Tohoku J. Exp. Med., Micronuclei
2005, 205, among
1-9 Newborns and Environmental Contamination
1
Monitoring of Lymphocyte Micronuclei among
Newborns from Kragujevac in Central Serbia before
and after Environmental Contamination
OLIVERA MILOŠEVIĆ-DJORDJEVIĆ, DARKO GRUJIČIĆ, SLOBODAN ARSENIJEVIĆ1 and
DRAGOSLAV MARINKOVIĆ2
Faculty of Science, University of Kragujevac, 1Clinic of Gynecology, University
of Kragujevac and 2Faculty of Biology, University of Belgrade, Serbia and
Montenegro
MILOŠEVIĆ-DJORDJEVIĆ, O., GRUJIČIĆ, D., ARSENIJEVIĆ, S. and MARINKOVIĆ, D. Monitoring of Lymphocyte Micronuclei among Newborns from Kragujevac in Central Serbia
before and after Environmental Contamination. Tohoku J. Exp. Med., 2005, 205 (1), 1-9
── The air strikes on “Zastava” complex in Kragujevac, in the spring of 1999, caused extensive environmental pollution with damage to soil, water and air. Since the main problem was the leakage of several tones of polychlorinated biphenyls (PCBs) as well as heavy
metals Cr and Ni into the environment and groundwater, we decided to evaluate influence
of the environmental contamination on eventual changes of genetic constitution of human
body cells. The subjects analyzed were 36 phenotypically healthy newborn babies, who
were born 12 months (n = 22) and 18 months (n = 14) after environmental contamination,
and 25 newborns in 1998 as a control group. For the assessment of mutagenic effects of
environmental pollutants in vivo, the cytokinesis-block micronucleus test was employed.
The results show significant increase of micronuclei (MN) in lymphocytes from newborns
born 12 months after contamination (9.36 ± 5.60), in comparison to controls (5.53 ± 3.02)
and newborns born 18 months after contamination (6.14 ± 3.57). Only 3 newborns (12%)
out of 25 controls showed more than 1 MN/1000 binucleated cells in respect to 8/22 after
12 months (36%) and 2/14 after 18 months from contamination (14%). The mean number
of binucleated cells with MN significantly varied in function of sex and environmental
changes in newborns born 12 months after contamination (as demonstrated by the analysis
of covariance F = 9.61, p < 0.003). After 18 months of contamination, environmental
components had no noticeable effects on MN frequency (F = 0.5, p > 0.48). These results
suggest that the exposure to environmental pollutants in utero affects genetic constitution
of fetus and increases MN values in their body cells, which is detectable right after birth in
peripheral blood lymphocytes. ──── environmental contamination; micronuclei; newborns; peripheral blood lymphocytes
© 2005 Tohoku University Medical Press
Received June 1, 2004; revision accepted for publication October 27, 2004.
Address for reprints: Olivera Milošević-Đorđević, Institute of Biology, Department of Genetics, Faculty of
Science, University of Kragujevac, Radoja Domanovića 12, P.O. Box 60, 34000 Kragujevac, Serbia, Serbia and
Montenegro.
e-mail: [email protected]
1
2
O. Milošević-Đjordjević et al.
In the analysis of micronuclei (MN) the cytokinesis-block method described by Fenech and
Morley (1985) is used both as an in vitro test for
genotoxicity and as in vivo assay for biomonitoring of genotoxic effects in humans (Falck et al.
2002). MN are formed from acentric chromosome or chromatid fragments and whole chromosomes or chromatids that lag behind in anaphase,
and are left in telophase outside the dauther nuclei
(Catalan et al. 2000; Falck et al. 2002). In normal
lymphocyte cultures MN are detected at a low
rate, but their frequency can be increased. Most
of MN may result from in vivo exposure to a
genotoxin (Norppa et al. 1993). The ability of the
MN assay to detect both clastogenic and aneugenic effects (leading to structural and numerical
chromosomal aberrations) is an advantage of the
MN technique (Fenech 2000).
Kragujevac, a central Serbian industrial
town, is the home of the “Zastava” car factory.
The factory is located on the right bank of the
Lepenica River, a small tributary of the Velika
Morava, and the left bank of the Zdralica River.
In 1999, the air strikes on “Zastava” complex,
caused extensive environmental pollution with
damage to soil, water and air. In the fires caused
by the bombing, 95,000 kg of different materials
and goods and 2,200 kg of sealing mass were
burnt. The main problem was the leakage of several tones of polychlorinated biphenyls (PCBs) as
well as heavy metals Cr and Ni, into the Morava
River and contamination of groundwater by PCBs
(BTF Technical Assesment Mission). According
to Zastava personnel, up to 3,500 kg of oil containing PCBs were released into the environment,
and underground water tanks below the factory
were polluted with transformer oil containing
PCBs. By the feasibility study finalized in April
2000, 27 clean-up projects were identified to
address the post conflict environmental and
humanitarian problems. Five of this 27 Projects
were attributed to the remediation activities in
Kragujevac (UNEP/BTF, Feasibility Study 2000).
It is known that PCBs are stable pollutants
which can be detected in the environment including the air, water, fish, human adipose tissue,
blood, and breast milk (Safe 1984). They enter
human organism in several ways: through food
chain, by inhalation/breathing, by ingestion, or in
a direct contact with skin. PCBs cause both acute
toxicity and secondary effects (Toborek et al.
1995). Kalina et al. (1991) studied the ability of
PCBs to induce chromosomal aberrations and sister chromatid exchanges in peripheral lymphocytes in a group of workers occupationally exposed to PCBs during production of the
Czechoslovakian PCB products. PCBs induced
clastogenic effect in observed group. On the contrary, Belpaeme et al. (1996) did not observe a
significant increase of MN frequency in human
lymphocytes treated with PCBs in vitro. Kholkute
et al. (1994) studied effects of PCBs on in vitro
fertilization in the mouse. The results suggested
that higher dosages of PCBs affected fertilisation
and caused an increased incidence of degeneration of oocytes and abnormality in the early
mouse embryos.
Heavy metals are Cd, Hg, Pb, As, Cr, Ni, Fe,
Co, Cu, Zn (Wronska-Nofer et al. 1994; Stohs and
Bagchi 1995), and they can be detected in environment including the soil, air, water, food and
tobacco-smoke. Their half-life is very long (20-30
years). Heavy metals are cytotoxic, genotoxic
and carcinogenic. Benova et al. (2002) studied
the cytogenetic and molecular effects of Cr(VI)
on peripheral lymphocytes and exfoliated buccal
cells employing a chromosomal aberration assay,
sister-chromatid exchanges and micronucleus test.
The data obtained from exposed works showed a
significant increase in the number of cells with
MN in lymphocytes as well as in the buccal cells.
Cr(VI) has both clastogenic and aneugenic effects. Wise et al. (2003) reported that Cr(VI) had
genotoxic and cytotoxic effects in human lung
cells. Clemens and Landolph (2003) reported that
Ni had carcinogenic effect.
Exposure to genotoxic agents in the environment can produce a wide variety of effects on human chromosomes and human health. The main
purpose of the present study was to determine
MN frequency in peripheral blood lymphocytes
of newborns in Kragujevac, born before and 12 or
18 months after environmental contamination.
Micronuclei among Newborns and Environmental Contamination
MATERIAL AND METHODS
This study was approved by the Ethic Committee of
Clinic of Kragujevac and consisted of 61 phenotypically
healthy newborn babies observed at the Department of
Obstetrics and Gynecology in Kragujevac in 1998 and in
2000. Control values of MN frequency were obtained by
the analysis of peripheral blood lymphocyte cultures
from 15 boys and 10 girls who were born in 1998 before
environmental (n = 25). The same analysis was carried
out 12 months after environmental contamination (in the
beginning of 2000) for 14 boys and 8 girls (n = 22), and
18 months after contamination for 9 boys and 5 girls (n =
14). Each newborn baby was 1 day old. All their mothers were non-smokers, without any therapy during the
pregnancy, and were not occupationally exposed to
known mutagenic agents.
The cytokinesis-block method described by Fenech
(1993) was used in our cytogenetic investigation. Under
sterile conditions, 20 drops of whole heparinized blood
from each newborn were incubated in 5 ml of Parker 199
(Institute Torlak, Belgrade) with 15% fetal calf serum
(Sigma) and 3% phytohemagglutinin (PHA INEP,
Belgrade). All the cultures were set-up in duplicate and
incubated at 37°C, for 68 hours. Cytochalasin B (Sigma)
at a final concentration of 6 μ g/ml was added to the samples after 44 hours of culture incubation. Sixty eight
hours after culture initiation, cells were centrifuged, then
washed with 0.9% NaCl, and treated with hypotonic solution (0.56% KCl) at 37°C. Cell suspension was fixed
in methanol/acetic acid 3:1 for 2 times, and dropped onto
specially prepared, clean, cold and lamp-dried slides.
Slides were stained in 2% Giemsa (Alfapanon, Novi
Sad).
The MN frequency was determined by the analysis
of 1000 binucleated cells (BN) per subject. The obtained
results were compared by the application of Student,s
t-test. Analysis of covariance (ANCOVA) was used to
compare variation of MN in function of sex and contamination.
RESULTS
Among the control newborns, the mean value of MN in peripheral blood lymphocytes was
5.53 ± 3.02 , i.e. 4.91 ± 3.01 for males with the
variation range from 1 to 13 MN/1000 BN, and
6.48 ± 2.89 for females with the variation range
from 2 to 11 MN/1000 BN (Tables 1 and 3, and
Fig. 1). Only 2 males out of 15, and only 1 fe-
3
male out of 10 (total 12%), showed more than 1
MN/1000 BN.
The results of individual and mean values of
MN in newborns born 12 or 18 months after environmental contamination are presented in Tables
2 and 3, and in Fig. 1.
The mean value of MN in peripheral blood
lymphocytes of newborns born 12 months after
environmental contamination was 9.36 ± 5.60, i.e.
8.21 ± 3.93 for males with the variation range
from 1 to 15 MN/1000 BN, and 11.38 ± 7.63 for
females with the variation range from 2 to 23
MN/1000 BN. Among males 4 newborns out of
14, and among females 4 out of 8 (total 36%),
showed more than 1 MN/1000 BN.
The mean value of MN in peripheral blood
lymphocytes of newborns born 18 months after
contamination was 6.14 ± 3.57, i.e. 4.67 ± 2.24
for males with the variation range from 1 to 8
MN/1000 BN, and 8.80 ± 4.21 for females with
the variation range from 6 to 16 MN/1000 BN.
Only one baby out of 9 boys, as well as 1 out of 5
girls (total 14%), showed more than 1 MN/1000
BN.
There were statistically significant differences in total MN values when controls were compared with newborns born 12 months after contamination (t = 2.86, p < 0.01), as well as with
newborns born 12 and 18 months after environmental contamination (t = 2.11, p < 0.05).
Significant difference in MN frequencies was observed among control males and males born 12
months after contamination (t = 2.52, p < 0.05),
as well as among males born 12 and 18 months
after contamination (t = 2.74, p < 0.05). No significant difference in MN was observed among
analyzed females (p > 0.05) in all analyzed
groups.
The summarised results for analysis of covariance are presented in Tables 4 and 5.
ANCOVA for MN in control newborns and
newborns born 12 months after contamination
(Table 4) shows that the mean number of binucleated cells with micronuclei significantly varied in
function of sex and environmental changes (F =
9.61, p < 0.003). Between group variance was
much greater than within group (MSBg = 178.92).
4
O. Milošević-Đjordjević et al.
TABLE 1. Frequency of micronuclei in peripheral blood lymphocytes from control male and female newborns
in Kragujevac, analyzed in 1998
Distribution of MN
Newborns
Age
(day)
Sex
Analyzed
cells
Frequency of
MN/1000 cells
1MN
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
F
F
F
F
F
F
F
F
F
F
1000
1000
1000
1000
1000
1000
1000
1000
1000
385
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
685
1000
8
7
4
5
5
2
3
8
13
2.60
4
1
4
3
4
5
6
9
6
3
5
9
2
8.76
11
8
7
4
5
5
2
3
8
7
1
2
1
4
3
4
5
6
9
6
3
5
9
2
6
5
ANCOVA for MN in newborns born 18 months
after contamination (Table 5) shows no significant
difference in MN variation (F = 0.5, p > 0.48).
Within group variance is greater than between
group (MSwg = 9.13).
DISCUSSION
Genomic mutations are directly or indirectly
responsible for several abnormalities or human
health problems. Many of these mutations are expressed as defects, which become evident before,
or right after birth. The aim of biomonitoring
studies in humans exposed to genotoxicants is
primarily to identify individuals, or populations,
2MN
3
1
3
at increased risk of developing chronic diseases
(Bonassi et al. 1995).
The cytogenetic damage in exposed humans
has been reported by many authors and most of
the published reports are restricted to occupational
exposures to ionizing radiation (Maluf 2004),
heavy metals (Benova et al. 2002; Clemens and
Landolph 2003; Wise et al. 2003), and PCBs
(Kalina et al. 1991; Joksić and Marković 1992).
Spontaneous frequency of DNA damages are
influenced by many exogenous and endogenous
factors such as age, viral infections, radiation, the
presence of genotoxicants in food and in environment. Children are the most convenient subjects
Micronuclei among Newborns and Environmental Contamination
5
TABLE 2. Frequency of micronuclei in peripheral blood lymphocytes from newborns born 12 and 18 months
after environmental contamination
Distributions
After
Newborns
Age
(day)
Sex
Analyzed
cells
Frequency of
MN/1000 cells
1MN
2MN
12 months
1
2
1
1
M
M
1000
1000
11
7
7
7
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
M
M
M
M
M
M
M
M
M
M
M
M
F
F
F
F
F
F
F
F
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
3
15
11
9
11
7
11
6
11
1
3
9
20
23
10
10
15
9
2
2
3
15
8
5
11
7
11
6
11
1
3
5
18
21
8
10
13
9
2
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
1
1
1
1
1
1
1
1
1
1
1
1
1
M
M
M
M
M
M
M
M
M
F
F
F
F
F
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1
7
5
5
6
2
4
4
8
6
7
6
16
9
1
7
5
3
6
2
4
4
8
6
7
6
12
9
18 months
3MN
1
2
2
1
1
1
1
1
2
6
O. Milošević-Đjordjević et al.
TABLE 3. Incidence of micronuclei in peripheral blood lymphocytes from controls and newborns born after
12 and 18 months from environmental contamination
Newborns
Males (M)
Females (F)
Total (M+F)
N0
Incidence
of MN
(mean ± S.D.)
N0
Incidence
of MN
(mean ± S.D.)
N0
Incidence
of MN
(mean ± S.D.)
Before environmental
contamination
15
4.91 ± 3.01
10
6.48 ± 2.89
25
5.53 ± 3.02
12 months after
environmental
contamination
14
8.21 ± 3.93
8
11.38 ± 7.63
22
9.36 ± 5.60
18 months after
environmental
contamination
9
4.67 ± 2.24
5
8.80 ± 4.21
14
6.14 ± 3.57
Student’s
t-test
t = 2.86, p < 0.01a t = 2.11, p < 0.05b
t = 2.52, p < 0.05c t = 2.74, p < 0.05d
Statistically significant difference between total mean values of MN in newborns born before and after
12 months from environmental contamination.
b
Statistically significant difference in total mean values of MN between newborns born 12 and 18
months after environmental contamination.
c
Statistically significant difference in mean values of MN between males born before and 12 months
after environmental contamination.
d
Statistically significant difference in mean values of MN between males 12 and 18 months after
environmental contamination.
a
Fig. 1. Frequency of micronuclei (MN) in peripheral blood lymphocytes of newborns born before and
12 and 18 months after environmental contamination.
*
Statistically significant difference between newborns born 12 and 18 months after contamination,
with probability p < 0.05.
**
Statistically significant difference between control and newborns born 12 months after contamination, which probability p < 0.01.
for studying the effects of environmental pollution. They are not affected by occupational exposure, unhealthy life style (smoking, alcohol,
drugs), or by diseases of adults (Rossner et al.
2002). Whyatt et al. (1998) reported that air pollution had increased DNA damage more in fetal
Micronuclei among Newborns and Environmental Contamination
7
TABLE 4. ANCOVA for micronuclei in peripheral blood lymphocytes from controls and newborns born after
12 months from environmental contamination
Source
SS
df
MS
F
p
Adjusted means (Bg)
Adjusted error (Wg)
Adjusted total
178.92
1028
1140.43
1
58
59
178.92
17.74
9.61
0.003
TABLE 5. ANCOVA for micronuclei in peripheral blood lymphocytes from controls and newborns born after
18 months from environmental contamination
Source
SS
df
MS
F
p
Adjusted means (Bg)
Adjusted error (Wg)
Adjusted total
4.57
328.54
333.11
1
36
37
4.57
9.13
0.5
0.48
than in maternal tissues. They concluded that the
DNA repair efficiency in the fetus was lower than
in the adult, so that the fetus was more sensitive
to the effects at the level of a genotype.
The data presented here show significant increase of MN values in newborns born 12 months
after contamination (exposed in utero) in comparison with control newborns. Comparing the newborns born 12 and 18 months after contamination
a decrease in MN frequency was observed.
Our results show that the environmental pollutants increased the number of BN cells containing more than 1 MN/1000 BN. Only 3 newborns
out of 25 controls (12%) showed more than 1
MN/1000 BN, in respect to 8/22 newborns born
12 months after contamination (36%) and 2/14
newborns born 18 months after contamination
(14%). This kind of difference has been also observed in individuals exposed to ionising radiation
(Joksić et al. 1999).
If we assume that MN are biomarkers for exposure, including industrial chemical exposure,
and urban air pollution effects (Rossner et al.
2002), the observed changes in mean values of
MN may be explained by substantial changes in
living environment during the observed periods.
Our results showed higher mean values of
MN in females in all analyzed groups of newborns in comparison with males. Obtained results
are in agreement with those reported by other authors. Namely, Fenech et al. (1999) and Bonassi
et al. (2001) reported that incidence of MN increased with the age, and that sex had significant
influence. Females had 19% higher level of MN
frequency. Similar results were reported by
Joksić et al. (2003) who studied the effect of sex
on MN frequency in human-fetal lymphocytes.
They concluded that X-chromosomes played an
important role in the increase of MN. Carere et
al. (1999) showed higher susceptibility of malsegregation of chromosome X in comparison to
autosomes. Catalan et al. (2000) have concluded
that possitive correlation between the frequency
of aneuploid cells and micronucleated cells indicats that X and Y are the chromosomes preferentially incorporated into the MN. Norppa and
Falck (2003) reported that the X chromosome especially tended to lag behind in female lymphocyte anaphase, and micronucleated more efficiently than autosomes. Nath et al. (1995) concluded
that newborns had a very low MN frequency and
in MNs did not show Y chromosome (no Y +
MN).
The level of genotoxic damages was changed
in both sexes during the observed periods.
Significant increase of MN in males born 12
months after contamination, and decrease of MN
in males born 18 months after contamination,
were observed.
In females born after contamination mean
MN values were higher than in controls, but significant difference was not observed. After 18
8
O. Milošević-Đjordjević et al.
months from contamination the MN frequency
was decreased, but no to the level of control females.
Among the lymphocytes of newborns before
contamination, individual variation in MN/1000
BN varied from 1 to 13; in a sample 12 months
after contamination MN varied from 1 to 23, and
after 18 months it ranged from 1 to 16.
The mean number of MN varied in function
of sex and environmental changes. Analysis of
covariance demonstrated that MN significantlly
varied in function of sex and contamination in
newborns born 12 months from contamination in
comparison to controls (F = 9.61, p < 0.003). The
presence of greater between group variance of
MN indicates that investigated groups of newborns were exposed in utero to different environmental conditions. Statistically significant variation of MN in newborns born 18 months after
contamination was not observed (F = 0.5, p >
0.48). The presence of greater within group variance in this period coud be attributed to interindividual variation of MN in males and females.
Some studies have analyzed the effects of
environmental pollution on children. Rosival et
al. (1983) and Rosival and Trnovec (1999) reported that fetal exposure to chlorinated pesticides
was shown to correlate with embryonal measurements of chlorinated pesticides. Higher level of
placental contamination by organochlorine compounds was observed in an industrial locality in
comparison to similar or even higher placental
contamination by heavy metals in a rural area.
Reichrtova (1995) reported that the presence of
heavy metals in the placenta could be detected in
particular in the syncytiotrophoblast. This refers
also to other chlorinated substances (e.g. PCBs).
In conclusion, exposure to environmental
pollutants in utero affected genetic constitution of
fetal body cells. Right after birth, newborns have
a low MN frequency, but when fetus was exposed
to environmental genotoxicans in utero, their frequency increased. The present study provides the
evidence for significant genetic damage in newborns born after 12 months from contamination,
as judged by the increase of lymphocyte MN frequency. MN frequency varies in function of sex
and the effects of environment.
Acknowledgements
This work was supported by the Serbian Ministry of Science and Technology, Grant No. 1214.
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