Study of Genetic Effects in Somatic Cells of Children Living on the ContaminatedTerritories in Belarus
Ludmila S. MIKHALEVICH
Institute of Genetics and Cytology, Academy of Sciences of Belarus
F.Skorina st. 27, 220072 Minsk, Republic of Belarus
Fax: +375-172-684917, e-mail: igc%bas32.basnet.minsk.by@demos.su
Due to the Chernobyl APS accident in April 1986, aseries of serious radiological consequences werebrought in Belarus: the pollution of her territory withradionuclides with gamma-, beta- and alpha-radiation,and “hot” particles found in human organisms [1]; thehigh irradiation doses obtained by children in first
period after the catastrophe; the chronic influence ofcomplex of radionuclides with various biologicaleffects on the organisms for many years. Constantexternal and internal exposure and their combined
action on organisms and their cells, including theblood formation system, required the development of
new approaches to evaluate mutation process inlymphocytes of peripheral blood of children [2].
The application of the conventional cytogenetic
method analysing chromosomal aberrations in
metaphase was necessary in the first stage of
examination of children population in 1986-1990
because it enabled to apply the biological dosimetry to
the estimation of radiation doses of individuals andgroups. In the course of time after the catastrophe, theinformation obtained by this method has decreased.
Besides, by this method of metaphase analysis, it isdifficult to study simultaneously both thechromosomal aberrations induced in a series of cellular
generations of lymphocytes and the hereditary
cellular-lethal effect, comparing the level ofchromosomal aberrations with that of gene mutations
based on comparatively small volumes of children
blood. We proposed and proved a new scheme
applying micronucleus technique to cytogenetic
examination. This method permits to take into accountall the above mentioned deficiencies of theconventional method [2].
We organised genetic monitoring of Belarus
children in the following two ways:
1. The monitoring (individual and group) was carriedout with the children in the same settlement (forexample, Bragin), which made possible to observe
the dynamics of mutation process in peripheral
blood cells.
2. The genetic examination (individual and/or group)
was carried out with the children in several
settlements with different radioecological situationin the same period of time (as it was in Komarinand Malejki settlements of the Bragin district),which made possible to fulfil comparative analysis
of mutation frequency.

1. Dynamics of in vivo mutation in lymphocytesof peripheral blood of the children in Bragin
town (1988-1994)
At the first stage of cytogenetic examination, bloodsmears were taken from the children in theradiocontaminated zones in order to study
non-proliferating lymphocytes in peripheral blood.
Presently, this method of blood analysis is one of themost popular laboratory tests used at the screeningstage without the process of cell cultivation. The
structural changes of lymphocyte populations obtainedby this method is considered a high-sensitive index ofirradiation at low doses [3,4].
It is known that cell division is necessary for
micronuclei to be formed. Consequently, we canconsider that the frequency of micronuclei registeredin circulating blood lymphocytes is governed by thedamages of blood-forming cells in vivo, possibly, as aresult of genetic injuries on the level of stem cells
and/or preceding cells of hemopoiesis which develop
to recognizable cells of peripheral blood.
We have been investigating structural changes inlymphocyte populations such as the level of mono- andpolynuclear cells with micronucleus, number ofmicronuclei in them, as well as of disturbances inblood cell morphology in all three groups oflymphocytes; small, wide-cytoplasmic lymphocytes
and lymphocytes with plasmatization of cytoplasm,
which represent the polymorphism of these cells inchildren [6,7].
In this connection, since 1988, the cytogenetic
monitoring of children in Bragin town has beenorganized based on two age groups: the first group (1-7
years old) and the second (7-15 years old). It isnecessary to note that there were different
characteristics of the same age groups examined indifferent years. The young group, examined in 1988,
consisted of equal number of children born before andafter the Chernobyl catastrophe, whereas the old group(7-15) wholly was born before the catastrophe. The
children of 1-7, examined in 1990, constituted a mixed
group, and the children of 7-15 were born before thecatastrophe. The groups, examined in 1994, were
uniform: the children in the young group were bornafter the catastrophe, and those in old group - before
the catastrophe.

184

Thus, comparison of the results of cytogenetic
examination of children “by horizontal” within one
year and “by vertical” in dynamics (1988-1994) allows
to answer the question concerning the differential
sensitivity of hereditary material of children
lymphocytes in dependence of age, period of birth
before or after the accident, period of living onradiocontaminated territory, and others.

qualitative but not quantitative characteristics [5]. Just
in 1976, Countryman and Heddel introduced
lymphocytes as the next cellular indicator of
micronuclei in man [8].
After the Chernobyl catastrophe, papers appeared
which indicated the increased micronuclei level inlymphocytes of peripheral blood of the affected people[9]. However, uncertainty of information about the
spontaneous level of in vivo mutation in lymphocytesof human peripheral blood restrains the advance of
population-genetic investigations.
Therefore, it is quite natural to do special studies
and analysis to know the spontaneous level ofmicronuclei in lymphocytes of children peripheral
blood. So, we carried out cytogenetic studies of
spontaneous level of structural and quantitative
chromosomal injuries in lymphocytes of the childrenof two principal age groups, living permanently in
Minsk. In 1988-1990-1994, we examined 139 childrenof age from 1 to 15 years. The summary data is shownin Table 1.
The analysis of cytogenetic structure oflymphocytes of the children in Minsk has shown thatpolynuclear lymphocytes represent only a small
fraction of the whole lymphocyte population. In thisconnection, mononuclear lymphocytes have beenchosen as the principal object of study. A complicated1.1. Study of the spontaneous level of in vivo
mutation in lymphocyte population of peripheral
blood of the children in the control group
The quantitative and qualitative assessment of
cytogenetic effects of ionizing radiation is impossible
without the knowledge of spontaneous level of in vivo
mutation observed in lymphocyte populations ofperipheral blood of the Belarus children. The
spontaneous level of mutation not only determines thereliability of increase of induced structural mutations
in lymphocytes of the children in Bragin town andother areas, but also create the basis to carry out the
population studies with the given method.
The first information about the spontaneous
frequency of micronuclei in erythrocytes in peripheral
blood of healthy people as compared with their level in
ill patients was obtained by hematologists. The
principal attention in those studies was paid to theTable 1. Dynamics of in vivo mutation mononuclear lymphocytes of peripheral blood of the children
in Minsk city.

Year

Age groupof children*/number of
examined

Number ofanalysed
lymphocytes

1 (n=20)
2 (n=18)
1 (n=15)2 (n=20)
1 (n=15)2 (n=18)

8705
3226
3974
1863
1339
769

1988
1990
1994

Number of cells withmicronuclei

Number of
micronuclei incells

Distribution of cells by
the number ofmicronuclei

Absolute
number(AN)

х ± Sx(%)

AN.

х ± Sx
(%)

1

2

3

4

5

61
28
21
25
21
15

0.7±0.1
0.9±0.2
0.5±0.1
1.3±0.3
1.6±0.3
1.9±0.5

61
28
21
25
21
15

0.7±0.1
0.9±0.2
0.5±0.1
1.3±0.3
1.6±0.3
1.9±0.5

61
28
21
25
21
15

-

-

-

-

*:1 - the young age group of 1-7 years, 2 - the old age group of 7-15 years.

Table 2. Dynamics of in vivo mutation in binuclear lymphocytes of peripheral blood of the children in
Minsk city.
Agegroup*

Number of
Number of
binuclear cells
analysed
lymphocytes
AN
x±Sx

Number ofcells withmicronuclei
ANx±Sx

1(n=20)2(n=18)

8705
3226

105
64

1.2±0.1
1.9±0.2-1(n=15)2(n=20)

3974
1863

24
43

0.6±0.1
2.3±0.3-1(n=15)2(n=18)

1339
759

38
38

2.8±0.4
4.9±0.8

-

1988
1990
1994
-

Number of
micronuclei in
cells
AN
x±Sx

1

2

3

4

5

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

*:1 - the young age group of 1-7 years, 2 - the old age group of 7-15 years.

185

Distribution of cells by the
number of micronuclei

picture has been revealed for mononuclear
lymphocytes with micronuclei. First of all, we are tonote that only cells with one micronucleus were foundduring the years examined. However, the variations in
the frequency of micronucleus cells were observed.
The significant difference in the level of micronucleus
cells between the young and the old groups was foundonly in 1990, whereas in 1988 and 1994 the levels ofsuch cells were approximately equal among two
groups. On the other hand, growth of the frequency of
micronucleus cells was revealed with time in bothgroups.
There were no substantial differences between thechildren born before and after the Chernobyl accident.
We also could not find dependence of micronucleus
induction on sex.
Regretfully, among all the analysed of lymphocyte
populations in vivo, a considerable number ofbinuclear cells were found, which are extremely rare in
healthy children (Table 2). In 1988, a increasing
tendency of binuclear lymphocyte frequency wasobserved in the old children groups compared with theyoung group. Then this difference turned out
statistically significant in the following years. The
highest indices were revealed in the children born
before the Chernobyl accident. As is seen in Table 2,cells with micronuclei were not found among binuclear
lymphocytes. Thus, lymphocytes populations inperipheral blood of the examined children in Minsk ischaracterized by increasing tendencies of the numberof mononuclear cells with micronucleus and thenumber of binuclear cells in vivo.
As we noted earlier, there are a limited number ofinvestigations dedicated to the quantitative andqualitative in vivo assessment of micronuclei inlymphocyte populations of human peripheral blood.
The data on the spontaneous level of micronuclei inlymphocytes were obtained in other researches inwhich a sufficient number of adults were examined in
China. Thus, when 183 persons of both sexes in the ageof 45.6±13.9 were examined, 0.16±0.35 micronuclei
were found per 1000 analysed lymphocytes [10].
Consequently, approximately equal frequency of
micronuclei was registered both in the children of
Minsk and in the adults of China. However, unlike in
the Chinese population, our investigations discoveredhigh occurrence of binuclear lymphocytes.
Apparently, the peculiarities of mutation process inlymphocyte populations of the children in Minsk arelinked to the unfavourable radioecological situation.
In 1996, one more article was published in which invivo micronuclei analysis of peripheral lymphocytes
was used in combination with an application of the

FISH method to an adult group of various ages [11].
Nevertheless, due to the fact that the number of
examined people is insufficient, it is not clear whether
the levels of micronuclei and polynuclear cells dependon age, sex and other factors. However, the dataobtained in the present study can be used to estimate
the levels of spontaneous and induced mutation in
lymphocytes of peripheral blood of the Belarus
children.
1.2. Dynamics of in vivo mutation in lymphocytes of
peripheral blood in the children of Bragin town
The cytogenetic monitoring of the children of
Bragin town in 1988-1994 discovered certain
regularities in the dynamics of mutation process inlymphoid cells in vivo (Tables 3-5).
The most substantial results of them are as follows:
1. Significant increase of the level of cytogenetic
injuries, as compared with corresponding control
groups, in lymphocyte populations of peripheral
blood of the children of all age groups regardlessof their birth date (before or after the Chernobyl
accident); the number of polynuclear lymphocytes,
the levels of micronucleus cells, and the number ofmicronuclei in them.
2. Statistically significant differences from thecontrol, not only in quantitative aspects - thefrequency of mutation in lymphocytes, but also in
qualitative one - distribution spectrum by the
number of registered injuries in one cell.
According to the results of micronuclei analysis,
the common phenomenon in the children groups in
the radiocontaminated areas is the presence of
subpopulation of cells with several micronuclei 2, 3, 4 and more, while they are absent in thecontrol preparations. This can serve as a proof ofcontact of the given children with dense ionizing
radiation.
3. Significant difference in the frequency of in vivo
mutation in lymphocytes of peripheral blood
between in the children born before the Chernobyl
catastrophe and in the children born onradiocontaminated areas of Bragin town after thecatastrophe. The number of cytogenetic injuries
grew with the course of time (i.e. depended on theaccumulated dose), and in 1994 it increased byabout one order as high as in 1986.
4. The level of mutation frequency registered in thechildren born after the Chernobyl accident also
increased with the course of time, or with theincrease of accumulated dose. But it is in a lesser
scale than in the children born before the accident.

186

Table 3. Dynamics of in vivo mutation in mononuclear lymphocytes of peripheral blood of thechildren in Bragin town.
Year

Age group ofchildren*/number of
examined

Number ofanalysed
lymphocyte

Number of cellswith micronuclei
AN

x±Sx

Number of
micronuclei in
cells
AN
x±Sx

1 (n=23)4444
113
2.5±0.2144
3.2±0.2
control (n=20)8705
61
0.7±0.161
0.7±0.11988
2 (n=22)7802
248
3.1±0.1345
4.4±0.2
control (n=18)3226
280.9±0.228
0.9±0.2
1 (n=18)6371
272
4.2±0.2397
6.2±0.3
control (n=15)3974
210.5±0.1
210.5±0.11990
2 (n=17)16622
677
4.0±0.1942
5.6±0.1
control (n=20)1863
25
1.3±0.325
1.3±0.31(n=15)1707
65
3.8±0.495
5.5±0.5
control (n=15)1339
21
1.6±0.321
1.6±0.3
1994
2 (n=25)1331
323
24.2±1.1 561 42.1±1.3
control (n=18)769
15
1.9±0.515
1.9±0.5*:1 - the young age group of 1-7 years, 2 - the old age group of 7-15 years.

Distribution of cells by the
number of micronuclei
1

2

3

4

5

91
61
175
28
180
21
484
25
41
21
164
15

16
55
64
131
18
98
-

4
12
23
54
6
48
-

1
6
5
6
8
-

1
2
5
-

Table 4. Dynamics of in vivo mutation in binuclear lymphocytes of peripheral blood of the children in
Bragin town.
Agegroup*

Number ofanalysed
lymphocyte

Number of
binuclear cells
AN

x±Sx

Number of
Number of cells
micronuclei inwith micronuclei
cells
AN
x±Sx
AN
x±Sx

1988
1(n=23)
4444
477
24
33
10.7±0.6
0.5±0.1
C(n=20)8705
108
1.2±0.12(n=22)7802
756
789.6±1.0
0.9±0.1 109
C(n=22)3225
64
1.9±0.1
1990
1(n=18)6371
629
98
9.8±0.1
1.5±0.1 122
C(n=15)3974
24
0.6±0.12(n=17)
16622
1515
282
9.1±0.2
1.6±0.0 396
C(n=20)1863
43
2.3±0.3
1994
1(n=15)1707
64553.7±0.4
0.2±0.0C(n=15)1339
38
2.8±0.02(n=25)
1331
247
36
44
18.5±0.8
2.7±0.4
C(n=18)769
38
4.9±0.7*:1 - the young age group of 1-7 years, 2 - the old age group of 7-15 years.

Distribution of cells by the
number of micronuclei
1

2

3

4

5

0.7±0.1
1.3±0.1
-

16
50
-

7
25
-

1
3
-

-

-

1.9±0.1
2.3±0.0
-

78
199
-

17
61
-

2
15
-

1
5
-

2
-

0.2±0.0
3.3±0.4
-

6
29
-

6
-

1
-

-

-

Table 5. Dynamics of in vivo mutation in trinuclear lymphocytes of peripheral blood of the children in
Bragin town.
Number of
micronuclei in
cells
AN
x±Sx

1

2

3

4

5

83
16
-

1.8±0.1
0.2±0
-

83
6
-

5
-

-

-

-

39
97
-

0.6±0
0.5±0
-

21
51
-

6
14
-

2
6
-

-

-

1(n=15)1707
28
1.6±0.3
C(n=15)1339
2(n=25)1331
9087
6.7±0.6 84
5.5±0.6C(n=18)769*:1 - the young age group of 1-7 years, 2 - the old age group of 7-15 years.

81
-

3
-

-

-

-

Age
group*

Number of
Number of
Number ofcells withanalysed trinuclear cells
micronuclei
lymphocyte
ANx±Sx AN x±Sx1(n=23)C(n=20)2(n=22)C(n=18)

4444
8705
8702
3226

83
130
49

1.8±0.1
1.6±0.1
1.5±0.483
11-

1(n=18)C(n=15)2(n=17)C(n=20)

6371
3974
16622
1863

198
455
-

3.1±0.2
2.7±0.1
-

29
71
-

1988
1.8±0.1
0.1±0
1990
0.4±0
0.4±0
1994
6.3±0.6

187

Distribution of cells by the
number of micronuclei

5. Significant differences found in the level and thespectrum of cytogenetic injuries of bi- andpolynuclear lymphocytes in the children born
before and after the Chernobyl catastrophe. It isnecessary to stress that the significant increase ofpolynuclear lymphocytes in peripheral blood of thechildren in Bragin town is also connected with thefactors of the radioecological situation.
From our point of view, in vivo cytogenetic analysis
of lymphocytes with use of peripheral blood smears
becomes an important part in the scheme of screening
examination of the children in the polluted areas ofBelarus. As it is seen from the results of our study, themost suffering part of the children population is those
who were born before the catastrophe. So, they are justto be the principal object of investigation andrehabilitation activities.

2. Ex vivo study of mutation in lymphocytes of
peripheral blood in the children of the Bragin
district (1992)(plus- or minus-trend) during the process of theirmitotic division?’.
In the same day of March of 1992, blood was takenfrom 2 children groups of 12-13 years old in Komarin
town and Malejki village in the Bragin district of theGomel region. The blood was transported to Minsk andanalysed immediately [2, 12]. In accordance with the
Law of Republic of Belarus of 12 November of 1991,
the territories polluted as the result of Chernobyl
accident were divided into zones in dependence of thedensity of soil pollution with radionuclides and thedegree of radiation effect on man [1]. Both settlements
belong to the zone of consequent resettlement, i.e. thezone on which the density of pollution is from 15 to 40
Ci/km2 with Cs-137 or 2-3 Ci/km2 with Sr-90, and theaverage annual effective equivalent dose canoverreach 5 mSv per year (over the natural andtechnogenic background). The density of pollution of
territory of Komarin town is 3.0 (Cs) and 2.8 Ci/km2(Sr), and that of Malejki village - 9.0 (Cs) and 2.8
Ci/km2 (Sr) [14].

The application of micronuclei analysis ofperipheral blood lymphocytes under the condition ofcytokinetic block during cell cultivation allows to
distinguish clearly the cells which have not passed themitosis and the cells after first and consequent mitoses[2, 12]. It is known that PHA reagent used for cell
cultivation stimulates mainly small T-lymphocytes tocell division [13]. Therefore, by analysing effects on
non-stimulated lymphocytes in cell culture, we canlook into a heterogeneity of examined lymphocyte
populations as compared with the results obtained by
the smear method. Besides, the micronuclei registeredin mononuclear T-lymphocytes in cell culture are also
the result of their expression in process of lymphoidcell divisions in vivo [2].
In this connection, micronuclei frequencies inmononuclear and binuclear T-lymphocytes of
peripheral blood were examined in order to reveal theeffect of low doses of external and internal irradiation
as well as of their combined effects, comparing theresults of in vivo condition with those of ex vivo ones.
Besides, such approach allowed us to follow to somedegree the transgenerational somatic effect. Both theelimination of cells from circulating blood after the“acute irradiation” in the early post-accident period
and the appearance of new ones containing unstable
aberrations were registered in our experiments in aseries of cell generations of lymphocytes. As a result ofsuch analysis, we tried to get an answer to the question:“what is the trend of selection for micronucleus cells

2.1. Ex vivo study of mutation in stimulated andnon-stimulated T-lymphocytes of peripheral
blood of the children in Komarin town
The results of micronuclei registered inmononuclear lymphocytes of the children in Komarin
town are presented in Table 6. A significant
inter-individual variation was observed in thefrequency of micronucleus cells and the number ofmicronuclei in mononuclear cells. The individual
cytogenetic examination showed that only in 3
children(No. 2, 4 and 9) significant increase of
micronucleus cells and of micronuclei frequency per
100 analysed cells was observed. These indices did not
differ from the control values in the rest of children.
However, the group average indices of mononuclear
cells with micronuclei and the indices of micronuclei
per 100 cells in children of Komarin town overreachedstatistical significant level over the corresponding
indices in the control (Minsk city).
It is substantial that cells with multiple micronuclei
were found in the children of Komarin town, whichappear in in vitro conditions under the quite high doses
or the action of dense ionizing radiation. It is necessary
to note also that the cells with chromatin pulverization
were found in the half of examined children. The groupaverage index of pulverization constituted 1.6±0.2%.
Such phenomena were never observed in the control.
This may be considered as confirmation of theirelimination and appearance de novo.

188

Table 6. Individual analysis of ex vivo mutation in non-stimulated mononuclear lymphocytes of
peripheral blood of the children in Komarin town (1992).
No.
1
2
3
4
5
6
7
8
9
10
11
12
Group total
Control

Numberofanalysedcells

Number of cells with Number of micronuclei
micronuclei
in mononuclear cells

Distribution of cells by the
number of micronuclei

AN

x±Sx

AN

x±Sx

1

2

3

4

>4

1
8
8
13
6
4
3
6
3
4
4
60
27

0.99±0.98
2.75 ±0.96
1.60 ±0.56
2.60 ±0.71
1.20 ±0.49
1.32 ±0.65
1.03 ±0.592.10 ±0.85
0.86 ±0.49
2.61 ±1.29
1.14 ±0.57
1.63 ±0.21
0.79 ±0.15

1
17
10
16
6
5
3
6
4
4
4
76
29

0.99± 0.98
5.84 ±1.37
2.00 ±0.63
3.20 ±0.79
1.20 ±0.49
1.64 ±0.73
1.03 ±0.592.10 ±0.85
1.15 ±0.57
2.61 ±1.29
1.14 ±0.57
2.06 ±0.23
0.85 ±0.16

7
6
12
6
6
3
3
6
2
4
4
54
25

2
1
1
4
2

-

1
-

1(10)
1
-

101
291
500
500
500
304
55
292
286
349
153
351
3682
3405

1
-

-

Table 7. Individual analysis of ex vivo mutation in binuclear lymphocytes of peripheral blood of thechildren in Komarin town (1992).
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Group total
Control

Number of Number of cells withmicronuclei
analysed
cells
AN
x±Sx
18
90
500
274
307
237
15
79
52
288
52
328
117
500
95
2952
16631

6
18
17
7
8
1
4
5
11
5
4
6
11
7
110
199

6.67± 2.63
3.60± 0.83
6.20± 1.46
2.28± 0.85
3.38± 1.17
6.67± 6.44
5.06± 2.47
9.62± 4.09
3.82± 1.139.62± 4.09
1.22± 0.60
5.13± 2.04
2.20± 0.66
7.33± 2.68
3.73 ±0.35
1.20± 0.08

Number of micronuclei
in binuclear cells
AN
x±Sx
6
19
23
7
9
1
4
8
11
5
5
8
15
8
129
217

Distribution of cells by the
number of micronuclei
1
2
3
4
>4

6.67± 2.63
3.80± 0.86
8.39± 1.66
2.28± 0.85
3.80± 1.24
6.67± 6.44
5.06± 2.47
15.38±5.0
3.82± 1.139.62± 4.09
1.52± 0.68
6.84 ±2.33
3.00± 0.76
8.42± 2.85
4.37± 0.38
1.30± 0.09

5
17
13
7
7
1
4
4
11
5
3
5
9
5
98
18

1
3
1
2
1
1
1
8
-

1
1
-

1
1
1
3
-

-

Table 8. Comparison of group average indices of ex vivo mutation in non-stimulated (mononuclear)and stimulated (binuclear) lymphocytes of peripheral blood of the children in Komarin town (1992).
Nuclearity oflymphocyte

Numberofanalysedcells

Number of cellswith micronuclei
AN

x±Sx

mononuclear
binuclear

3682
2952

60
110

1.63± 0.21
3.73±0.35

mononuclear
binuclear

3405
16631

27
199

0.79± 0.15
1.20 ±0.08

Number of
micronuclei inbinuclear cells
AN
x±Sx
Komarin
76 2.05± 0.23
129 4.37± 0.38
Control
29 0.85± 0.16
217 1.30± 0.09

189

Distribution of cells by
the number ofmicronuclei
1
2 3 4
>4

Morphologicalchanges ofnucleus
AN
x±Sx

54
98

4
8

- 1
1 3

66 1.79 ± 0.22
66 2.24 ±0.27

25
181

2
18

-

-

1(10)
-

1
-

0.03
-

The results of micronucleus analysis of binuclear
lymphocytes of peripheral blood with use ofcytocholasin B in cell culture of the same children [12]are adduced in Table 7. As shown in the Table, thegroup average indices of micronucleus in binuclear
cells are approximately three times as high as thecorresponding indices in the control (the difference isstatistically significant). However, the results ofindividual cytogenetic examination indicate only atendency of increase of mutation level in binuclear
lymphocytes in the majority of children of this group.
Two children of the experimental group (No. 5 and 12)
did not differ in the mutation level from the control,and in one child (No. 9) the index of micronuclei rate
per 100 analysed cells was significantly higher than thecontrol.
Regretfully, the obtained material does not allow tocarry out an exact quantitative assessment oftransgenerational somatic effect and individual
sensitivity because the proliferative activity oflymphocytes of the children in Komarin town turned
out to be extremely low. However, the comparison ofgroup average indices of the level of micronucleus
cells indicate their significant increase in binuclear
lymphocytes as compared with mononuclear ones, i.e.
as a result of one cell division in culture (Table 8).
The fact of unusually large decrease of proliferative
response of T-lymphocytes to PHA is worth attention.
It was not observed in our experiments with thechildren in other settlements of the Bragin district withhigher indices of territory pollution with cesium andstrontium, such as Malejki village of the Bragin
district. This lymphocyte reaction is apparently
connected with the factors of radioecological situationof this settlement which is situated immediately close
to the Chernobyl power station. Besides, thepulverization of genetic material was discovered andmorphologically
reproductive
perishingoflymphocytes (1.5% of cells) was registered as well.
Taking into account these high indices of mono- andbinuclear cells perishing, we can suppose that a
considerable share of micronuclei registered inbinuclear cells arose de novo.

mononuclear lymphocytes of peripheral blood of theexamined children with the data obtained by analysing
this cell populations with in vivo smear method. As
shown in Table 9, the level of micronucleus
lymphocytes in vivo in the children in Malejki village
is 5 times as high as the corresponding indices in thechildren in Minsk city, and that of micronuclei in them- more than one order. In both cases the differences are
statistically significant.
The comparison of micronucleus frequency in exvivo non-stimulated lymphocytes of the children of theexperimental group with the control also showedsignificant increase. But it is only two times higher
than the control in this case.
Taking into consideration the presence, among theexamined in vivo and ex vivo lymphocyte populations,of cells with various morphological changes of their
nuclei (apoptosis, necrosis, pulverization and others),
in other words, the strong selection in minus-trend of
the micronuclei frequency at mitotic divisions, we can
suppose that the micronuclei analysed in binuclear
cells, like in the respective experiments withlymphocytes of the children in Komarin town, wereexpressed de novo.
Table 10 shows the data of individual cytogenetic
examination of 15 children in Malejki village. The
results of cytogenetic examination allow to divide thisgroup into 2 subgroups: the 1st subgroup consists ofthe children in which the frequency of binuclear cellswith micronuclei and of micronuclei in binuclear cells
are on the level of control, and the 2nd subgroupconsists of the children in which the lymphocytes havethe frequency of binuclear cells with micronuclei on
the level of control whereas the second index micronuclei number per 100 cells - is statisticallyhigher than the control.
The 1st subgroup is also unequal by the spectrum ofregistered micronuclei. In children No. 3 and 4, thecells with 3-4 micronuclei were registered among 500
analysed cells, the child No. 10 had one cell with 15
micronuclei among 1000 analysed cells.
It is necessary to note a change observed in the
results of cytogenetic examination of lymphocyte
populations of peripheral blood in the children of theBragin district. According to our ex vivo individual
examination in 1986-1988, 100% of children showedsignificantly increased levels of mutation. In 1992,
however, only in vivo analysis at the individual level
allowed to register significant disturbances.
In theory of cytogenetic analysis, under thecondition of uniform exposure, the distribution ofchromosomal injuries in cells is considered to follow
the Poisson distribution [15]. The preliminary analysis
of our results showed that the hemopoietic tissue in the
majority of the children in Komarin and Malejki
received not uniform but non-uniform dose ofradiation.

2.2. Ex vivo analysis of micronuclei in stimulated
and non-stimulated T-lymphocytes of thechildren in Malejki village
At the cultivation of lymphocytes of peripheral
blood of the children in Malejki village, the contrary
picture was revealed referring to the proliferative
indices as compared with cell cultures of the children
in Komarin town. The proliferative response oflymphocytes to PHA was so high that we could analyse
only about 50 mononuclear among bi- and polynuclear
lymphocytes per each child. In this connection, we
could assess only the group average indices, and theresults are adduced in Table 9. However, we cancomplement the picture of mutation process in
190

Table 9. Comparison of group average indices of in vivo and ex vivo mutation in lymphocytes of
peripheral blood of the children in Malejki village (1992).
Nuclearity
Number Number of cellsofofwith micronuclei
lymphocytes analysed
cells
AN
x±Sx

Number of
micronuclei inbinuclear cells
AN
x±Sx

Distribution of cells by
the number ofmicronuclei
1
2 3 4 >4

Morphologicalchanges ofnucleusANx±Sx
584.76± 0.56

in vivo
MalejkiControl
Mononuclear
MalejkiMononuclear
Binuclear
Control
Mononuclear
Binuclear

1429

54

3.78± 0.50

156

10.91± 0.82

16

19

7

14

7945

57

6.72± 0.09

58

0.73± 0.10
ex vivo

56

1

-

-

-

-

-

1250
10000

27
126

2.16± 0.41
1.26± 0.1134230

2.72±0.46
2.30± 0.15

24
103

2
12

2

2

1(6)
7

23
23

1.84 ±0.38
0.23± 0.05

3405
16631

27
199

0.79± 0.15
1.20± 0.0829217

0.98± 0.16
1.30± 0.09

25
181

2
18

-

-

-

1
-

0.03
-

Table 10. Individual analysis of ex vivo mutation in binuclear lymphocytes of peripheral blood of
the children in Malejki village (1992).
No.

Number ofanalysed
cells

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Total
Control

500
800
500
500
1000
700
1000
800
500
1000
500
700
500
500
500
10000
7580

Number of cellswith micronuclei
AN

x±Sx

10
11
7
8
10
11
6
9
6
6
11
7
3
11
10
126
102

2.00± 0.63
1.38± 0.41
1.40± 0.52
1.60± 0.56
1.00± 0.31
1.57± 0.47
0.60± 0.24
1.12± 0.37
1.20± 0.49
0.60± 0.24
2.20± 0.66
1.00± 0.38
0.60± 0.34
2.20± 0.66
2.00± 0.63
1.26± 0.11
1.35± 0.13

Number of
micronuclei inbinuclear cells
AN
x±Sx
63
13
10
12
10
39
8
10
6
20
20
7
3
13
13
245
109

12.60±1.48
1.62± 0.45
2.00± 0.63
2.40± 0.68
1.00± 0.31
5.57± 0.87
0.80± 0.28
1.25± 0.39
1.20± 0.49
2.00± 0.44
4.00± 0.88
1.00± 0.38
0.60± 0.34
2.60± 0.45
2.60± 0.45
2.45± 0.15
1.44± 0.14

Thus, the results of cytogenetic analysis of thechildren in the Bragin district testify at present that,apparently, the leading dose-forming factor is internal
irradiation with complex of radionuclides including α-,β- emitters.

Distribution of cells by the number ofmicronuclei
1

2

3

4

>4

5
9
6
5
10
9
4
8
6
5
8
7
3
7
7
103
95

2
2
2
2
1
3
3
12
7

1
1
2
-

1
1
2
-

3(30)-1 c.
2(15)
1(15)
1(5)
7
-

chronic radiation at low doses. Therefore, beside of theindividual and group cytogenetic monitoring, it isimportant to pay attention to the problems of
mutagenesis connected with gene mutations.
The scheme of genetic examination of children,
proposed and developed by the Institute of Genetics
and Cytology of the Academy of Sciences of Belarus,
is based on the Norman’s method [16] which detects
mutations
in
locus
of
hypoxanthine-guanine-phosphoribosiltransferase(HPRT).
The short-term Norman’s method was chosen for
detecting mutant T-lymphocytes due to the next
reasons: the potential possibility to apply it to themonitoring of populations because of its relative
simplicity and lesser cost (as compared with othermethods of gene mutations account), the possibility to

3. Genetic examination of the children in
Komarin and Malejki settlements of the Bragin
district of the Gomel region
The ionizing radiation causes a whole range ofgenetic changes in somatic and sex human cells: genemutations, chromosomal aberrations, and genome
mutations. In this connection, the assessment ofgenetic risk of the children living on theradiocontaminated territories in Belarus depends onthe completeness of study about genetic effects of191

Table 11. Individual data of the frequency of TG-resistant lymphocytes in cells culture of the children
in Komarin town of the Bragin district
Group/child No.

Number of TG-resistant cells

Control
Komarin
1
2
3
4
5
6
7
8
Group average

6.1 * 10 - 6
260.2 * 10 - 6
211.2 * 10 - 6
485.9 *10 - 6
315.8 *10 - 6
214.3 *10 - 6
374.0 *10 - 6
410.7 *10 - 6
306.4 *10 - 6
314.4 *10 - 6

Table 12. Individual data of the frequency of TG-resistant lymphocytes in cells culture of thechildren in Malejki village of the Bragin district
Group/child No.

Number of TG-resistant cells

Control
Malejki
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Group average

6.1 * 10 - 6
2.2 *10 -5
4.2 *10 - 5
3.6 *10 - 4
8.0 *10 - 5
7.4 *10 - 5
1.6 *10 - 4
1.6 *10 - 4
1.8 *10 - 4
6.8 *10 - 5
5.8 *10 - 5
1.0 *10 - 3
3.9 *10 - 4
3.8 *10 - 4
1.6 *10 - 4
7.4 *10 - 5
2.2 *10 -4

radiocontaminated zones with the index in the control
group of donors in Minsk city reveals substantial
excess of mutant cells in the experimental group(approximately two degrees).
The obtained results conform the data by otherauthors of the examination of persons irradiated as aresult of the accident in Goiania (Brazil) in September
1987. The conditions of irradiation in Goiania werequite similar to those of some population groups due tothe Chernobyl catastrophe. In both cases, the people
were subjected to the external and internal irradiation.
The papers of Natarajan et.al. [17, 18] indicate theincrease of TG-resistant lymphocytes in irradiatedpersons 10 to 100 times higher as compared with thecontrol group.
Thus, the results of genetic examination of thechildren of 12-13 years old who live permanently on
radiocontaminated territories of Komarin and Malejki
settlements in the Bragin district of the Gomel region
have shown a necessity and a possibility to evaluate
radiation dose to individuals and groups after many
years since the Chernobyl catastrophe, based on thefrequency of gene mutations in HPRT locus.

analyse simultaneously the gene mutations and themicronuclei as chromosomal aberrations analog, thepossibility to use a series of parallel cultures because of
the difficulty to obtain from children big volumes ofvein blood as well as the possibility of full automationof research.
Tables 11 and 12 present the results of TG(thioguanine) -resistant induction in lymphocytes inperipheral blood of the children in Komarin andMalejki settlements as compared with these indices in
the control group of Minsk city.
As are shown in the results of Tables, the frequency
of TG-resistant T-lymphocytes in the control groupconstituted 6.1 x 10-6 cells. In 100% of examinedchildren from the given settlements, the level of mutant
lymphocytes was significantly differing from thecontrol. The significant quantitative inter-individual
variations were also observed within the groups of
examined children. So, the number of TG-resistantlymphocytes in the culture of cells of the children in
Malejki village varied from 2.2 x 10-5 to 1.0 x 10-3, thatof the children in Komarin town - from 2.1 x 10-4 to 4.8
x 10-4. The comparison of group average index of
TG-resistant lymphocytes frequency in the children in

192

Conclusion

Literature

The general conclusion of our study, which has tobe addressed at first, is the seriousness of discovered
genetic disturbances in the examined children in
Bragin town and other settlements in the Bragin
district of the Gomel region.
The results of seven-year monitoring of children
with use of in vivo micronucleus analysis oflymphocytes have shown that the highest level ofmutation was found in the children born before theChernobyl catastrophe. Consequently, the principle ofradiation protection according to the level of averageannual radiation dose is not acceptable to protect thechildren in the Bragin district because it does not take
into account the total radiation dose since 1986 which
conditions the radiation consequences for children
health.
The analysis of the results of 1988-1994 indicates
that, under the chronic action of ionizing radiation,complicated interactions between mutation pressure
and selective process against cells with genetic injuries
have been taking place in lymphocyte populations of
the children in the Bragin district. Substantial
differences between the examined children and thecontrol were found in the level of mutations registeredin peripheral blood lymphocytes both in vivo and ex
vivo. The micronuclei level in lymphocyte populations
in vivo did not decrease during 1988-1994. On thecontrary, it increased approximately one order,
whereas one mitotic division ex vivo in cell cultureindicated substantial changes in different trends.
The cells with gene mutations capable to continue
their life activity, apparently, undergo the selection inminus-trend to some extent but, probably, also
contribute to the plus-trend selection both in vivo andex vivo. As a result, in the last years we observe in exvivo examination the high level of gene mutations
against the background of relatively low level ofchromosomal injuries.
The results of genetic monitoring of the children
subjected to the long-term non-controlled radiation
action with different intensity and duration in the
Bragin district have shown that the frequency of genemutations in HPRT locus can be a highly efficientbioindicator to evaluate individual and group
irradiation. The obtained results confirm a principal
necessity to carry out genetic monitoring of Belarus
children with obligatory use of this method to accountgene mutation . Simultaneous registration of mutation
spectrum and frequency obtained with in vivo and exvivo examination of peripheral blood lymphocytes inchildren will give more reliability to the bioindicator ofchronic radiation effects.

1. Consequences of Chernobyl APS Catastrophe for The
Republic of Belarus//Chernobyl Catastrophe: Causes andConsequences. Part III. in Russian. Minsk, “Skaryna”, 1992.
P. 207.
2. Fenech M., Perepetskaya G.A., Mikhalevich L.S. - A More
Comprehensive Application of The Micronucleus Techniquefor Biomonitoring of Genetic Damage Rates in Human
Populations - Experiences from the Chernobyl Catastrophe.
Environmental and Molecular Mutagenesis (in press). 1997.
3. Bebeshko V. G., Klimenko V. I. Clinical Aspects Of
Chernobyl Catastrophe. Blood Forming System//Chernobyl
Catastrophe. in Russian. Kiev, “Navukova Dumka”, 1995, P.
449-454.
4. Manual Of Radiation Hematology. in Russian. Ìoscow,“Meditsina”, 1974, P. 231-239.
5. Muller W.U., Streffer C. Advances in Mutagenesis Research.
5. Ed.by G.Obe. 1994.-Micronuckleus Assay.- P. 1-187.
6. Pavlova M.P. Manual of Hematological Diseases in Children.
in Russian.Ìinsk,“Vyshehjshaya Shkola”,1988, 271 p.
7. Mosyagina E. N., Torubarova N. A., Vladimirskaya E. B.
Diseases of Blood in Children. in Russian, Moscow,“Meditsina”, 1981, 179p.
8. Countryman P.I., Heddle J.A. The Production of Micronuclei
from Chromosome Aberrations in Irradiated Cultures of
Human Lymphocytes. Mutat. Res. 1986.- V. 41.- P. 321-332.
9. Chernobyl Catastrophe. In Russian. Kiev, “Navukova
Dumka”, 1995, 547 p.
10. Xue K.X., Wang S., Ma G.J. et al. Micronucleus Formationin Peripheral Blood Lymphocytes from Smokers and the
Influence of Alcohol - and Tea-Drinking Habits. Intern. J.
Cancer. - 1992.V. 50.- P. 702-705.
11. Surralles J.,Falk G., Norppa H. In Vivo Cytogenetic
Damage Revealed by FISH Analysis of Micronuclei in
Uncultured Human T Lymphocytes. // Cytogenetics And Cell
Genet.75.-N 2-3.-P. 151-154.
12. Fenech M.,Morley A.A. Measurement Of Micronuclei In
Lymphocytes // Mut.Res. 1985.-147.-P.29-36.
13.Evans H. Human Peripheral Blood Lymphocytes for the
Analysis of Chromosome Aberrations in Mutagen Tests //Handbook of Mutagenicity Test Procedures. 1984 .- P. 427.
14. Data On the Radioactive Pollution of Settlements of
Byelorussian SSR With Caesium-137 and Strontium-90 (byJune 1989). in Russian, Ìinsk, “Polymya”, 176 p.
15. Dolphin C.W. Handling of the Radiation
Accidents.1969.-Vienna.- P. 215-224
16. Norman A., Mitchell J.C., Iwamoto K.S. A Sensitive Assayfor
6-Thioguanine-Resistent
Lymphocytes//
Mut.Res..-1988.-208.- P. 17-19.
17.Natarajan A.T., Vyas R.C., Weigman J., Ñurado M.P. A
Cytogenetic Follow-Up Study of the Victims of a Radiation
Accident in Goiania ( Brasil ) // Mut.Res..- 1991.- 247 .- P.
103-111.
18. Natarajan A.T., Ramalho A.N., Vyas R.C., Bernini
L.F.,Tates A.D., Ploem J.S., Nascimento A.C.H., Curado
M.P. Goiania Radiation Accident : Results of Initial Dose
Estimation and Follow Up Studies // New Horisonts in
Biological Dosimetry. Progress in Clinical and BiologicalResearch .- 1991.- Vol. 372 .-P.145-153.

193