Carcinogenesis vol.28 no.4 pp.
816–822, 2007
doi:10.1093/carcin/bgl175
Advance Access publication October 27, 2006
Increased health risk in Bangkok children exposed to polycyclic aromatic
hydrocarbons from traffic-related sources
Jantamas Tuntawiroon1, Chulabhorn Mahidol2,
Panida Navasumrit1, Herman Autrup3 and
Mathuros Ruchirawat1,4,
1 PAHs including benzo(a)pyrene (BaP) have been classified
Laboratory of Environmental Toxicology, 2Laboratory of Chemical
Carcinogenesis, Chulabhorn Research Institute, Bangkok, Thailand,
3
Department of Environmental and Occupational Medicine, Institute of
Public Health, University of Aarhus, Aarhus, Denmark and 4Department of
Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand
To whom correspondence should be addressed at: Laboratory of
Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210,
Thailand. Tel: +66 2 574 0615; Fax: +66 2 574 0616;
Email: mathuros@cri.or.th
The aim of this study is to assess potential health risk
of exposure to particle-associated polycyclic aromatic
hydrocarbons (PAHs) in children living in a megacity with
traffic congestion such as Bangkok. The study population
comprised 184 Thai schoolboys (aged 8–13 years) attend-
ing schools adjacent to high-density traffic areas in
Bangkok and schools located in the provincial area of
Chonburi. The ambient concentration of total PAHs
at roadsides in proximity to the Bangkok schools was
30-fold greater than at roadsides in proximity to the
provincial schools (30.39 ± 5.80 versus 1.50 ± 0.28 ng/m3;
P < 0.001). Benzo(g,h,i)perylene (BghiP), an indicator of
automobile exhaust emission, was the predominant PAH.
Personal exposure to total PAHs and the corresponding
benzo(a)pyrene (BaP) equivalent concentrations in Bang-
kok schoolchildren were 3.5-fold higher than in provincial
schoolchildren (4.13 ± 0.21 versus 1.18 ± 0.09 ng/m3;
P < 0.001 and 1.50 ± 0.12 versus 0.43 ± 0.05 ng/m3;
P < 0.001, respectively). The concentration of urinary
1-hydroxypyrene (1-HOP) was significantly higher in
Bangkok schoolchildren. Bulky carcinogen–DNA adduct
levels in peripheral lymphocytes were also significantly
higher (0.45 ± 0.03 versus 0.09 ± 0.00 adducts/108 nt;
P < 0.001). Finally, a significantly higher level of DNA
strand breaks and a significantly lower level of DNA repair
capacity were observed in Bangkok schoolchildren
(P < 0.001). This study indicates that Bangkok schoolchil-
dren exposed to a high level of genotoxic PAHs in ambient
air may be more vulnerable to the health impacts associated
with the exposure to genotoxic pollutants than children
in provincial areas and may have increased health risks
for the development of certain diseases such as cancer.
Introduction
Current urban air pollution problems in megacities such as
Bangkok are mostly the result of heavy traffic congestion (1).
Polycyclic aromatic hydrocarbons (PAHs), a major group of
Abbreviations: PAHs, polycyclic aromatic hydrocarbons; 1-HOP, 1-hydro-
xypyrene; BaP, benzo(a)pyrene; BghiP, Benzo(g,h,i)perylene.
# The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
genotoxic carcinogens found in urban atmospheric pollution
from motor vehicle emissions, are generated through the
incomplete combustion of fossil fuels and oil products. Many
by IARC as probable human carcinogens (2). Exposure to
these compounds is a public health concern particularly in
children, who are one of the most susceptible groups of the
population. Reasons for the greater susceptibility are both
behavioural and physiological in nature. In many cases,
children are thought to have greater exposure to airborne
pollution per body weight than adults, because children
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generally tend to spend more time outdoors, have higher
physical activity and have a higher ventilation rate than
adults (3,4). Thus, they are exposed proportionally to higher
doses of the toxic compounds (5,6). Moreover, exposure to
genotoxic carcinogenic compounds at a young age may
represent a health risk, i.e. by causing genetic damage
(mutation, sister chromatid exchanges and other genetic
disruption) (7–9) that may increase the risk of cancer later in
life (10,11). Epidemiological and experimental data reported
increased cancer risks following childhood exposure to
carcinogens as compared with exposure occurring at a
mature age (12). Exposure to motor vehicle exhaust and the
evidence for increased risk of childhood cancer has been
established in case–control studies performed in Sweden and
United Kingdom (13,14). These studies showed elevated
risks of childhood cancer, including leukemia and central
nervous system tumor among children living near heavily
travelled streets or highways with high levels of ambient air
pollution.
In order to assess the health risks from the exposure to
pollutants, it is important to quantify the actual dose that gets
into the body as well as the resultant changes that are
effected through the use of various biomarkers associated
with the different toxicants. For the assessment of environ-
mental exposure to PAHs, urinary 1-HOP a metabolite of
pyrene, is a widely used biological indicator of internal dose
of exposure in the occupational environment and in smokers
(15) and to low level of PAHs in non-occupationally exposed
people (16,17). Carcinogenic PAHs will, following meta-
bolic activation, form adducts with cellular macromolecules.
PAH–DNA adducts have been used to assess exposure
to PAHs as a biomarker of biologically effective dose. The
32
P-postlabelling has been used extensively to detect bulky
carcinogen–DNA adducts in peripheral lymphocytes from
occupationally exposed people (18–20). DNA adducts may
also be capable of detecting early biological effects in
children and newborns exposed to environmental pollutants
(21). Several recent studies indicate that bulky adducts are a
good indicator of cancer risk (22,23).
The health risks from exposure to genotoxic carcinogenic
pollutants can be assessed through the measurement of DNA
damage, DNA repair capacity and chromosomal aberrations,
all of which are considered to be biomarkers of early
816

biological effects. These biomarkers were included as
intermediate end-points related to the carcinogenesis process.
The study of Perera et al. (24) suggested an exposure to
genotoxic air pollutant was associated with an increased
genetic alteration (i.e. DNA adducts, chromosomal aberra-
tions), which was relevant to the increased cancer risk in
humans. The associations between the increased level of
genetic damage and environmental exposure to air pollutions
have been reported in young children living in polluted areas
(25,26).
This study provides evidence of potential adverse health
effects of exposure to particle-associated PAHs in schoolchil-
dren through the measurement of ambient exposure levels as
well as biomarkers of exposure and early biological effects.
The target group consisted of schoolchildren who attended
different schools located in a high-density traffic area in
Bangkok (high exposure) and schoolchildren attending
schools in the provincial area, Chonburi (low exposure).
Materials and methods
Study subjects
A total of 184 healthy primary school boys were enrolled in the study. The
group was divided into two subgroups according to the suspected exposure.
The high exposure group consisted of 115 boys (mean age 10.17 years; range
8–12 years) randomly selected from four primary schools located at different
sites in areas of high traffic density in Bangkok. The low exposure group
consisted of 69 boys (mean age 10.81 years; range 9–13 years) randomly
selected from two primary schools located in provincial area of Chonburi
province.
Through information provided in the questionnaires, factors such as age
and lifestyle (i.e. environmental tobacco smoke exposure, transportation,
medication, type of diet, etc.) were taken into consideration to ascertain that
they were well matched between Bangkok and Chonburi schoolchildren. The
study was approved by the Institutional Ethical Committee in agreement with
the Helsinki Declaration. All parents were informed about the study and have
signed the consent form.
Study locations
Six primary schools were selected as study sites. Four of these primary
schools were located at four different sites in areas of high traffic density in
Bangkok within 500 m of the main roads, namely Chuckawat, Rama I, Rama
IV and Sripraya, which represented Sites 1, 2, 3 and 4, respectively. The
other two primary schools were located in Bangphra and Vornapa districts in
Chonburi (the provincial area located
110 km from Bangkok), which
represented Sites 5 and 6, respectively.
Sample collection
Samples were collected during the winter season (January–February, 2004
and 2005), a period when high exposure to PAHs was expected. The
meteorological conditions during this period in Bangkok were temperature
28.4 C (24.8–33.3 C); relative humidity 73.1% (61.0–81.0%) and surface
wind speed 4.8 km/h (3.3–7.6 km/h). In Chonburi province mean temperature
was 28.8 C (33.2–25.4 C), relative humidity, 74.0% (63.0–80.0%); and
surface wind speed, 4.5 km/h (3.0–6.1 km/h). All samplings were carried out
on a Wednesday, which preliminary studies (unpublished data) indicated to
be representative for average traffic activity and thus weekly PAHs exposure.
Environmental measurements and personal exposure were conducted at
the roadsides close to schools, in the school areas and in the children’s
breathing zone. Air particulates were collected for 8 h (8 a.m.–4 p.m.) on
glass fiber filter (37 mm) using personal air samplers attached to a battery-
operated SKC air check samplers (model 224). For environmental air
sampling, the pumps were fixed at a height of
150 cm, while personal air
samples were collected in the breathing zone. Air samples were collected at a
flow rate of 2l/min. After sampling, the filters were wrapped in aluminium
foil and kept at 70 C until analysis.
Biological samples were collected at the day of air sampling. At the end
of school hours, whole-blood samples (7 ml) were collected, which were
transferred to the laboratory, and lymphocytes extraction was processed
within 4–6 h of collection (27). Whole-blood samples for the Comet assay
were processed immediately in the laboratory to minimize the decline in
DNA damage due to DNA repair and/or the loss of heavily damaged cells or
Traffic-related pollutants and increased health risk to children
by an increase in DNA damage associated with storage conditions. Spot
urine samples were collected in the morning on the day of air sampling (prior
to the start of school day; Day 0) and then in the next morning (Day 1) and
stored at 20 C until analysis.
Quantification of PAHs in ambient air
The extraction of PAHs was performed according to a slight modification of
Garivait (28). The filter samples were extracted by ultrasonication with
dichloromethane (10 ml). The extracts were concentrated under a gentle
nitrogen stream. All processes were carried out without direct exposure
to light. After a solvent exchange step to acetonitrile, the extract was
analysed by HPLC (Hewlett Packard Series 1100) coupled with fluorescence
detector. The PAHs were separated on a LiChrospher PAH (250 · 3 mm)
column at 18 C using 60–100% acetronitrile gradient and a flow rate of
0.56 ml/min. Ten PAHs with variable wavelength fluorescence detection
were quantified: anthracene (AN), excitation 246 nm, emission 400 nm;
fluoranthene (FA), excitation 237 nm, emission 460 nm; pyrene (PY),
excitation 234 nm, emission 387 nm; benzo(a)anthracene (BaA), excitation
278 nm, emission 392 nm; chrysene (CHR), excitation 262 nm, emission
379 nm; benzo(b)fluoranthene (BbFA), excitation 256 nm, emission 437 nm;
benzo(k)fluoranthene (BkFA), excitation 240 nm, emission 417 nm;
benzo(a)pyrene (BaP), excitation 263 nm, emission 410 nm; dibenzo(a,h)an-
thracene (DBahA), excitation 291 nm, emission 400 nm; and benzo(g,h,i)-
perylene (BghiP), excitation 288 nm, emission 415 nm, as target PAHs.
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The concentration of each PAH was quantified by its peak area. The con-
centrations of the PAHs were transformed to BaP equivalent using the toxic
equivalency factors (TEFs) for PAHs according to Nisbet and Lagoy (29).
Determination of urinary 1-hydroxypyrene
Reverse-phase HPLC was used for the quantitative analysis of 1-HOP in
urine using a modification of the procedure described by Hansen et al. (30).
An aliquot of urine (10 ml) was diluted with equal amount of sodium acetate
(0.2 M; pH 5.0), adjusted to pH 5.0 and incubated with b-glucuronidase and
sulphatase overnight at 37 C. C18 cartridges were used to extract 1-HOP that
was eluted twice with 500 ml of acetonitrile (recover 96.7–100.9%). The
eluate was separated by HPLC using Zobrax XDB-C18 column and a linear
acetonitrile gradient (30–100%, 14 min) and a flow rate of 1.5 ml/min. The
metabolite was detected by fluorescence (excitation 242 nm; emission 390
nm). The concentration of 1-HOP was quantified by its peak area. The results
are expressed as micromoles per mole (mmol/mol) creatinine to account for
difference in urine dilution. Urinary creatinine was determined by a standard
colorimetric method following the picric acid reaction using a creatinine kit
(Human GmbH, Germany).
32
P-Post-labelling of DNA adducts
DNA, extracted from lymphocytes by enzymatic digestion followed by
chloroform/phenol extraction, was precipitated with ice-cold ethanol. For
32
P-postlabelling assay, DNA (4 mg) was digested with micrococcal nuclease
(MN) and spleen phosphodiesterase (SPD), and adducted nucleotides were
then enriched by using butanol extraction as described by Nielsen and Autrup
(31) with minor modification. After enzymatic hydrolysis of DNA by MN
and SPD, the digested material was dried and resuspended in 7.5 ml of H2O.
The resuspended DNA was then added to an equal volume of T4
polynucleotide kinase mixture containing [g-32P] in a total of 15 ml. The
labelled samples were spotted and developed on polyethyleneimine–cellulose
thin layer chromatography plates. The DNA adduct spots were quantified
using a PhosphorImager (PI; Molecular Dynamics Sunnyvale, CA). BaP-
diolepoxide–DNA adduct standard was prepared from calf thymus DNA
treated with BaP-diolepoxide and was included in the analysis to correct for
assay variability. The reported carcinogen–DNA adduct level was the
average of at least two completely independent assays. The adduct level was
expressed as adduct/108 nucleotides.
Determination of DNA strand breaks
DNA strand breaks were determined by way of the alkaline Comet assay, as
described according to Singh et al. (32) and Marcon et al. (33) with a slight
modification. Briefly, 20 ml of whole-blood was mixed with LMP agarose,
and embedded into an agarose precoated slide. Slides were submerged in
cold lysis solution for at least 1 h at 4 C. Subsequently, slides were
transferred to an electrophoresis chamber and covered with alkaline solution
(pH 13) for 20 min before electrophoresis at 300 mA, 24 V for 20 min. After
electrophoresis, slides were neutralized with 1 M ammonium acetate and
stained with 50 ml Sybr
solution (1:5000) (Molecular Probes, USA). A total
of 50 cells from each of the duplicated slides were examined randomly under
an epi-fluorescence microscope (Axioplan 2, Zeiss, Germany). The extent of
DNA stand breaks was measured quantitatively using the CometScan image
analysis software (Metasystems), and expressed as tail length (the distance of
817
J.Tuntawiroon et al.

cellular DNA migration form head end position to tail end position) and
olive tail moment (the product of the proportion of DNA in the tail by the Table I. Ambient concentrations of total PAHs at various locations
distance between head and tail centres of gravity).
School locations Total PAHs (ng/m3)
Determination of DNA repair capacity by the cytogenetic challenge assay
The challenge assay used in this study was carried out according to the Roadside School area
methods that have been previously described (34,35). At 24 h after blood
Bangkok
culture, the cells were irradiated with 100 cGy using a 137Cs source at a dose
Site 1 68.35 ± 7.67a,,$ 6.85 ± 1.41,$$
rate of 5 Gy/min. Cells were blocked, 50 h after culture initiation, with
67.49 (48.23–99.95)b 5.64 (2.79–12.40)
Colcemid (final concentration of 0.1 mg/ml) for 1.5 h and harvested using the
(n ¼ 6)c (n ¼ 6)
standard procedure. Cytological preparations were made, coded and stained
Site 2 21.55 ± 4.99,$ 4.88 ± 1.21,$
with a 10% Giemsa solution for 15 min. Fifty metaphase cells were analysed
19.59 (11.58–35.43) 4.40 (2.64–8.08)
from each of the duplicated slides under the microscope. The presence of
(n ¼ 4) (n ¼ 4)
dicentric chromosomes and chromosome deletions per metaphase were
Site 3 9.43 ± 2.22,$ 5.89 ± 2.83,$$
determined.
7.58 (4.63–23.78) 3.11 (2.10–25.54)
Statistical analysis (n ¼ 8) (n ¼ 8)
Site 4 24.23 ± 6.96,$ 4.85 ± 0.28,$
The Mann–Whitney two-tailed test was used to compare the biomarker
23.32 (11.73–38.54) 4.79 (4.26–5.59)
levels in the different groups. Pearson correlation was used to evaluate the
(n ¼ 4) (n ¼ 4)
association between the biomarker levels. Wilcoxon sign ranks test was used
All sites in Bangkok 30.39 ± 5.80### 5.78 ± 1.08###
to compare each individual at the different times. All the statistical analyses
19.59 (4.63–99.95) 4.79 (2.10–25.54)
were performed with SPSS Statistical package (SPSS Inc-version 12.0).
(n ¼ 22) (n ¼ 22)
P-value <0.05 was considered statistically significant.
Provincial (Chonburi)
Site 5 1.77 ± 0.32 1.23 ± 0.25

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1.86 (0.31–2.92) 1.36 (0.28–2.31)
Results (n ¼ 9) (n ¼ 10)
Site 6 0.69 ± 0.22 0.85 ± 0.19
Ambient air pollution is significantly higher in metropolitan 0.60 (0.37–2.08) 0.91 (0.34–1.23)
Bangkok due to the high traffic density. In this study, (n ¼ 3) (n ¼ 4)
environmental and personal air samplings were used to All sites in Chonburi 1.50 ± 0.28 1.12 ± 0.19
assess the exposure to particle-associated PAHs in ambient 1.35 (0.31–2.92) 1.07 (0.28–2.31)
(n ¼ 12) (n ¼ 14)
air. Internal dose and biologically effective dose were
assessed by the measurement of urinary 1-HOP and bulky a
Values are expressed as mean ± SE.
carcinogen–DNA adduct respectively, as biomarkers. The b
Median (minimum–maximum).
c
potential health risks from exposure were assessed through Number of samples.
,Significant differences from Site 5 at P < 0.01, 0.001 respectively.
the use of the biomarkers of early biological effects by the $,$$
Significant differences from Site 6 at P < 0.05, 0.01 respectively.
measurement of DNA strand breaks and DNA repair ###
Significant difference from all sites in Chonburi at P < 0.001.
capacity.
Ambient PAHs concentrations at various sites and personal Table II. PAHs exposure concentrations and biomarkers of exposure in
schoolchildren
exposure to PAHs
The concentrations of total PAHs, based upon the sum of 10 Parameters School locations
PAHs, measured from the ambient air at various study sites
Bangkok Provincial
in Bangkok as well as the provincial area of Chonburi are (Chonburi)
summarized in Table I. The ambient total PAHs concentra-
tion measured at the roadsides in close proximity to the four Individual exposure (ng/m3)
Bangkok schools was
30-fold higher than that of the Total PAHs 4.13 ± 0.21a, 1.18 ± 0.09
3.48 (1.01–10.57)b 1.03 (0.11–3.67)
provincial schools (P < 0.001). Similarly, the ambient (n ¼ 114)c (n ¼ 68)
total PAHs concentration in the school areas in Bangkok BaP equivalent 1.50 ± 0.12 0.43 ± 0.05
was 5-fold higher than that of provincial schools (P < 0.001). 1.31 (0.15–8.76) 0.23 (0.00–1.85)
Children in Thailand spend at least 8 h (8 am to 4 pm), (n ¼ 114) (n ¼ 68)
Urinary 1-HOP (mmol/mol creatinine)
approximately a third of a day, at school. The home Day0 0.18 ± 0.01 0.10 ± 0.01
environments for the Bangkok and Chonburi schoolchildren 0.15 (0.04–0.55) 0.09 (0.02–0.47)
were similar in nature; however, Bangkok children are (n ¼ 105) (n ¼ 61)
exposed to higher concentrations of ambient PAHs as a result Day1 0.22 ± 0.02 0.12 ± 0.01
of traffic congestion. Personal exposures to total PAHs and 0.16 (0.03–0.99) 0.11 (0.02–0.43)
(n ¼ 100) (n ¼ 61)
their corresponding BaP equivalent exposures in schoolchil- DNA adducts 0.45 ± 0.03 0.09 ± 0.00
dren are summarized in Table II. It can be seen that personal 8
(adducts/10 nucleotides) 0.34 (0.13–1.04) 0.09 (0.05–0.16)
exposures to total PAHs were
3.5 times greater in Bangkok (n ¼ 107) (n ¼ 69)
schoolchildren (P < 0.001). When the concentrations of a
Values are expressed as mean ± SE.
carcinogenic PAHs were converted to BaP equivalents, b
Median (minimum–maximum).
schoolchildren in Bangkok were exposed to
3.5-fold greater c
Number of samples.
levels than schoolchildren in the provincial area (P < 0.001). Significant difference from Chonburi at P < 0.001.
The pattern of the 10 PAHs measured at the roadsides, in
the school areas and at the breathing zone of schoolchildren (range 0.86–1.28 ng/m3, representing 24–37% of total PAHs
obtained from each school is illustrated in Figure 1. For burden). BghiP was also the predominant PAH in ambient air
children in the four Bangkok schools, the predominant at the roadsides (2.67–21.25 ng/m3, 34–45%) as well as in
PAH in air samples from the breathing zone was BghiP the school areas (0.97–2.44 ng/m3, 29–41%). A similar
818

Fig. 1. Pattern of ten PAHs at different locations. The median concentration
of each PAH was determined from different sites in Bangkok and Chonburi
province. Ten PAHs: anthracene (AN), fluoranthene (FA), pyrene (PY),
benzo(a)anthracene (BaA), chrysene (CHR), benzo(b)fluoranthene (BbFA),
benzo(k)fluoranthene (BkFA), benzo(a)pyrene (BaP), dibenzo(a,h)anthracene
(DBahA) and benzo(g,h,i)perylene (BghiP) in air samples were collected for
a period of 8 h. Sites 1, 2, 3 and 4 were located in Bangkok, while Sites 5 and
6 were located in the provincial (Chonburi) area.
pattern of distribution of PAHs was also observed in air
samples at different locations in the provincial area. BghiP
was the predominant PAH in air samples from the breathing
zone of Chonburi schoolchildren (range 0.19–0.28 ng/m3,
representing 20.93–22.17% of total PAHs burden) as well as
at the roadside ambience (0.18–0.49 ng/m3, 26.27–27.71%),
but in school areas the concentrations of DBahA (0.09–
0.15 ng/m3, 12.96–19.06%) and CHR (0.12–0.19 ng/m3,
15.79–26.09%) appeared to be higher than that of BghiP
(0.10–0.11 ng/m3, 14.44–15.25%).
Urinary 1-hydroxypyrene
The concentrations of urinary 1-HOP in schoolchildren for
both Day 0 and 1 of urine samples collection are summarized
in Table II. Urinary 1-HOP has been reported in unit of
mmol/mol creatinine to standardize the results (36). Similarly
to the ambient PAHs exposure, the concentrations of urinary
1-HOP in Bangkok schoolchildren for both Day 0 and 1 of
urine collection were found to be significantly higher (P <
0.001) than those in the provincial schoolchildren, indicating
a greater level of PAHs exposure. Urinary 1-HOP concen-
tration on Day 0 did not correlate with total PAHs exposure
Traffic-related pollutants and increased health risk to children
in Bangkok schoolchildren (r ¼ 0.145, P ¼ 0.141); however,
a weak, but significant correlation could be established
between Day 1 concentration and total PAHs exposure (r ¼
0.244, P ¼ 0.015).
Bulky carcinogen–DNA adducts
Bulky carcinogen–DNA adduct levels in Bangkok
schoolchildren compared with provincial schoolchildren are
shown in Table II. The bulky carcinogen–DNA adduct levels
in peripheral lymphocytes of Bangkok schoolchildren were
5-fold higher than those in provincial schoolchildren (P <
0.001), reflecting a greater level of exposure to genotoxic air
pollutants. These results demonstrated that children attending
schools in Bangkok, in areas with serious traffic congestion
problems, were exposed to a higher level of genotoxic PAHs
compounds. In Bangkok schoolchildren, negative correlation
between formation of DNA adduct and total PAHs (rs ¼
0.620, P ¼ 0.000) or BaP equivalent exposure (rs ¼
0.442, P ¼ 0.000) were observed; however, no correlation
between DNA adduct level and total PAHs exposure was
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seen in Chonburi schoolchildren (r ¼ 0.066, P ¼ 0.595).
DNA strand breaks and DNA repair capacity
Biomarkers of early biological effects in schoolchildren;
DNA strand breaks and DNA repair capacity are summarized
in Table III. Tail length and olive tail moment were used to
determine the levels of DNA strand breaks through the
Comet assay. The levels of DNA strand breaks in peripheral
blood samples from Bangkok schoolchildren were
1.5-fold
higher than those in provincial schoolchildren (P < 0.001).
Furthermore, DNA repair capacity, as measured by the
cytogenetic challenge assay, was also significantly reduced in
Bangkok schoolchildren compared with schoolchildren from
the provincial area. It can be seen that the number of
radiation-induced aberrant chromosomes determined as
frequencies of dicentrics or deletions per metaphase were

1.7-fold higher in Bangkok schoolchildren compared with
those of provincial schoolchildren (P < 0.001). The data from
the challenge assay demonstrated that Bangkok schoolchil-
dren may have more problems in the repair of DNA damage
which produced more aberrations and deletions than the
schoolchildren in the provincial area. DNA strand breaks
levels did not correlate with either total PAHs (r ¼ 0.042,
P ¼ 0.654) or BaP equivalent exposure (r ¼ 0.134, P ¼
0.156) in Bangkok schoolchildren. However, the levels of
DNA strand breaks and DNA adduct revealed significant
correlations with the reduction of DNA repair capacity (DNA
strand breaks r ¼ 0.288, P ¼ 0.006; DNA adduct r ¼ 0.443,
P ¼ 0.000) in these children.
Discussion
A previous study carried out in Bangkok showed that the
concentration of total PAHs on the roadside was in the range
of 7.10–83.04 ng/m3 (37), which was quite similar to the
concentration of total PAHs at the various roadsides
measured in this study (4.63–99.95 ng/m3). The differences
in PAHs concentrations at the roadsides in Bangkok can
partly be explained by different traffic intensity, different
composition of traffic and street configurations. The relative
distance from the main road also affected the concentration
of measured total PAHs in ambient air. From data in Table I,
a significant decrease in levels of PAHs with increasing
819
J.Tuntawiroon et al.

excretion of 1-HOP may be a consequence of PAHs exposure

Table III. Biomarkers of early biological effects in schoolchildren
from the previous day. A number of studies have been
Parameters School locations reported a relatively long half-life for urinary excretion of
1-HOP in adults. In workers exposed to coke oven, half-life
Bangkok Provincial (Chonburi) for urinary excretion of 1-HOP is reported as
18 h (41) or
DNA strand breaks up to 35 h (42). No study has reported on the half-life of
Tail length (mm) 1.93 ± 0.09a, 1.28 ± 0.12 1-HOP in children. The concentrations of urinary 1-HOP in
1.94 (0.65–6.77)b 1.13 (0.00–6.29) Bangkok schoolchildren were lower than those reported in
(n ¼ 115)c (n ¼ 69) children living in urban areas of other Asian countries such
Olive tail moment (mm) 0.23 ± 0.01 0.16 ± 0.01
0.21 (0.03–0.81) 0.13 (0.00–0.52) as China (43) and in some European countries such as the
(n ¼ 115) (n ¼ 69) Netherlands (36) and Poland (44).
DNA repair capacity The potential impact from PAHs exposure on children’s
Dicentric/metaphase 0.34 ± 0.01 0.21 ± 0.00 health can be demonstrated by a significantly greater number
0.36 (0.14–0.48) 0.20 (0.12–0.28)
(n ¼ 91) (n ¼ 55)
of bulky carcinogen–DNA adduct observed in Bangkok
Deletion/metaphase 0.45 ± 0.01 0.26 ± 0.01 schoolchildren when compared with that of the provincial
0.46 (0.18–0.76) 0.26 (0.18–0.36) schoolchildren which was 5-fold higher levels. The increase
(n ¼ 91) (n ¼ 55) of this precarcinogenic lesion may suggest an increased
a health risk for the development of certain diseases such as
Values are expressed as mean ± SE.
b
Median (minimum–maximum). cancer as a result of genotoxic PAHs exposure during
c childhood (10). An association between DNA adducts and

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Number of samples.
Significant difference from Chonburi at P < 0.001.
distances from the main roads may be explained on the basis
that PAHs are traffic-related pollutants and are predominantly
particle-bound, their concentrations being highest near the
source i.e. roadsides. In comparison with other urban areas,
the concentrations of ambient PAHs at Bangkok roadsides
were considerably higher than what has been reported in
tropical and temperate urban areas, i.e. Australia (38), but
still lower than those reported in other Asian countries such
as Indonesia and Korea (38).
BghiP was the predominant PAH detected in air samples
collected at the roadsides and breathing zone of all school-
children. This compound is considered an indicator of PAHs
derived from automobile traffic emission (39) indicating that
traffic emission may be the predominant source of PAHs
exposure in these children. However, the carcinogenic
potency of BghiP is only 1/100 compared with that of BaP
(29). BaP accounted for
10% of the total carcinogenic
PAHs.
Personal exposure to total PAHs and the corresponding
BaP equivalent concentrations in schoolchildren in Bangkok
were
3.5-fold higher (P < 0.001) than in children attending
in the provincial school. Most studies have used BaP as a
reference substance for carcinogenic PAHs because this
compound has a carcinogenic potency 10–100 times greater
than many other PAHs. The total personal PAHs exposure of
schoolchildren in Bangkok transformed to BaP equivalent
corresponded to 1.50 ng/m3 (range 0.15–8.76 ng/m3),
therefore the children were exposed to PAHs at levels
exceeding the WHO risk estimate guideline value for PAHs
in air. A consideration of the health based evidence and
acceptance that the lifetime risk to lung cancer of 8.7 · 105
would correspond to the exposure of 1.0 ng/m3 BaP in
air (40).
The urinary 1-HOP concentration was significantly higher
in samples collected from Bangkok schoolchildren for
both two time points (Day 0 and 1) of sample collection
(P < 0.001), which reflects higher levels of PAHs exposure in
Bangkok schoolchildren. A significant correlation between
total PAHs exposure and 1-HOP concentration was observed
at Day 1, but not Day 0, which may imply that the urinary
820
cancer risk established in recent epidemiological studies
demonstrated a high level of bulky carcinogen–DNA adduct
was associated with an increased risk of developing lung
cancer (22,23). Nevertheless, it is not possible to conclude
that the greater number of carcinogen–DNA adducts detected
in Bangkok schoolchildren may be from traffic-related
sources alone. The alternative sources for PAHs exposure in
schoolchildren other than traffic (i.e. cooking, grilling,
consumption of contaminated food, activity related to
burning) should also be considered. However, the informa-
tion obtained from questionnaires indicated that the pattern of
lifestyle and food consumption in both groups was similar.
Therefore, the contribution of PAHs exposure, if any, from
other lifestyle-related sources and food, should be the same
for both groups.
PAH–DNA adducts have been detected in umbilical cord
blood from newborns of mothers who lived in regions of high
concentrations of carcinogenic PAHs in ambient air (45)
and from smoking mothers (46). DNA adducts have also
been found in adolescents living in highly polluted areas
(47). The levels of DNA adduct formation may be influenced
by individual variation in the activity of metabolic enzymes,
induction of DNA repair processes or other mechanisms.
Therefore, the genetic differences in xenobiotic metabolism
of each individual, which may affect the functions of these
enzymes and therefore influence DNA adduct levels, should
not be overlooked (48). Since this is a human study the
possibility of concurrent exposure to other genotoxic envi-
ronmental toxicants cannot be ruled out. With respect to our
observation that DNA adducts negatively correlated with total
PAH and BaP equivalent exposures, an inconsistency has
been observed in the literature with regards to the association
between exposure to PAHs and level of DNA adducts formed
(49–51). This negative correlation is indeed surprising and
needs to be explored further. At the same time, we acknow-
ledge certain limitations in the correlation of these two
measurements, including the fact that DNA adduct formation
is an effect of cumulative exposure while individual
exposures to total PAHs were measured once during the
school period of
8 h to represent patterns of daily exposure
in these children. Additionally, there are factors that may
affect the correlation of PAH exposure levels and DNA
adducts formation, including PAH-metabolizing enzyme

kinetics and genetic differences in these metabolizing
enzymes and DNA repair in human subjects.
Human biomonitoring using the Comet assay is a novel
approach for the assessment of genetic damage in exposed
populations (52). The increased levels of genetic damage in
children could have important implications for biologically-
based evidence of the potential health effects from exposure
to genotoxic pollutants (21). In our study, the results showed
the levels of DNA strand breaks including tail length and
olive tail moment were significantly higher (
1.5-fold) in
Bangkok schoolchildren than those in provincial schoolchil-
dren. These results may indicate that Bangkok schoolchildren
attending schools close to areas with high traffic density may
be more prone to the initiation of genetic damage such as
DNA strand breaks which may be the earliest step in
carcinogenesis. However, the induction of DNA damage may
not be due to PAHs exposure alone. The DNA strand breaks
may be modified by concurrent interaction of other DNA
reactive agents that can also injure cells through direct or
indirect damage to DNA (53). Significantly increased DNA
damage assessed by the Comet assay was also observed in
young children and adolescents exposed to air pollutants in
Mexico City, Mexico (25) and in Wilrijk, Belgium (47),
respectively.
The cytogenetic challenge assay in which whole-blood
cultures were irradiated to challenge cells to repair the
radiation-induced DNA damage can be used to document
biological effects from exposure to mutagens. The effect is
an abnormal DNA repair response (54). In this study, a
significant decrease in DNA repair capacity measured by an
increase in the radiation-induced dicentric chromosomes
(chromosome-type translocation) and chromosome deletions
per metaphase was observed in Bangkok schoolchildren
when compared with that of provincial schoolchildren. The
apparent decrease of DNA repair capacity related to PAHs
exposure may reflect any defects in DNA repaired mecha-
nism. Au et al. (55) suggested that the problem in DNA
repair process may be caused by mutation in genes that code
for DNA repair enzymes or by blockage of repair processes
on DNA (i.e. DNA adduct). In addition, it remains to be
resolved whether a threshold exists in the DNA repair
capacity induction, and repeated long-term exposure could
exhaust this induction. However, as mentioned previously,
the influence of genetic variations in metabolism of
chemicals and DNA repair which affects the function of
DNA repair enzymes on each individual for the manifestation
of chromosome aberrations and DNA damage in this study
cannot be ruled out. In our study, the levels of DNA adduct
and DNA strand breaks showed significant correlations with
the reduction of DNA repair capacity in the Bangkok
schoolchildren. The manifestation of an increase in DNA
adduct and cellular DNA damage, if not repaired, can
interfere with important cellular functions and increase risks
for development of diseases such as cancer later in life.
Hagmar et al. (56) have shown that an elevated chromosomal
aberration frequency in peripheral blood lymphocytes is
related to the increased frequency of malignant disease, and it
has a predictive value for increased cancer risk. In addition,
the study of Peluso et al. (22) and Bak et al. (23) also
suggested that bulky DNA adducts may be used as a
predictive indicator of lung cancer risk.
In conclusion, the present study indicates that schoolchil-
dren who attended schools located in proximity to high traffic
Traffic-related pollutants and increased health risk to children
density areas in Bangkok are exposed to higher levels of
genotoxic PAHs present in vehicle emissions. A significant
increased in levels of bulky carcinogen–DNA adducts and
DNA strand breaks, as well as a decrease of DNA repair
capacity in Bangkok schoolchildren was observed. These
results provide an indication that children who spend a
significant amount of time close to traffic-related sources
may be more vulnerable to health impacts associated with the
exposure to genotoxic environmental pollutants and may
have increased health risks for the development of certain
diseases such as cancer later in life due to this exposure.
Acknowledgements
We wish to thanks Mr. Duy Anh Dang Department of Environmental and
Occupational Medicine, Aarhus University, Denmark for DNA adducts
measurement. This project was supported by research grants from the
Chulabhorn Research Institute and the Post-Graduate Education, Training and
Research Program in Environmental Science, Technology and Management
under Higher Education Development Project of the Commission on Higher
Education, Thailand.
Downloaded from http://carcin.oxfordjournals.org/ by guest on August 26, 2014
Conflict of Interest Statement: None declared.
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Received May 26, 2006; revised August 28, 2006;
accepted September 2, 2006