chernobyl reactor accident: temporal correlation or causal relation? Br. Med. J. 309 (1994) 158– 162.
[15] T. Harjulehto-Mervaala, R. Salonen, T. Aro, L. Saxen, The accident at Chernobyl and trisomy 21 in Finland, Mutat. Res. 275 (1992) 81–86. [16] C.N. Ramsay, P.M. Ellis, H. Zealley, Down’s syndrome in the Lothian region of Scotland—1978 to 1989, Biomed. Pharmacother. 45 (1991) 267–272. [17] H. Mocan, H. Bozkaya, M.H. Mocan, E.M. Furtun, Changing incidence of anencephaly in the eastern Black Sea region of Turkey and Chernobyl, Paediatr. Perinat. Epidemiol. 4 (1990) 264–268. [18] The EUROCAT Working Group: preliminary evaluation of the impact of the Chernobyl radiological contamination on the frequency of central nervous system malformations in 18 regions of Europe, Paediatr. Perinat. Epidemiol. 2 (1988) 253–264. [19] Y.E. Dubrova, A.J. Jeffreys, A.M. Malashenko, Mouse minisatellite mutations induced by ionizing radiation, Nat. Genet. 5 (1993) 92–94. [20] M. Kodaira, C. Satoh, K. Hiyama, K. Toyama, Lack of effects of atomic-bomb radiation on genetic instability of tandem-repetitive elements in human germ-cells, Am. J. Hum. Genet. 57 (1995) 1275–1283. [21] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellite mutation rate after the Chernobyl accident, Nature 380 (1996) 683–686. [22] A.J. Jeffreys, A. MacLeod, K. Tamaki, D.L. Neil, D.G. Monckton, Minisatellite repeat coding as a digital approach to DNA typing, Nature 354 (1991) 204–209. [23] J. Buard, A. Bourdet, J. Yardley, Y.E. Dubrova, A.J. Jeffreys, Influence of array size and homogeneity on minisatellite mutation, EMBO J. 17 (1998) 3495–3502. [24] K. Tamaki, C.A. May, Y.E. Dubrova, A.J. Jeffreys, Extremely complex repeat shuffling during germline mutation at human minisatellite B6.7, Hum. Mol. Genet. 8 (1999) 879–888. [25] G. Vergnaud, F. Denoeud, Minisatellites: mutability and genome architecture, Genome Res. 10 (2000) 899–907. [26] A.J. Jeffreys, K. Tamaki, A. MacLeod, D.G. Monckton, D.L. Neil, A.J.L. Armour, Complex gene conversion events in germline mutation at human minisatellites, Nat. Genet. 6 (1994) 136–145. [27] A.J. Jeffreys, N.J. Royle, V. Wilson, Z. Wong, Spontaneous mutation rate to new length alleles at tandem-repeat hypervariable loci in human DNA, Nature 332 (1988) 278– 281. [28] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko, G. Vergnaud, F. Giraudeau, J. Buard, A.J. Jeffreys, Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident, Mutat. Res. 381 (1997) 267–278. [29] Y.E. Dubrova, G. Grant, A.A. Chumak, V.A. Stezhka, A.N. Karakasian, Elevated minisatellite mutation rate in the post-Chernobyl families from Ukraine, Am. J. Hum. Genet. 71 (2002) 801–809. [30] L.A. Livshits, S.G. Malyarchuk, E.M. Lukyanova, Y.G. Antipkin, L.P. Arabskaya, S.A. Kravchenko, G.H. Matsuka, E. Petit, F. Giraudeau, P. Gourmelon, G. Vergnaud, B. Le
Y.E. Dubrova / Mutation Research 544 (2003) 433–439
[31]
[32]
[33]
[34]
[35]
[36]
Guen, Children of Chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles, Radiat. Res. 155 (2001) 74–80. A. Kiuru, A. Auvinen, M. Luokkamaki, K. Makkonen, T. Veidebaum, M. Tekkel, M. Rahu, T. Hakulinen, K. Servomaa, T. Rytomaa, R. Mustonen, Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers, Radiat. Res. 159 (2003) 651–655. M.I. Balonov, Overview of doses to the Soviet population from the Chernobyl accident and the protective actions applied, in: S.E. Mervin, M.I. Balonov (Eds.), The Chernobyl Papers, vol. 1, Research Enterprises, Richland, Washington, 1993, pp. 23–46. F. Darroudi, A.T. Natarajan, Biological dosimetric studies in the Chernobyl radiation accident, on populations living in the contaminated areas (Gomel regions) and in Estonian clean-up workers, using FISH technique, in: A. Karaoglou, G. Desmet, G.N. Kelly, H.G. Menzel (Eds.), The Radiological Consequences of the Chernobyl accident, European Commission, Luxembourg, 1996, pp. 1067– 1072. N.A. Maznik, V.A. Vinnikov, D.C. Lloyd, A.A. Edwards, Chromosomal dosimetry for some groups of evacuees from Prypiat and Ukrainian liquidators at Chernobyl, Radiat. Protec. Dosim. 74 (1997) 5–11. L.S. Mikhalevich, D.C. Lloyd, A.A. Edwards, G.A. Perepetskaya, N.A. Kartel, Dose estimates made by dicentric analysis for some Belarussian children irradiated by the Chernobyl accident, Radiat. Protec. Dosim. 87 (2000) 109– 114. H. Sato, T. Takatsuji, J. Takada, S. Endo, M. Hoshi, V.F. Sharifov, I.I. Veselkina, I.V. Pilenko, W.A. Kalimullin, V.B. Masyakin, I. Yoshikawa, T. Nagatomo, S. Okajima, Measuring the external exposure dose in the contaminated area near the Chernobyl nuclear power station using the thermoluminescence of quartz in bricks, Health Phys. 83 (2002) 227–236.
439
[37] I.A. Likhtarev, L.N. Kovgan, P. Jacob, L.R. Anspaugh, Chernobyl accident: retrospective and prospective estimates of external dose of the population of Ukraine, Health Phys. 82 (2002) 290–303. [38] V.A. Pitkevitch, V.K. Ivanov, A.F. Tsyb, M.A. Maksyoutov, V.A. Matiash, N.V. Shchukina, Exposure levels for persons involved in recovery operations after the Chernobyl accident: statistical analysis based on the data of the Russian National Medical and Dosimetric Registry (RNMDR), Radiat. Environ. Biophys. 36 (1997) 149–160. [39] M.F. Lyon, R.J.S. Phillips, H.J. Bailey, Mutagenic effects of repeated small doses to mouse spermatogonia. I. Specific-locus mutation rates, Mutat. Res. 15 (1972) 185–190. [40] H.S. Weinberg, A.B. Korol, V.M. Kirzhner, A. Avivi, T. Fahima, E. Nevo, S. Shapiro, G. Rennert, O. Piatak, E.I. Stepanova, E. Skvarskaja, Very high mutation rate in offspring of Chernobyl accident liquidators, Proc. Roy. Soc. B 268 (2001) 1001–1005. [41] A.J. Jeffreys, Y.E. Dubrova, Monitoring spontaneous and induced human mutation by RAPD PCR, Proc. Roy. Soc. B 268 (2001) 2493–2494. [42] Y.E. Dubrova, R.I. Bersimbaev, L.B. Djansugurova, M.K. Tankimanova, Z.Zh. Mamyrbaeva, R. Mustonen, C. Lindholm, M. Hultén, S. Salomaa, Nuclear weapons tests and human germline mutation rate, Science 295 (2002) 1037. [43] A.M. Matsuschenko, G.A. Tsyrkov, A.K. Chernyshov, Y.V. Dubasov, G.A. Krasilov, V.A. Logachev, S.G. Smagulov, Y.S. Tsaturov, S.A. Zelentsov, Chronological list of nuclear tests at the Semipalatinsk Test Site and their radiation effects, in: C.S. Shapiro, V.I. Kiselev, E.V. Zaitsev (Eds.), Nuclear Tests, Long-Term Consequences in the Semipalatinsk/Altai Region, Springer, Berlin, 1998, pp. 89–97. [44] B.I. Gusev, Zh.N. Abylkassimova, K.N. Apsalikov, The Semipalatinsk nuclear test site: a first assessment of the radiological situation and the test-related radiation doses in the surrounding territories, Radiat. Environ. Biophys. 36 (1997) 201–204.