Early and Late Cytogenetic Effects Study in Groups Exposed Due to theChernobyl Accident Using Conventional Chromosome Analysis andFluorescence In-Situ Hybridisation (FISH) Technique
Nataliya A. Maznyk
Institute of Medical Radiology named by S.P Grigiriev, Academy of Medical Science of Ukraine
Pushkinskaya St., 82, Kharkiv, 61024 Ukraine: lrcg.imr@mail.ru

Introduction
During last two decades a large body of scientific data have been accumulated by observations
targeted to assessing and quantifying various biomedical effects in humans exposed to ionizing radiation
due to the catastrophe at the Chernobyl nuclear power plant (Ukraine, former USSR) in 1986. It wascommonly believed that Chernobyl-related radiation doses for the majority of affected individuals wereclinically low, particularly not exceeding 1 Gy of equivalently acute low-LET rradiation, and that was
confirmed by the absence of stable deterministic sindroms. But whatever low, those doses must be taken
into account when late stochastic effect risk is calculated for the exposed populations, which are generally
subdivided into three main categories: (i) the population evacuated from 30-km exclusive zone around theChernobyl NPP and some nearby regions heavily contaminated with radionuclides, (ii) clean-up workers(“liquidators”) who were irradiated during their duties in the Chernobyl zone and (iii) people who
continue to live in areas with increased levels of radioactivity [1].
The cytogenetic analysis based on chromosomal aberration scoring in cultured human peripheral
lymphocytes appeared to be one of the most demanded techniques for monitoring the Chernobyl criticalgroups. Firstly, post-Chernobyl cytogenetic research provided fundamental radiobiological data about
spectrum and magnitude of genetic damage caused in human somatic cells by Chernobyl genotoxic factors,
amongst which ionizing radiation dominated, but chemical agents also played quite a noticeable role [2-5].
But more importantly, the cytogenetic method was applied so intensively because of its ability to serve as
the most powerful tool for biological dosimetry [6]. It should be fairly noted that initial steps ofchromosomal dosimetry in the post-Chernobyl critical groups sometimes suffered from limitations due tospecific irradiation scenarios (chronic or protracted exposure) and low number of cells scored per person
when huge number of cases required rapid cytogenetic screening. With time pass after the accident anatural process of elimination of lymphocytes carrying unstable chromosomal aberrations comprised
another big problem for conventional chromosomal biodosimetry, and stable chromosome aberration
analysis using fluorescence in situ hybridization (FISH) technique also didn’t provide an ideal alternativeat the beginning of its application into real biodosimetry practice. It took about 20 years to develop proper
methodological approaches which allow satisfactory overcoming the majority of listed problems [4, 7-10].
Regarding cytogenetics, evacuees from the 30-km Chernobyl exclusive zone have remained lessinvestigated in compare with liquidators and inhabitants of radioactively contaminated areas. That could
be a result of the lack of specialized cytogenetic laboratories in research institutes and hospitals, where
initial examinations of those persons took place. Nevertheless cytogenetic data in evacuees were obtainedby research groups from Minsk [11], St. Pitersburg [12], Moscow [2, 13] and Kharkiv (Ukraine) [3]. But
only the Radiation Cytogenetics Laboratory of Kharkiv Institute for Medical Radiology started thecytogenetic survey in this group nearly immediately after the accident (since 28th April 1986). We carriedout our study during long period of time, with several time-effect points gathered in randomly sampledcohort between the first few days and 14 years after evacuation. The summary results of theseinvestigations performed by conventional cytogenetic analysis and additional data concerning the- 116 -

possibilities of retrospective biological dosimetry using FISH technique in Chernobyl evacuees andchronically exposed residents of radioactively contaminated areas are presented in this paper.
Materials and Methods
Study groups
In total 112 evacuees had been investigated by the conventional chromosome analysis. They were
50 adult males and 52 adult females, age ranged from 23 to 66 years, and also 10 children 4 to 17 years
old. Blood samplings for cytogenetic assay were done in the time range from 2 days to 14.8 years after the
departure from the Chernobyl exclusive zone. Amongst them there were 18 individuals (7 males and 11
females, age ranged from 16 to 55 years), for whom FISH analysis was also carried out in period 12.814.8 years after evacuation. The studied evacuees group did not contain cases of acute radiation syndrome,
local skin and soft tissue injuries or cancer.
Another examined group consisted of 21 residents of radioactively contaminated regions ofBelarus (6 males and 15 females). They were children at the time of the Chernobyl accident and continued
to live in areas with increased levels of radionuclide deposition. Their age varied from 15 to 26 years at
the time of blood sampling which was performed 12.8-14.8 years after the Chernobyl accident. This group
was investigated by FISH technique only. Blood samples from the Belorussian residents group werecollected at the Institute of Genetics and Cytology (Minsk, Belarus) and passed to the National
Radiological Protection Board of the United Kingdom (NRPB, currently HPA-RPD) for cell culturing,and then coded metaphase preparations were transferred to the KhIMR for further FISH analysis.
Control group established for conventional chromosomal assay consisted of 50 healthy persons(19 males and 31 females), unexposed inhabitants of Kharkiv region aged from 19 to 58 years (mean 33
years). Amongst them a subgroup of controls for FISH study was formed, comprising 5 males and 7
females, aged from 19 to 58 years, randomly selected in trying to cover the age ranges in both exposedgroups.
Cell culturing, conventional analysis, FISH painting and aberration scoring
The details of techniques and aberration scoring criteria used at KhIMR Radiation Cytogenetic
Laboratory during investigations in post-Chernobyl human cohorts were published earlier [3-5, 8-10].
Briefly, throughout all the period of investigations the unified method of peripheral blood lymphocyte
culturing was used with PHA-stimulated lymphocyte cultures set up for 48-50 h, metaphases harvesting
after 4 h colchicin treatment and fixing in methanol/acetic acid mixture, that well corresponds to thestandard technique described in IAEA manual [6]. From each sample replicated slides were prepared,
coded and either stained by Giemsa for conventional analysis or processed by FISH technique according
to the protocol [14].
For conventional assay all cytogenetic abnormalities recognised without special karyotyping wererecorded, i.e. dicentrics and centric rings, both accompanied by acentric, excess acentric fragments,
chromatid breaks and exchanges (combined below into total chromatid aberrations), hyper- and polyploids(combined below into total genomic abnormalities; all polyploids found didn’t contain replicatedaberrations; virtually all hyperploids represented threesomics, i.e. 47 chromosomes).
For FISH assay slides were FITC painted, highlighting chromosome combinations 1, 2 and 4(Cambio), other chromosomes were counterstained with DAPI (diamidino-2-phenylindole),pancentromere probes (Oncor) that fluoresced red were also applied. Slides were examined underfluorescence microscopes (Nikon, Zeiss) equipped with filter sets for FITC, DAPI and all three
fluorochromes vizualizing. Aberrations were counted in cells containing 46 centromeres and diploidamount of painted material from FITC-highlighted chromosomes. Translocations were recorded using themodified hybrid of conventional/PAINT descriptive nomenclature as complete tcomp or incomplete tincAb
and tincBa. The latter were subdivided into three subgroups: involving an unshorterned painted- 117 -

chromosome – tincBa*, accompanied by a fragment from the painted chromosome – tincBa+ac, involving amarkedly shorterned chromosome with no missing painted fragment present somewhere in the cell –tincВаМР (“missing part”). Insertions of Aba and Bab-types were pooled into one category. Each exchange,
either complete or incomplete, was accounted as an entity. Deleted painted chromosomes with a segment
absent, dicentrics and centric rings accompanied by fragment and excess acentrics in painted
chromosomes were also recorded for data completeness.
Statistical analysis
From 50 to 1200 metaphases were analyzed per person. Numbers of actual cells scored by FISH
assay were converted into genome equivalents by monocolour version of Lucas’ formula for the sum of
DNA content in highlighted chromosomes [15, 16].
When individual data were pooled, the randomness of the individual aberration yield distribution
within the group was checked and weighted mean yields of cytogenetic damage were estimated. Standard
errors for the mean were calculated from the observed dispersion of the cytogenetic damage yields
amongst individuals that nearly always was close to Poisson statistics. For intergroup data comparison
Student’s t-test was applied.
Results and discussion
The yield of aberrations measured by conventional analysis: general characteristics and time course
Kharkiv region remained non-polluted by the Chernobyl fallout and therefore comprised one of
the main destinations for the routs of evacuation of citizens from town Pripiat’ and nearby villages locatedwithin 30-km exclusive zone around the Chernobyl NPP. Thus initial decontamination procedures andbiomedical examinations of exposed persons were carried out by specialised departments and laboratoriesof the Kharkiv Institute for Medical Radiology (KhIMR). Chernobyl-related cytogenetic research started
at KhIMR 28th April 1986, i.e. just 2 days after the catastrophe, and continued for the following 15 years.
Individual cytogenetic data in evacuees were combined depending on time gap between departurefrom the Chernobyl zone and investigation (Table 1). Within each group the individual aberration yields
appeared at random. The individual values of total aberration frequency varied from 1.2 to 12.0 per 100
cells in groups studied up to 1 year after exposure; later that range narrowed to 0.0-5.0 per 100 cells.
Dicentrics and centric rings accompanied by fragment represented a significant proportion amongst
chromosome type aberrations: 30-40 % at the beginning of the observation, 18-29 % later, in comparewith 13.4 % in the control group. Cells with more than 1 chromosome exchange were not found, thus
aberration-per-cell distribution in that cohort was in a good agreement with Poisson statistics.
Table 1. Total cytogenetic damage yields in evacuees sampled at various times after their departure fromthe 30-km Chernobyl exclusive zone
Time after evacuation
range

mean ± SE

1-9 days
4.25±0.65 d.
0.2-1.0 y.
0.73±0.03 y.
1.4-3.7 y.
2.13±0.11 y.
4.6-10.7 y.
7.99±0.87 y.
12.8-14.8 y. 14.32±0.15 y.
Controls

Mean cytogenetic damage yield per 100 cells ±SE
Number of Cells
Chromosome Chromatid
Genomic
persons scored Aberrant cells
aberrations
aberrations abnormalities

20
40
20
14
18
50

2316
4221
2065
3603
9314
19289

4.66±0.54
5.19±0.39
4.65±0.60
2.66±0.38
1.48±0.21
1.24±0.11

2.94±0.36
3.61±0.29
2.52±0.40
1.72±0.26
0.64±0.14
0.62±0.09

1.77±0.34
1.85±0.21
2.61±0.51
1.17±0.22
0.91±0.12
0.66±0.07

0.48±0.15
0.31±0.08
0.34±0.13
0.17±0.10
0.14±0.04
0.08±0.02

Here and in other tables: SE is a standard error for the mean; chromosome aberrations don’t include abnormal
monocentrics resulted from translocations and insertions; genomic abnormalities comprise the sum of hyper- andpolyploids.

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Up to 2 years after exposure the mean frequency of aberrant cells remained on the plateau of the
4-fold excess above control, the mean yields of chromosome type aberrations and genomic abnormalities
were 4-6 times higher and chromatid type aberrations – 3-4 times higher than the respective spontaneous
values (p<0.001 for all end-points). Later an elimination of lymphocytes carrying chromosome aberrations
took place, and the difference between evacuees and controls became statistically insignificant for
genomic abnormalities approximately 8 years after exposure, for chromosomal rearrangements – 14 years
after departure from the Chernobyl zone.
According to well known mechanisms of cytogenetic damage formation in mature human
lymphocytes, the over-spontaneous excess of chromosome type aberrations, particularly dicentrics andcentric rings in evacuees can be attributed to ionizing radiation exposure, but elevated yield of chromatidaberrations, polyploids and aneuploids should be considered as fingerprints of chemical genotoxic action
or non-specific, stress-related endogenous mutagenesis (for details see [7]). In our opinion, the mostpossible source of chemical genotoxic action could be various compounds released from the reactor andalso some reagents used during firefighting and decontamination procedures. The conclusion about
appearance of those chemical factors in the environment due to the the Chernobyl accident is confirmedby the finding of the same spectrum and very similar quantitative outcome of cytogenetic damage
inspecific to radiation in Chernobyl clean-up workers [17, 18].
The time course for dicentric and centric rings yield in evacuees is presented in Fig. 1. The generaltendency for this end-point was gradual disappearence. However, the phenomenon of an initial rise of thedicentrics plus rings frequencies over one year was found, that can be explained by continuing exposuresfrom short-lived radionuclides. After that aberration yield declined quite rapidly and in 14 years after
irradiation it nearly reached the control level.
Dependence on age, gender and departure time
Regarding these results, the following analysis of cytogenetic data in relevance to age and gender
was concentrated on 60 persons examined within 1 year after evacuation, when the dicentrics plus centricrings yield didn’t start a decrease (Table 2). In control donors subdivided into appropriate subgroups nodifference for cytogenetic parameters was found between males and females, and the same occurred for
non-exposed individuals of different age, with the only exception of excess chromosome acentricFig. 1. Time effect relationship for the mean dicentrics plus centric rings yield in evacuees from the
30-km Chernobyl zone (1) in compare with controls (2). Y axis – aberration frequency per 100 cells;
X axis – time after departure, years. Vertical bars represent standard errors of the mean.
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Table 2. Early cytogenetic damage levels in evacuees depending on age and gender
Mean cytogenetic damage yield per 100 cells ±SE
Group
Numberformation
Groupof
Excess
Chromatid
Genomic
Dic+CR fr
parameter
persons
acentrics
aberrations abnormalities
4-17 years
10
0.92±0.26
1.30±0.32а
1.53±0.28
0.31±0.19bAge23-35 years
30
1.31±0.20
2.18±0.20
1.96±0.30
0.50±0.11c1.78±0.29
0.20±0.11
36-66 years
20
1.39±0.21
2.52±0.35
Females28
1.26±0.19
2.22±0.24
1.89±0.29
0.50±0.13
GenderMales
32
1.25±0.18
2.02±0.23
1.76±0.22
0.26±0.08
Dic+CR fr are dicentrics and centric rings accompanied by fragment; a, b & c – the spontaneous level ofexcess acentrics in the respective age subgroups in control, that were a = 0.09±0.09; b = 0.37±0.11; c =0.91±0.22 per 100 cells.
fragments yield (for acentrics see footnote for Table 2; for other cytogenetic damage data not shown, as
they didn’t differ from the mean control values presented in Table 1 and Fig. 1). No changes in chromatidaberrations and genomic abnormalities levels were observed with evacuees’ age. The difference fordicentrics plus centric rings yield between the youngest and the oldest age subgroups didn’t reach thestatistical significance (р>0.05), but the tendency for lower yield of radiation-specific chromosome
exchanges in children may reflect some attempts to minimize the radiation load for youth, probably bylimiting their outdoors activity between the accident and evacuation. The positive age dependence forexcess acentric yield in evacuees was obviously related to the similar tendency for the spontaneous levelin control donors. The statistical difference between respective evacuees and controls age subgroups foreach cytogenetic end-point was equally highly significant (p<0.001).
No obvious influence of gender on cytogenetic parameters was initially found in evacuees, butwhen children were excluded from the data analysis a statistically higher yield of chromatid exchanges
was observed in young adult males in compare with young adult females (p<0.05), and the latters also had
an increased outcome of poliploids in compare with three other age/gender subgroups (Fig. 2). The
possible reasons for those differencies, particularly active smoking status in young males and higher
intensity of endogeneous mutagenesis resulted from more reactive neuro-humoral response topsichological stress in young females, were discussed earlier [5].
The initial phase of the time course for unstable aberrations yields was compared between

Chromatid exchanges

Polyploids

Fig. 2. The yields of chromatid exchanges and poliploids in lymphocytes of adult evacuees samples
within 1 year after departure from the Chernobyl zone: 15 females (1) and 15 males (2) aged 23-35
years, 10 females (3) and 10 males (4) aged 36-66 років years. Y axis – cytogenetic damage
frequency per 100 cells. Vertical bars represent standard errors of the mean.
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Table 3. Chromosomal rearrangements in evacuees with different time of departure from the
Chernobyl zone
Time between Time between
explosion and evacuation andevacuation
blood sampling
4.2±0.8 d
2 days
0.79±0.03 y
2.20±0.14 y
5.7±1.2 d
3-11 days
0.66±0.05 y
2.07±0.17 y

Number of
persons
13
22
10
7
18
13

Mean aberration yield per 100 cells ±SE
Excess
Chromatid breaks
Dic+CR fr
acentrics
0.87±0.16
2.60±0.36
1.60±0.42
1.30±0.24
1.95±0.22
1.18±0.19
0.58±0.24
1.75±0.61
1.36±0.62
0.98±0.35
0.98±0.29 a
1.48±0.41
1.65±0.33
2.45±0.36b
1.82±0.31
0.87±0.29
1.83±0.40
3.47±0.57 а,bThe difference is statistically significant (р<0.05): a – between subgroups “2 days” and “3-11 days” at similar
time after exposure; b – in compare with the first time point inside the subgroup “3-11 days”.

evacuees with different time of departure from the Chernobyl zone (Table 3). During the first two years
after exposure chromatid break yield slightly declined in persons evacuated in 2 days after the accident but
increased in those who left the Chernobyl zone later. These changes appeared to be associated withchromosome acentrics behaviour that was expessed as elimination in the former evacuees’ subgroup andstability in the latter. The initial increase of dicentrics plus centric rings yields during 1 year after
irradiation appeared to be a common feature for all evacuees but was more pronounced in persons who
were evacuated from 3 to 11 days after the accident compared with those who left sooner; just 2 days after
the explosion, and this tendency could be traced during 2 years after exposure. As the duration of exposurein evacuees had such a remarkable influence on the aberration outcome, it was judged as a main factor tobe considered during biological dose estimating, in addition to the known role of exposure protraction inderiving an appropriate dose-response equation from a standard acute calibration curve for chromosomal
dosimetry [6].
The observation of significantly increased level of chromatid aberrations in evacuees, apart fromdetection of chemical impact, provided a serious implication for biodosimetry: the list of end-points
suitable for dose reconstruction must be restricted to dicentrics and rings only, with all excess acentrics
excluded, because the latter could be caused with quite high probability by chemical mutagens either
directly or as derived from chromatid breaks in lymphocyte precursors.
Biological dose estimations based on conventinal analysis data
To carry out the radiation dose estimation the individual dicentrics plus centric rings yields from
evacuees investigated during non-elimination phase of those aberrations dynamics (i.e. within 1 year postexposure) were pooled in two subgroups depending on when they left the exclusion zone – 2 days or 3-11
days (Table 4). In both groups, chromosome aberrations appeared at random amongst individuals, and theparameters of distribution were close to Poisson statistics that indicated an absence of persons
significantly overexposed in compare with others inside the groups. Thus, carrying out group
biodosimetry was methodologically correct.
According to the IAEA Chromosomal Biodosimetry Manual [6], mean doses for the evacuees
groups were calculated by referring their mean dicentrics plus centric rings yields to intralaboratory invitro dose response curve constructed for acute γ-exposure within low dose range [7], with quadraticcoefficient reduced by the Lea & Catchiside G-function, which takes into account the exposure duration.
Thus the dose response equation Y = с + α • D + β • G • D2 was solved using the following parameters: thespontaneous level of aberrations c=0.08 per 100 cells and linear term α = 2.98 per 100 cells per Gy forboth groups; initial acute quadratic term β = 8.05 per 100 cells per Gy2 was reduced to 1.5 per 100 cells
per Gy2 for persons evacuated in 2 days after the accident and to 0.52 per 100 cells per Gy2 for those who
left later (average departure time was 7 days after the explosion).

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Table 4. Individual dicentrics plus centric rings yields distributions and mean biological dose estimations in former citizens of t. Pripiat’ and 30-km
Chernobyl exclusive zone depending on time of their evacuation
Individual Dic+CR yield per 100 cells
Mean Dic+CR yield
Biological dose
Departure after the accident
0
1
2
3
4-5
per 100 cells
estimation, mGy(number of persons)
Number of individuals
2 days (35)
9
15
9
2
0
1.13±0.17300±29
3-11 days (25)
5
10
7
2
1
1.44±0.20420±39Total (60)
14
25
16
4
1
1.25±0.13
360±16

Table 5. Aberration levels measured by FISH technique in Chernobyl groups in compare with controls
GenomeAberration frequencies ±SE per 100 genome equivalents (actual numbers are given in parenthesis)Group
equivalents Dicentrics
Acentric
tcomp
tinc (Ba*),
Deleted(number of persons)tinc (Ab)tinc (BaMP)
Insertions
scored+Ringsfragments
chromosomes(Ba+ac)a(Ab+Ba)0.10±0.04
0.66±0.11
0.37±0.05
0.42±0.06
0.07±0.03
0.29±0.06
0.07±0.04
1.44±0.12
Controls
4088
(4)
(27)
(15)
(17)
(3)
(12)
(3)
(59)
(12)
Evacuees0.17±0.05
0.98±0.13
0.57±0.07
0.63±0.10
0.15±0.07
0.44±0.10
0.13±0.06
2.08±0.27
5282
(18)
(9)
(52)
(30)
(33)
(8)
(23)
(7)
(110)
Residents of RCA
0.10±0.03
0.71±0.12
0.29±0.05
0.44±0.07
0.10±0.03
0.21±0.05
0.09±0.037916
1.20±0.14 (95)
(21)
(8)
(56)
(23)
(35)
(8)
(17)
(7)
Spontaneous levels for inhabitants
0.10±0.04
0.54±0.16
0.22±0.02
0.27±0.02
0.00
0.18±0.02
0.02±0.01
1.17±0.04(mean age 21 yrs)b

RCA – radioactively conraminated territories. a – one tinc (Ba+ac) was detected in the control group, one in evacuees group and six in residents group. b – applying ageeffect regressions for complete translocations, insertions and deleted chromosomes and real yields of acentrics and incomplete translocations observed in a subgroup ofyoung control donors [6].

122The resulted dose estimate of protracted irradiation appeared to be 1.4 times higher in those
persons who were evacuated during the period 3-11 days after the accident that was well in line with
irradiation scenario. To the best of our knowledge, these are the only results of direct (i.e. early dicentric)
biodosimetry obtained for the Ukrainian Chernobyl evacuees in accordance with methodologicalrequirements of IAEA Manual [6]. A group of Belorussian children evacuated from contaminated areas
soon after the reactor explosion and examined cytogenetically in short time after exposure by authors [11]showed quite similar dicentric yields and similar resultant dose estimates to that of in our study.
However, any biodosimetry data limited to group mean dose only, whatever useful, seems to be
not informative enough for epidemiologists, who also need a picture of individual dose distribution within
exposed cohort. It turned to be a real challenge due to very large statistical uncertainty of individual dose
assessments occurred with low number of aberrations (up to 5) found in limited number of cells (typically,100) scored per person in early post-accident period. Moreover, the significant proportion of all cases
were those with zero dicentrics observed, so according to classic biodosimetry the zero should be set asmean radiation dose for such individuals, but statistics showed that with zero dicentrics in 100 cells we
can not exclude doses up to 0.5 Gy.
To solve this problem we applied a probabilistic approach, namely Bayesian analysis, that allows
looking at aberrations as stochastic events, which occur with some probability and moreover can bedetected (observed) also with some probability. The Poisson distribution of aberrations amongst cells seenin all exposed individuals in our study provided an opportunity to apply this function as a main part of apriori – a posteriori equation, which links the probability of existence of “true” number of aberrations inthe sample in case when any particular number of aberrations is observed. The “true” number ofaberrations divided by number of cells scored results into the “true” yield of aberrations, and that can beconverted into absorbed radiation dose using an appropriate dose response curve. Initially we applied thisapproach and accordant mathematical algorithm to the group of Chernobyl clean-up workers that showedvery satisfactory results [4, 10].
The probability distributions of absorbed dose were constructed for every evacuee in our study. It
was done in two variants: using dose response relationship for protracted exposure but also for acuteexposure. The later provided dose values which can be used in risk assessment without necessity ofcorrecting data for relative biological effectiveness of protracted irradiation. All individual distributions
were pooled together by combining the probability density in short dose intervals – 50 mGy, and in thisFig. 3. Probability density distributions of protracted and equivalently acute doses of γirradiation estimated using Bayesian analysis applied to cytogenetic data in evacuees from t.
Pripiat’ and 30-km Chernobyl exclusive zone.
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manner the total probabilistic distribution of protracted and equivalently acute radiation doses were finallyobtained (Fig.3).
Mean dose of protracted γ-irradiation estimated from the total probability density distribution
appered to be equal to that of obtained by classic biodosimetry data treatment. Modal protracted doses in
evacuees fall within 200-400 mGy. Probability density up to 1 Gy of protracted exposure was the mainpart of total dose range, but about 20 % of protracted doses in evacuees exceeded this 1 Gy limit. The
distribution of probability density for equivalently acute radiation doses within the interval from zero to
750 mGy appeared to be nearly symmetrical around modal values (tha same 200-400 mGy, as for
protracted doses), and 99.8 % of all probability density of acute doses was contained by the dose range up
to 1 Gy. Such data were in total agreement with clinical observations in those persons, particularly with
absence of acute radiation syndromes.
It should be noted, that physical dose calculations for citizens of t. Pripiat’, which represented thebasis for “official” opinion about radiation doses to that population, resulted into estimations of average
effective dose about 11.5 mSv, the maximum of individual dose about 114 mSv and 0.75 % of Pripiat’population exceeded the accidental permissible limit 50 mSv for public [19]. Obviously, these results wereobtained by modeling involved many assumptions concerning irradiation conditions, and some factors
seem to be not taken into account (e.g. remarkable external radioactive contamination of those evacuees),
therefore the mentioned dose values were in controversy to our results of cytogenetic dosimetry carriedout in the group of evacuees from t. Pripiat’. However, there is another set of physical dose calculations
for this category of persons affected by the Chernobyl [20], where the estimations of accumulated doses
appeared to be up to 1 Gy for inhabitants of certain villages around the reactor, and that was well in excess
of estimates in publication [19], but in a good agreement with our data presented here.
Cytogenetic data and biodosimetry estimations resulted from the FISH assay
Regarding the debates around true radiation doses to evacuees from Chernobyl, who (in contrast
to clean-up workers) were non-monitored by physical dosimetry, and for whom modeling dosimetry data
appeared to be unsure, the necessity occurred for additional verification of chromosomal biodosimetry
results. From the studies of time course of conventionally scored aberrations (see Fig. 1) it became
obvious, that any attempts of applying the conventional cytogenetics based on unstable aberrations
analysis several years after the accident wouldn’t give sufficient results for biological dosimetry due toelimination of cells carrying dicentrics from the circulating lymphocyte pool [3, 6, 8]. Therefore thealternative approach based on stable chromosome rearrangements quantification should be used for
estimating the yield of cytogenetic damage in lymphocytes of exposed persons. To solve this task, the lastgroup of evacuees amongst five mentioned in Table 1 (12.8-14.8 years after exposure) was examined byboth conventional cytogenetic analysis and FISH technique, specifically applied for stable aberrations
visualizing. Simultaneously, a group of young residents of radioactively contaminated areas of Belarus’
was also surveyed by FISH to check whether this method can serve as biodosimetry tool for quantifying
low doses of not past, but long lasted chronic exposure.
Actual numbers of unstable and stable chromosome aberrations and their yields per 100 genome
equivalents in studied groups are shown in Table 5. Cytogenetic parameters in evacuees were compareddirectly to those of in total control group due to similarity of mean age values. The spontaneous levels oftranslocations, insertions, acentrics and deleted chromosomes for inhabitants were calculated using ageeffect regressions generated for our control group earlier [21]. Metaphases with complex rearrangements
were rare and all aberrations from the complexes (apart from insertions) were included into appropriate
columns of Table 5. Individual aberration yields were randomly distributed in consistence with Poisson
statistics within both groups.
The average levels of dicentrics plus centric rings and acentric fragments in evacuees wereslightly increased above control, but no statistical difference was observed (р>0.05). That was in a very
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good agreement with cytogenetic effects outcome measured by conventional method, confirming again
the fact of elimination of lymphocytes with unstable aberrations from the circulating pool during years
post-irradiation. The level of unstable aberrations in residents was even more close to spontaneous values
than that of in evacuees that probably reflected very low dose rates during their living in contaminatedareas.
In contrast to unstable aberrations, the average yields of stable rearrangements were markedly
increased in exposed groups above control. However that was related mainly to exchanges, but not todeleted chromosomes. This type of chromosomal abnormalities was rarely reported to be measured in
exposed persons, because they obviously resulted from the lost of chromosome acentrics or chromatid
breaks during mitosis of the lymphocyte precursors, where fragments were initially induced. Both types of
unstable aberrations have no exclusivity to radiation, so a low sensitivity of deleted chromosomes as an
exposure marker after protracted or chronic irradiation to low doses could be concluded. Unlike deleted
chromosomes, the stable chromosome exchange yields were increased above control, and the meaningful
difference with spontaneous level occurred for tcomp in evacuees (р<0.05), tincАb, tincBa* plus tincBa+ac andinsertions in inhabitants (p<0.05-0.01). The total level of incomplete translocations was statisticallyelevated in both exposed groups (p<0.01-0.001). Noteworthy, the sum of incomplete translocations was
1.9-2.6 times higher than the level of complete ones. The ratio of tincАb to total tincBa translocations had
similar values in evacuees (1 : 0.9) and residents (1 : 0.7).
The cytogenetic parameter applied for biological dosimetry was the yield of stable exchanges withactual or assumed full presence of chromosomal material in “stable” cells. That represented a combinationof complete translocations, insertions, incomplete translocations tincBa* and virtual proportion of tincАb
involving unshortrened chromosome – tincАb*. The reasons of choosing this particular combination ofparameters instead of total translocation level for practical purposes of retrospective biodosimetry were
fully explained earlier [7].
Particularly, it was suggested that incomplete translocations with “missing part” had no
exclusivity to ionising radiation and may occur as a result of segregations of chemically-induced balanced
chromatid exchanges in dividing lymphocyte precursors [22]. The presence of increased level ofchromatid exchanges in post-Chernobyl critical groups (including residents of contaminated areas) was
reported in several independent studies [17, 18], and particularly for evacuees it can be seen in early
conventional analysis results presented here. Thus for preciseness of radiation exposure detection the data
analysis in Chernobyl groups has to be restricted to chromosome exchanges in cells with full presence ofchromosomal material. Additionally, incomplete translocations accompanied by acentric fragments werealso withdrawn due to instability of their wholeness during mitotic divisions of lymphocyte precursors.
Elimination of acentrics would result in a lack of genetic material following by cell death or arising ofincomplete translocations with “missing part” in daughter lymphocytes. It should be noted, that amongst
three exposed groups a significant number of tincBa+ac was detected only in inhabitants of radioactively
contaminated areas that obviously reflected the radiation induction of this type exchanges directly inmature cells during lymphocyte lifetime. Therefore, the cytogenetic assessment of radiation doses
accumulated years ago or during long term chronic exposure has to be based on the yield of chromosome
exchanges formed without accompanying acentrics. Amongst incomplete translocations these were
unshortened chromosomes with joined counterstained material that probably represented reciprocal
exchanges involving a small telomeric region beyond the limits of visual resolution by FISH. Aassuming
the identity of the mechanisms of tAb and tBa exchanges formation, the yield of tincАb* was calculated bymultiplying the number of total tincАb by the respective fraction of tincBa* within total tincBa in studiedgroups. The obtained values were rounded off to the integer numbers and applied for deriving the fullgenome yields of tincАb*, which was used for calculating the total yield of stable chromosome exchanges
as described above (the most full version of this approach was patented [23]).

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The calibration dose-response curve for the mentioned end-point was constructed in vitro within alow dose range (up to 1 Gy) and fitted to a linear-quadratic model [7]. Taking into account the protracted
exposure conditions in both Chernobyl groups the biological dose assessment was performed using only
the initial linear slope of the curve, that was expressed by equation Y = c + α • D, where α=1.401 per 100
genome equivalents per Gy, and the background incidence of aberrations for evacuees was established in
the total control group (c=0.55±0.09 per 100 genome equivalents), and that for young residents wascalculated from the empirically generated regression YSp=0.11+2.68 • 10-4 •А2, where A is age in years(for A=21 years c=0.23±0.11 per 100 genome equivalents) [7, 21].
The mean yields of stable exchanges of 0.97±0.14 per 100 genome equivalents in evacuees and
0.44±0.06 per 100 genome equivalents in residents corresponded to protracted dose estimations about
300±130 mGy and 150±90 mGy, respectively (errors for mean doses were calculated applying Poisson
standard errors for the excess of aberration level above control).
The results of retrospective FISH biodosimetry in evacuees were in a good agreement with earlydose estimates based on conventional aberration scoring (see Table 4). In residents of contaminated areas
the yield of dicentrics measured by FISH technique appeared to be unsuitable even for distinguishing
exposed group from the control. It is well known that chromosomal biodosimetry utilizing unstable
aberrations in chronically irradiated persons is a highly challenging task, which particularly can be solved
using an elegant approach suggested by M. Sasaki [24]. We were happy to succeed with FISH-detected
stable chromosome exchange yield, which provided another tool for identification of the exposed
population and quantitative measurement of the low radiation dose.
From data present in literature concerning dose estimations by FISH analysis in Chernobyl
cohorts one can see that biodosimetry performed by other authors were based on measuring the yield of
either complete translocations alone or total translocations (including those with missing part ofchromosomal material) [25-33]. Our approach with splitting translocations into “full presense ofchromosomal material” and “missing part” categories seems to be unique in application to biodosimetry invivo. Therefore our data have to be better compared with other authors’ results regarding totaltranslocation levels or, even better, overspontaneous excess for this end-point in Chernobyl groups, rather
than for dose estimations made by other laboratories. The literature analysis showed that in general our
results of measuring the translocation yields were in a good agreement with other data presented forsimilar categories of persons exposed to ionizing radiation due to the Chernobyl accident [25, 28, 32, 33].
Thus, in total, retrospective FISH biodosimetry confirmed our early assessments of radiation doses
accumulated by common population due to the Chernobyl NPP accident.
Conclusions
The cytogenetic survey carried out in population exposed to ionising radiation due to thecatastrophe at the Chernobyl NPP showed the significantly increased level of unstable chromosomal
aberrations in blood lymphocytes of individuals sampled early after the accident, and also the presence ofmarkedly increased yield of stable chromosomal rearrangements was detected by FISH technique latetime after irradiation. The time-course changes of chromatid type aberrations, chromosome type fragments,
hyperploidy and polyploidy levels in evacuees were displayed as a gradual decline of chromosomal
rearrangements and genome abnormality frequencies from the statistically elevated level in the first 1-2
years after the accident to the subcontrol meanings at the end of the 14-years period. The increased levelof these cytogenetic damages indicated the role of the combination of mutagenic factors acted in theaccidental situation at Chernobyl zone.
In Ukrainian evacuees from the Chernobyl zone both early dicentric assay and late FISH
translocation measurement resulted in very similar mean dose estimates in range 300-400 mGy of
protracted γ-irradiation. The yield of stable chromosome exchanges in Belorussian inhabitants ofradioactively contaminated areas corresponded to doses of chronic exposure about 150 mGy.
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According to our data, both evacuees from the 30-km Chernobyl exclusive zone and residents ofradioactively contaminated areas should be considered as a category of exposed persons, who’s radiation
doses (especially if expressed in acute exposure equivalents) were below the threshold of induction ofdeterministic radiation syndromes, but high enough for expecting an increased risk for late effects
occurrence on population level. Thus, development of advanced technologies in chromosomal analysis
and, more importantly, gradual improvement of methodology of cytogenetic data interpretation allows
obtaining meaningful practical results for biological dosimetry of past and chronic radiation exposure to
low doses. Therefore medico-biological observations in Chernobyl groups have to be continued and earlydata can also be retreated and discussed using knowledge and experience obtained during post accident
period by various scientific groups throughout the world.
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