International Journal of Hygiene and Environmental Health 216 (2013) 533540

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International Journal of Hygiene and

Environmental Health
journal homepage: www.elsevier.com/locate/ijheh

Health impact of air pollution to children

Radim J. Sram a, , Blanka Binkova a , Miroslav Dostal a , Michaela Merkerova-Dostalova b ,
Helena Libalova a , Alena Milcova a , Pavel Rossner Jr. a , Andrea Rossnerova a ,
Jana Schmuczerova a , Vlasta Svecova a , Jan Topinka a , Hana Votavova b
a
b

Institute of Experimental Medicine AS CR, Prague, Czech Republic

Institute of Hematology and Blood Transfusion, Prague, Czech Republic

a r t i c l e

i n f o

Article history:
Received 25 July 2012
Received in revised form
23 November 2012
Accepted 6 December 2012
Keywords:
PM2.5
Carcinogenic polycyclic aromatic
hydrocarbons
Pregnancy outcome
Respiratory morbidity
Asthma bronchiale
DNA adducts
Micronuclei
Transcriptomics

a b s t r a c t
Health impact of air pollution to children was studied over the last twenty years in heavily polluted
parts of the Czech Republic during. The research program (Teplice Program) analyzed these effects in the
polluted district Teplice (North Bohemia) and control district Prachatice (Southern Bohemia).
Study of pregnancy outcomes for newborns delivered between 1994 and 1998 demonstrated that
increase in intrauterine growth retardation (IUGR) was associated with PM10 and c-PAHs exposure (carcinogenic polycyclic aromatic hydrocarbons) in the rst month of gestation. Morbidity was followed in
the cohort of newborns (N = 1492) up to the age of 10 years. Coal combustion in homes was associated
with increased incidence of lower respiratory track illness and impaired early childhood skeletal growth
up to the age of 3 years. In preschool children, we observed the effect of increased concentrations of
PM2.5 and PAHs on development of bronchitis.
The Northern Moravia Region (Silesia) is characterized by high concentrations of c-PAHs due to industrial air pollution. Exposure to B[a]P (benzo[a]pyrene) in OstravaRadvanice is the highest in the EU.
Children from this part of the city of Ostrava suffered higher incidence of acute respiratory diseases in
the rst year of life.
Gene expression proles in leukocytes of asthmatic children compared to children without asthma
were evaluated in groups from OstravaRadvanice and Prachatice. The results suggest the distinct molecular phenotype of asthma bronchiale in children living in polluted Ostrava region compared to children
living in Prachatice.
The effect of exposure to air pollution to biomarkers in newborns was analyzed in Prague vs. Ceske
Budejovice, two locations with different levels of pollution in winter season. B[a]P concentrations were
higher in Ceske Budejovice. DNA adducts and micronuclei were also elevated in cord blood in Ceske Budejovice in comparison to Prague. Study of gene expression proles in the cord blood showed differential
expression of 104 genes. Specically, biological processes related to immune and defense response were
down-regulated in Ceske Budejovice.
Our studies demonstrate that air pollution signicantly affect child health. Especially noticeable is
the increase of respiratory morbidity. With the development of molecular epidemiology, we can further
evaluate the health risk of air pollution using biomarkers.
2012 Elsevier GmbH. All rights reserved.

Introduction
It is generally accepted that exposure to air pollution has
negative effects on human health including increased risk of mortality and morbidity from respiratory and cardiovascular diseases
(Dockery et al., 1993; Pope et al., 2002; Pope, 2007). It has been

Corresponding author at: Institute of Experimental Medicine AS CR, Videnska

1083, 142 20 Prague 4, Czech Republic. Tel.: +420 241 062 596;
fax: +420 241 062 785.
E-mail address: sram@biomed.cas.cz (R.J. Sram).
1438-4639/$ see front matter 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ijheh.2012.12.001

shown that long-term exposure to particulate matter (PM), a prevailing component of air pollution onto which other pollutants
are adsorbed, increases mortality from lung cancer, cardiovascular
and respiratory diseases (Pope et al., 2002; Pelucchi et al., 2009),
while acute exposure to PM is linked particularly to various cardiovascular events including hospital admissions due to myocardial
infarction and heart failure (Mills et al., 2009), as well as cardiorespiratory and all-cause mortality (Ren and Tong, 2008).
Analysis of biomarkers has been proposed as a method of
evaluation of biological effects from exposure to environmental
pollutants on human health (Castano-Vinyals et al., 2004; Moller
and Loft, 2010; Sorensen et al., 2003). Although the results from

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different laboratories vary depending on the method used, it

has been concluded that exposure to air pollutants (e.g. PM and
polycyclic aromatic hydrocarbons, PAHs) is associated with elevated levels of biomarkers, including PAHDNA adducts (Binkova
et al., 2007), oxidative stress markers (Moller and Loft, 2010) and
chromosomal aberrations (Sram et al., 2007). These data conrm
deleterious effects of air pollutants on human health and underline
the importance of biomarker analysis as a preventive measure in
populations living in polluted areas.
PAHs such as benzo[a]pyrene (B[a]P) are carcinogenic environmental pollutants resulting from incomplete combustion that are
commonly found in tobacco smoke, ambient and indoor air, and
charbroiled food. They are released to the air from local heating,
trafc and industrial sources. Exposure to PAHs represents a health
risk due to their mutagenic, genotoxic and carcinogenic activity,
which was detected in humans only during the last two decades.
They are metabolized to form phenolic products and reactive epoxides, which have the capacity to bind to DNA, forming PAHDNA
adducts (Binkova et al., 1995; Whyatt et al., 1998). Binkova et al.
(2003) observed, that the genotoxicity of respirable particulate
matter is related to the content of PAHs. A growing body of evidence suggests that exposure to B[a]P at levels over 1.0 ng/m3
induces DNA damage (WHO, 2010). Personal exposure to B[a]P over
1.0 ng/m3 predicted greater genomic frequency of translocations
(Sram et al., 2007), micronuclei (Rossnerova et al., 2009) and DNA
fragmentation in sperm (Rubes et al., 2010).
Effect of air pollution seems to be signicant to children, who
are more sensitive than adults as their organism is in the stage
of development. New knowledge about respiratory particles and
complex mixtures as represented by PAHs adsorbed on their surface and the use of biomarkers of exposure and effect during the
last twenty years were substantial to better understand how air
pollution may affect children health already from the beginning of
fetal life (Lewtas, 2007).
PAHs induce DNA adducts in placenta (Topinka et al., 1997).
It has been shown that PAHs can cross the placenta. Perera et al.
(2005) suggested that the developing fetus may be 10 times more
susceptible than mother to PAH-induced DNA damage. Prenatal
exposure to ambient air PAHs has been associated with intrauterine growth restriction (IUGR) and with increased likelihood of low
birth weight (Dejmek et al., 2000; Choi et al., 2008, 2012; WHO,
2010). B[a]P concentrations in umbilical cord blood were correlated
with reduced neonatal height and gestational age (Guo et al., 2012).
Birth weight may be also affected by consuming barbecued meat
(Jedrychowski et al., 2012). In a study of nonsmoking women from
Beijing detectable concentrations of PAHs in breast milk, placenta
and umbilical cord blood were found (Yu et al., 2011).

Teplice Program
Very specic pollution problem from the use of brown coal for
local heating and power plants in the late eighties in the Czech
Republic is of major health concern. It becomes the reason for an
international study, the Teplice Program. The study focused on the
impact of air pollution to pregnancy outcomes, sperm quality, bronchitis in children, children genome and the genomic frequency of
chromosome translocation.
The mining districts of Northern Bohemia were considered to
be one of the most polluted regions in all of Europe in the late
1980s, due to environmental pollution in the form of sulphur dioxide (SO2 ), nitrogen oxides (NOx ), polycyclic aromatic hydrocarbons
(PAHs) and heavy metals (Moldan and Schnoor, 1992).
Brown coal from open pit surface mines, very high in sulfur
and low in energy quality, was used in power plants to produce
energy for the industrialization of all Czechoslovakia. The impact

Fig. 1. Map of the Czech Republic.

of sulfur dioxide on forests was already recognized in the 1960s.

During the 70s the rst health effects that were suspected to be the
consequences of environmental pollution were recognized.
The rst signs of deteriorating human health in the mining districts of Northern Bohemia were related to an increase in allergies,
immunodeciencies and respiratory illnesses in children. Simultaneously, an increase in birth defects and the rising prevalence
of children with low birth weight was observed (Sram et al.,
1990; Sram, 1991); especially striking was the shortening of life
expectancy for inhabitants of this region as compared to the rest of
the country, especially in males with an increase in mortality rates
for cancer and cardiovascular diseases.
In November 1990, the Czech government initiated program to
improve the ecological situation in the mining districts of Northern
Bohemia. In this regard, the district of Teplice was recommended as
a model district for a newly established research program: Impact
of Air Pollution on Human Health (Teplice Program). The district of
Prachatice, a signicantly less polluted region in Southern Bohemia,
was chosen as a control district (Fig. 1) (Sram et al., 1996).
The main task of the Teplice Program has been to provide
scientically valid information on the impact of environmental
pollution, especially air pollution, on human health, particularly
on respiratory effects, behavioral endpoints, pregnancy outcomes,
mortality and cancer incidence.
Pregnancy outcome study
Pregnancy outcome study was followed on newborns delivered
in the period 19941998 on pregnancies in the district of Teplice
(polluted) and the district of Prachatice (matched). Teplice district
(approximately 120,000 inhabitants and 1100 births per year) lies
in the brown-coal basin of Northern Bohemia and has a chemical
industry, surface mining, and large coal power plants. Prachatice
district (approximately 50,000 inhabitants and about 450 births
per year) is an agricultural region in Southern Bohemia without
heavy industry and with moderate levels of air pollution. During the 4-year period, there were 3378 live births in Teplice, and
1505 in Prachatice (N = 4883). Detailed personal data were obtained
via questionnaires and medical records. Mean PM10, PM2.5 and
c-PAHs during the 9 gestational months were estimated for each
mother.
The results showed that concentrations of PM10 > 40 g/m3 in
the rst gestational month signicantly increased IUGR (Dejmek
et al., 1999). In addition, IUGR was increased when concentrations
of c-PAHs in the rst gestational month >15 ng/m3 (B[a]P 2.8 ng/m3 )
(Dejmek et al., 2000).
Morbidity in the Teplice-Prachatice cohort
Among the newborns from both districts, 1492 were followed
up to the age of 10 years for their morbidity:

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When 452 children were followed for 3 years to determine the

incidence of lower respiratory track illness, it occurred more frequently in homes heated by coal (RR = 1.45; 95% CI, 1.071.97), and
particularly in children not breast-fed (RR = 2.77; 95% CI, 1.455.27)
(Baker et al., 2006).
Ghosh et al. (2011) examined in the group of 1133 children followed from birth to age 36 months for relationship between the
indoor coal combustion for heating and early childhood growth.
Using z score for height for age and sex at age 36 months, they
observed a reduction of height about 1.34 cm (95% CI, 0.512.16)
for boys and 1.30 cm (95% CI, 0.502.10) for girls raised at homes
that used coal. Those results indicate that pollution from indoor
coal use may impair early childhood skeletal growth.
In 1133 preschool children up to 4.5 years, bronchitis rates
increased with rising pollutant concentrations. Below 2 years of
age, increments in 30-day averages of 100 ng/m3 PAHs and of
25 g/m3 PM2.5 resulted in RRs for bronchitis of 1.29 (95% CI,
1.071.54) and 1.30 (95% CI, 1.081.58), respectively; from 2 to
4.5 years of age, these RRs were 1.56 (95% CI, 1.222.00) and 1.23
(95% CI, 0.941.62), respectively. Ambient PAHs and ne particles
were associated with early-life susceptibility to bronchitis. Associations were stronger for longer pollutant-averaging periods and,
among children >2 years of age, for PAHs compared with ne particles (Hertz-Picciotto et al., 2007).
The marker of oxidative DNA damage 8-oxodeoxyguanosine
(8-oxodG) was investigated in the urine of 894 children aged
610 years. 8-oxodG was determined by ELISA. The exposure to
PM10 and PM2.5 as measured by stationary monitors during a 7day period as well as to c-PAHs as measured during 3-day periods
13 and 79 days were identied as factors affecting 8-oxodG levels in collected urine according to multivariate models. Short-term
exposure to ne particles and c-PAHs also induced oxidative stress,
and therefore may be a starting point for respiratory and allergic
morbidity in children (Svecova et al., 2009).
Rossner et al. (2011) studied associations between the levels of
8-oxodG in placental DNA, exposure to air pollutants during pregnancy, genetic polymorphisms in 94 selected genes, and pregnancy
outcomes in 891 newborns who were born between the years
19941998. Levels of 8-oxodG in placental DNA were associated
with the risk of IUGR as well as LBW. Newborns sex, gestational age,
maternal smoking, and genetic polymorphisms in the promoter
region of the MBL2 gene were associated with LBW incidence.
The frequency of micronuclei (MN, binucleated cells) in
peripheral blood lymphocytes (PBLs) was analyzed to assess the
cytogenetic effects in children and mothers living in the two districts. From each area, two groups of children from a total of 24
families (mean age: 6.0 0.6 and 9.0 1.2 years) in a total of 47
children and 19 mothers (mean age: 33.6 3.9 years) participated.
Signicantly higher frequencies of MN were found in the younger
children living in the Teplice area as compared with those living
in the Prachatice area (7.0 2.3 per thousand versus 4.9 2.0 per
thousand, p = 0.04). Higher levels of MN were also measured in the
older children and the mothers from the Teplice area (9.2 3.7 per
thousand versus 6.6 4.4 per thousand) and (12.6 3.4 per thousand versus 10.1 4.0 per thousand). The increased MN frequency
may be associated with elevated c-PAHs concentration of PM2.5
measured in the ambient air during winter (Pedersen et al., 2006).
The same groups of individuals were investigated for response
to air pollution, using genome-wide oligonucleotide microarrays.
For a considerable number of genes, the expression differed signicantly between the children from the two areas. When gene
expression was analyzed also in parents, more genes or gene groups
and pathways signicantly differed between children from both
regions than between parents. The two most important biological
processes or molecular functions that were signicantly modulated
in children were related to nucleosome and immune response (van

535

Leeuwen et al., 2006, 2008). These were two rst reports using
transcriptomics as a biomarker to air pollution and environmental
carcinogenesis.
Morbidity up to 10 years of age
Lists of all illnesses (using ICD-10 codes) from birth to 10 years
of age (the children were born in 19941998) were obtained from
the medical records of 960 children. Multivariate negative binomial
regression was used to compare the incidence of upper respiratory
infections (URI), bronchitis, laryngitis and tracheitis, pneumonia,
inuenza, tonsillitis and otitis media in children living in the town
of Teplice (urban environment, TE-1, N = 242), in the rest of the
district of Teplice (industrial environment, TE-2, N = 272), and with
those in the rural district of Prachatice (PRA, N = 446).
Children born and living in TE-1 had, up to the age of 3 years
(ys), a signicantly higher cumulative incidence of laryngitis and
tracheitis, pneumonia and otitis media, and a lower incidence of
bronchitis and tonsillitis than children in PRA. At older ages, the
most signicant differences were a higher incidence of laryngitis
and tracheitis and inuenza, and a lower incidence of URI and bronchitis in TE-1 than in PRA. The children living in TE-2 had a lower
rate of URI (410 ys) and of bronchitis (16 ys) than children in
PRA. Within the district of Teplice, there was a higher incidence
of laryngitis and tracheitis (110 ys) and inuenza (110 ys) and
a lower incidence of URI (16 ys) in TE-1 than in TE-2. The aggregate incidence of all respiratory illnesses (including otitis media)
of children in their 10th year of age was approximately 50% lower
than in children of preschool age, and did not exceed the value of
1.4 per child/year. Any relevant covariates included in the multivariate models did not confound the statistical signicance of the
association of area of residence with the incidence of respiratory
illnesses.
Inuenza, laryngitis and tracheitis occurred with the highest
incidence in children living in the urban environment of the town
of Teplice, and the incidence of upper respiratory infections was
highest in the rural environment of the district of Prachatice. Upper
respiratory infections dominated the aggregate morbidity, which
decreased with age in school children (Dostal et al., 2009).
Results of this cohort study shows that valuable new information can be obtained when the health of children is followed from
the time of pregnancy to the school age. When such study is related
to the air pollution, a necessary prerequisite should be the stationary monitoring of pollutants such as PM10, PM2.5, c-PAHs as well as
a cooperation with obstetricians and pediatricians, to get not only
questionnaires from mothers, but also medical records.
The hot spot of B[a]P exposure (Program Ostrava)
In the Northern Moravia Region (Silesia, regional city of
Ostrava), air pollution came especially from industrial sources
as steel industry, coke-oven. It differs from all other parts of
country by high concentrations of c-PAHs, e.g. in the year 2011,
PM2.5 concentrations were 50100% higher than in Prague,
but concentrations of B[a]P in OstravaPoruba (which is understood as a clean part of Ostrava) were 3.4 4.5 ng/m3 , in
OstravaRadvanice 10.1 10.6 ng/m3 , in Karvina 7.4 11.4 ng/m3
vs. in Prague 0.9 1.1 ng/m3 (Table 1) (CHMI, 2012). Exposure to
B[a]P in OstravaRadvanice is the highest in EU (Fig. 2), probably
similar to pollution in industrial Chinese cities such as Beijing (Liu
et al., 2007), Guiyu (Guo et al., 2012), or Tianjin (Jiao et al., 2009).
The concentration of B[a]P in OstravaRadvanice increased in
the period 20032007. The district pediatrician simultaneously
observed the increased incidence of asthma bronchiale in children
from 10% in the year 2001 up to 30% in the year 2007. It became

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Table 1
Air pollution in the selected localities of the Czech Republic 2011 (CHMI, 2012).
Locality

PM 10 g/m3
X

OstravaPoruba
OstravaRadvanice
Karvina
Havirov
PragueSmichov
PragueLibus
Ceske Budejovice

34.0
49.4
44.7
43.9
35.9
27.5
27.6

27.1
27.7
34.6
34.2
19.6
17.2
19.0

PM2.5 g/m3
XG

(26.9 1.9)
(43.7 1.6)
(35.8 1.9)
(35.2 1.9)
(31.1 1.7)
(23.6 1.7)
(22.4 1.9)

27.6
36.0

17.9
17.3
20.3

B[a]P ng/m
XG

23.9
22.9

10.5
11.7
14.5

(20.3 2.2)
(30.9 1.7)

(15.3 1.8)
(14.1 1.9)
(16.2 2.0)

X
3.4
10.1
7.4

0.9
1.3

XG
4.5
10.6
11.4

1.1
1.5

(1.1 5.4)
(5.8 3.2)
(2.7 4.6)

(0.3 4.6)
(0.5 5.5)

() not measured, Annual average: X arithmetic mean SD; XG geometric mean SD.

the reason to study the impact of air pollution to human health in

this region more thoroughly, under the project Program Ostrava.
In vitro studies
PM2.5 particles were collected by high volume samplers in the
most polluted area of the Czech Republic-Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting
a low level of air pollution Trebon a small town in the
non-industrial region of Southern Bohemia. PM2.5 particles were
extracted (extractable organic matter EOM) and c-PAHs contents
in the EOMs were determined. As markers of genotoxic potential,
DNA adduct levels and oxidative DNA damage levels (8-oxo-7,8dihydro-2 -deoxyguanosine, 8-oxodG) induced by EOMs in an acellular assay of calf thymus DNA coupled with 32 P-postlabeling (DNA
adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct
levels (17.20 adducts/108 nucleotides/m3 vs. 8.49 adducts/108
nucleotides/m3 ) were induced by EOM from OstravaBartovice
(immediate proximity of heavy industry) compared with that
from OstravaPoruba (mostly trafc emissions). Oxidative DNA
damage induced by EOM from OstravaBartovice was more than
fourfold higher than damage induced by EOM from Trebon (8oxodG/108 dG/m3 : 0.131 vs. 0.030 for OstravaBartovice vs. Trebon,
respectively). c-PAH contents in EOMs are the most important factors relating to their genotoxic potential (Topinka et al., 2011).
Morbidity in children
In 10 pediatric districts in Ostrava, morbidity of children born
20012004 up to 5 years of age (N = 1888) was followed. The pediatricians abstracted medical records in ICD-10 codes. Comparisons
of detailed age-specic morbidity of 1655 children of the Czech ethnicity born and living in the district of OstravaRadvanice/Bartovice

Fig. 2. Monthly concentrations of benzo[a]pyrene Bartovice (Ostrava) 2009.

had signicantly higher incidence of acute illnesses than children

in other parts of Ostrava. They suffered higher incidence of acute
respiratory diseases in the rst year of life (Fig. 3) and higher prevalence of asthma bronchiale (37.1%, N = 170) vs other parts of Ostrava
(10.213.2%, N = 1287) (Dostal et al., 2011). Prenatal exposure
to PAHs may be associated with altered lymphocyte immunophenotypic distribution in cord blood and possible changes in cord
serum immunoglobulin E levels as proposed by Hertz-Picciotto
et al. (2008). We hypothesize that high concentrations of PAHs
affected maturation of the immune system. Therefore, children
from a more polluted region suffered higher respiratory morbidity
especially during the rst year of their life.
Asthma bronchiale
The task of this study was to evaluate the impact of air pollution
to gene expression in children and to analyze, if there is any specic effect to the origin and the development of asthma bronchiale.
Specically, we used gene expression proles in leukocytes of asthmatic children compared with those in children without asthma,
using Illumina HumanHT-12 BeadChip in a group of 200 children living in OstravaRadvanice/Bartovice (100 asthmatic and 100
healthy children, age 615 years) and a control group of 200 children living in Prachatice (100 asthmatic and 100 healthy children)
(Libalova et al., 2011).
Comparing the rst signs of asthma bronchiale, the prevalence
in Ostrava was approx. 60% cases diagnosed up to the age of
3.5 years, and in Prachatice only 25% (Fig. 4).
Gene expression was analyzed in 368 samples, RNA was
hybridized on the whole genome chips with 48 thousand
genes/chip. Samples were evaluated according to disease and
locality (Ostravaasthma, Ostravacontrol, Prachatice-asthma,
Prachatice-control). Differences in gene expression were checked

Fig. 3. Children respiratory morbidity (URI + bronchitis + pneumonia + tonsillitis +

otitis media).

R.J. Sram et al. / International Journal of Hygiene and Environmental Health 216 (2013) 533540

Fig. 4. First signs of asthma bronchiale. Relationship to age.

537

by t-test and ANOVA. When children are compared according to

locality and the change in the gene expression >1.5, we observed
64 deregulated genes (Fig. 5). When we compare Ostrava-asthma
vs. Ostrava-control, we observed 12 deregulated genes, in Prachatice 17 deregulated genes. Using Venns diagrams, genes specic for
asthma in Ostrava and asthma in Prachatice completely differ, no
one gene was observed in both localities (Fig. 6). In Ostrava, MAPK
signaling pathway (p < 0.01, 1.5-fold) and in Prachatice cytokinecytokine receptor interaction pathway (p < 0.01, 1.5-fold) were
affected.
Selected genes were veried using qPCR method (Fig. 7). Results
show in asthmatic children from Prachatice increased expression
of genes SIGLEC8, CLC, CCL23 and CACNG6 (relationship to the presence of eosinophils; eosinophilic inammation is related to the
allergic type of asthma) corresponding to the allergic phenotype.
On the other hand, in asthmatic children from Ostrava, increased
gene expression corresponded to the non-allergic phenotypes
(DEFA4 relationship to the presence of neutrophils; neutrophilic

Fig. 5. Comparison of no asthma or asthma. Results from the two regions. (There is a signicant amount of differentially expressed genes comparing the 2 no asthma or
the 2 asthma groups, respectively, indicating a strong region effect. The Venn diagrams show the number of signicant transcripts obtained with these two comparisons
and with the disease-independent comparison control vs. Ostrava.)

Fig. 6. Comparison of no asthma vs. asthma t-test results. (In the Venn diagrams shown, the t-test results obtained using all experiments either with a p-value cutoff of
0.01 or a p-value cutoff of 0.01 and at least a 1.5-fold change are compared.)

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Table 2
DNA adducts in newborns (cord blood) Prague vs. Ceske Budejovice.
N

Adducts/108 nucl.
B[a]P like

Prague
Ceske Budejovice
***

74
75

Total

Mean SD

Med (minmax)

Mean SD

Med (minmax)

0.21 0.14
0.43 0.23***

0.18 (0.000.95)
0.40 (0.001.38)

0.99 0.43
1.45 0.65***

0.90 (0.002.71)
1.32 (0.003.61)

p < 0.001.

Table 3
Micronuclei in newborns (cord blood) Prague vs. Ceske Budejovice.
N

MN frequencies
MN/1000 BNC

Prague
Ceske Budejovice
***

86
92

% AB.C.

Mean SD

Med (minmax)

Mean SD

Med (minmax)

2.17 1.32
3.82 2.43***

2.00 (0.006.50)
3.00 (1.0015.50)

0.21 0.12
0.37 0.23***

0.20 (0.000.65)
0.30 (0.001.50)

p < 0.001.

inammation is related to the non-alergic type of asthma, AHSP

stabilization of hemoglobin , HBG2 part of fetal heamoglobin
(subunits 2 and 2), higher afnity to oxygen). We may ask, if
the increased expression of genes HBG2 and AHSP is related to
the hypoxia in Ostrava children or if it is related to the changes
in hematopoiesis. The signicant difference in the gene expression was observed comparing children from Ostrava and Prachatice,
which is probably related to dissimilarity of air pollution between
those two regions.
This study is unique because it is the rst time when whole
genome chips were used to analyze the relationship between air
pollution and asthma bronchiale. The results suggest the distinct
phenotype of asthma in children living in polluted Ostrava region
comparing to children living in Prachatice.
Changes in the genome of newborns
The effect of exposure to air pollution to biomarkers in newborns
was analyzed in two locations with different level of pollution:
Prague vs. Ceske Budejovice in winter season 2008/2009. The levels of B[a]P, benzene and PM2.5 for both locations for the years
2008 and 2009 were obtained from the Czech Hydrometeorological Institute. The mean concentration of these pollutants 3 months
before birth were calculated to estimate the individual exposure
of each mothernewborn pair: B[a]P concentration in Prague was
1.9 0.5 ng/m3 vs. 3.2 0.2 ng/m3 in Ceske Budejovice (p < 0.001),
PM2.5 27.0 2.5 g/m3 vs. 24.5 0.7 g/m3 (p < 0.001), benzene
2.5 0.5 g/m3 vs. 2.1 0.1 g/m3 , respectively (Rossnerova et al.,
2011).

As biomarkers, umbilical cord blood (UCB) was used to determine DNA adducts by 32 P-postlabeling (Binkova et al., 2007) and
micronuclei using automated image analysis (Rossnerova et al.,
2009) (Table 2). DNA adducts were analyzed as B[a]P-like adducts
and total adducts, both categories were signicantly higher in Ceske
Budejovice vs. Prague (p < 0.001). Higher frequencies of micronuclei in newborns in Ceske Budejovice vs. Prague (p < 0.001) were
observed. Multivariate logistic regression showed a signicant
impact of 3 months mean B[a]P exposure before birth (Table 3)
(Rossnerova et al., 2011).
The changes in the transcriptome of newborns from UCB
were studied (Votavova et al., 2012). Non-smoking mothers
were selected: 52 from Ceske Budejovice and 35 from Prague.
Total RNA was isolated from leukocytes, gene expression proles
were determined by HumanRef-8 Expression BeadChips (Illumina,
San Diego, CA, USA) containing 24,526 transcript probes. Genes
with/log FC/ > 0.58 (binary logarithm of fold change) and p < 0.01
were considered as differentially expressed between Ceske Budejovice and Prague. Leukocytes from newborns showed different
expression of 104 genes (37 up-regulated and 67 down-regulated
genes). Down-regulated biological processes were immune and
defense response (KIR2DL3, KIR3DL3, KIR3DL4, KIR2DS5, KLRC3,
CLTA4), negative regulation of proliferation (CNDKN1A, CTLA4,
TGFBR3), apoptosis (PRF1, NR4A2, GZMB, TNFAIP3, PP2R2B, DDIT4),
response to oxygen levels, cell migration, organ regeneration, signal transduction (RGS1, SOCS1, THBS) and cell differentiation (FLT3,
ZBTB16), up-regulated gene encoding SERPINA1 (which is considered as biomarker of exposure to genotoxic agents). Down
regulated signaling pathways were natural killer cell mediated
cytotoxicity, antigen processing and presentation, autoimmune
thyroid disease, graft vs. host disease, up-regulated MAPK signaling
pathway (Merkerova-Dostalova et al., in preparation).
The results were surprising because air pollution in Prague was
understood to be higher than in Ceske Budejovice. Results of analysis of DNA adducts, micronuclei and transcriptome indicate in the
same direction the signicance of exposure to B[a]P in Ceske Budejovice to induce genetic damage in newborns, when PM2.5 and
benzene was higher in Prague. It seems to be a coincidence that
changes observed in Ceske Budejovice were observed when exposure to B[a]P was 3.2 ng/m3 , corresponding to the effect of exposure
inducing IUGR 2.8 ng/m3 (Dejmek et al., 2000).
Discussion

Fig. 7. qPCR verications. Asthma bronchiale in Prachatice and Ostrava.

Studies in the Czech Republic substantiate the previous hypothesis that exposure to high air pollution during pregnancy can affect

R.J. Sram et al. / International Journal of Hygiene and Environmental Health 216 (2013) 533540

539

Table 4
Overview of studies in the Czech Republic.
Factor
Pregnancy outcome

Morbidity

Air pollution
PM10 > 40 g/m3 /rst month of pregnancy
B[a]P > 28 g/m3 /rst month of pregnancy
Homes heated by coal
Indoor coal combustion for heating
Air pollution PM2.5, PAHs
Living in Teplice vs. Prachatice

Air pollution - B[a]P, PM2.5

Biomarkers

Air pollution c-PAHs

Air pollution c-PAHs
Air pollution PM10, PM2.5, c-PAHs
8-oxodG in placental DNA
Ostrava vs. Prachatice

Air pollution B[a]P

Air pollution B[a]P

functional changes, which are signicant for morbidity during the

whole life of such subjects (Barker, 1995; Painter et al., 2005;
Varvarigou, 2010). In our study, air pollution signicantly affect
children especially the increase of respiratory morbidity. With the
development of molecular epidemiology, we can further evaluate
the health risk using biomarkers (Table 4).
The latter approach is certainly owed to the pioneer work of F.
P. Perera for her studies in New York (USA), Cracow (Poland) and
China (Perera et al., 2003, 2004, 2005; Choi et al., 2006; Millman
et al., 2008). Her group used DNA adducts and personal monitoring as biomarkers of PAH exposure. Data from New York and
Cracow indicate that exposure to PAHs during pregnancy signicantly affected child development as IQ or behavior (Perera et al.,
2011, 2012). PAHs can be found in the air and from eating barbecued meat where were associated with increased genetic damage
as DNA adducts and micronuclei in newborns (Pedersen et al., 2012)
and decreasing birth weight (Jedrychowski et al., 2012).
The spectrum of diseases and functional changes induced by
air pollution in children is more varied than we expected e.g. two
decades ago. We should also realize that the impact of induced
changes do not affect only the exposed child, but is transferred
through gametes to next generation. Studies of Somers et al. (2002)
and Yauk et al. (2008) observed increased germline mutations and
DNA methylation in mice by ambient air in a polluted industrial
area near steel mills. This germ-line DNA damage and epigenetic
modications have the potential to affect disease incidence in the
descendents of exposed individuals (Yauk et al., 2008). Indeed, the
concentrations of B[a]P in polluted Hamilton, Ontario, Canada,
are similar to that in Prague, Czech Republic approx. 1 ng/m3 .
Air pollution in hot spots as e.g. Teplice and Ostrava in the
Czech Republic, Cracow in Poland, or Tianjin and Chongqing in
China are examples of regions, where a very high pollution signicantly inuence children health. New knowledge about the
consequences of air pollution to human health should stimulate
long-term cohort studies, which could be also be used to check the
use of new biomarkers. Such studies would indicate they can be
used by regulatory agencies and for disease prevention.
Children morbidity, especially in preschool age, signicantly
affect, family life. It represents also a signicant economic burden.

Effect

References

IUGR
IUGR
Lower respiratory tract
illness
Reduction of height
at 3ys of age
Bronchitis up to 4.5 ys
of age
Laryngitis & tracheitis,
Pneumonia, otitis media
in children up to 3 ys
Acute respiratory diseases
in the 1st year of life
Micronuclei in children
and mothers
Gene expression in children

Dejmek et al. (1999)

Dejmek et al. (2000)
Baker et al. (2006)

8-oxodG in urine children

Aged 610 ys
IUGR, LBW
Different deregulation
of genes in children
with asthma bronchiale
DNA adducts in newborns,
Micronuclei in newborns
Deregulation of genes in newborns

Svecova et al. (2009)

Ghosh et al. (2011)

Hertz-Picciotto et al. (2007)
Dostal et al. (2009)

Dostal et al. (2011)

Pedersen et al. (2006)
van Leeuwen et al. (2006, 2008)

Rossner et al. (2011)

Libalova et al. (2011)

Rossnerova et al. (2009)

Votavova et al. (2012)

We should therefore use such ndings to develop exposure and

disease preventive measures.
Acknowledgements
Critical comments by Prof. William Au are highly appreciated.
This work was supported by the Czech Ministry of the Environment (SP/1b3/8/08), the Czech Ministry of Education (2B08005)
and by the Grant Agency of the Czech Republic (P503/11/0084 and
P503/11/0142).
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