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Carcinogenesis Advance Access originally published online on October 27, 2006
Carcinogenesis 2007 28(4):816-822; doi:10.1093/carcin/bgl175
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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 Laboratory of Environmental Toxicology, Chulabhorn Research Institute Bangkok, Thailand
2 Laboratory of Chemical Carcinogenesis, Chulabhorn Research Institute Bangkok, Thailand
3 Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus Aarhus, Denmark
4 Department 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{at}cri.or.th


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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) attending 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 Bangkok 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 schoolchildren 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.

Abbreviations: PAHs, polycyclic aromatic hydrocarbons; 1-HOP, 1-hydroxypyrene; BaP, benzo(a)pyrene; BghiP, Benzo(g,h,i)perylene.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
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 genotoxic carcinogens found in urban atmospheric pollution from motor vehicle emissions, are generated through the incomplete combustion of fossil fuels and oil products. Many PAHs including benzo(a)pyrene (BaP) have been classified 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 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 environmental 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 metabolic 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 32P-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 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 aberrations), 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 schoolchildren 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
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
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 2 l/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 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 x 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)anthracene (DBahA), excitation 291 nm, emission 400 nm; and benzo(g,h,i)perylene (BghiP), excitation 288 nm, emission 415 nm, as target PAHs. The concentration of each PAH was quantified by its peak area. The concentrations 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 ß-glucuronidase and sulphatase overnight at 37°C. C18 cartridges were used to extract 1-HOP that was eluted twice with 500 µl 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 (µmol/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).

32P-Post-labelling of DNA adducts
DNA, extracted from lymphocytes by enzymatic digestion followed by chloroform/phenol extraction, was precipitated with ice-cold ethanol. For 32P-postlabelling assay, DNA (4 µg) 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 µl of H2O. The resuspended DNA was then added to an equal volume of T4 polynucleotide kinase mixture containing [{gamma}-32P] in a total of 15 µl. 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 µl 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 µl 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 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 distance between head and tail centres of gravity).

Determination of DNA repair capacity by the cytogenetic challenge assay
The challenge assay used in this study was carried out according to the methods that have been previously described (34,35). At 24 h after blood culture, the cells were irradiated with 100 cGy using a 137Cs source at a dose rate of 5 Gy/min. Cells were blocked, 50 h after culture initiation, with Colcemid (final concentration of 0.1 µg/ml) for 1.5 h and harvested using the standard procedure. Cytological preparations were made, coded and stained with a 10% Giemsa solution for 15 min. Fifty metaphase cells were analysed from each of the duplicated slides under the microscope. The presence of dicentric chromosomes and chromosome deletions per metaphase were determined.

Statistical analysis
The Mann–Whitney two-tailed test was used to compare the biomarker levels in the different groups. Pearson correlation was used to evaluate the association between the biomarker levels. Wilcoxon sign ranks test was used to compare each individual at the different times. All the statistical analyses were performed with SPSS Statistical package (SPSS Inc-version 12.0). P-value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Ambient air pollution is significantly higher in metropolitan Bangkok due to the high traffic density. In this study, environmental and personal air samplings were used to assess the exposure to particle-associated PAHs in ambient air. Internal dose and biologically effective dose were assessed by the measurement of urinary 1-HOP and bulky carcinogen–DNA adduct respectively, as biomarkers. The potential health risks from exposure were assessed through the use of the biomarkers of early biological effects by the measurement of DNA strand breaks and DNA repair capacity.

Ambient PAHs concentrations at various sites and personal exposure to PAHs
The concentrations of total PAHs, based upon the sum of 10 PAHs, measured from the ambient air at various study sites in Bangkok as well as the provincial area of Chonburi are summarized in Table I. The ambient total PAHs concentration measured at the roadsides in close proximity to the four Bangkok schools was ~30-fold higher than that of the provincial schools (P < 0.001). Similarly, the ambient total PAHs concentration in the school areas in Bangkok was 5-fold higher than that of provincial schools (P < 0.001).


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  		Table I Ambient concentrations of total PAHs at various locations

 
Children in Thailand spend at least 8 h (8 am to 4 pm), approximately a third of a day, at school. The home environments for the Bangkok and Chonburi schoolchildren were similar in nature; however, Bangkok children are exposed to higher concentrations of ambient PAHs as a result of traffic congestion. Personal exposures to total PAHs and their corresponding BaP equivalent exposures in schoolchildren are summarized in Table II. It can be seen that personal exposures to total PAHs were ~3.5 times greater in Bangkok schoolchildren (P < 0.001). When the concentrations of carcinogenic PAHs were converted to BaP equivalents, schoolchildren in Bangkok were exposed to ~3.5-fold greater levels than schoolchildren in the provincial area (P < 0.001).


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  		Table II PAHs exposure concentrations and biomarkers of exposure in schoolchildren

 
The pattern of the 10 PAHs measured at the roadsides, in the school areas and at the breathing zone of schoolchildren obtained from each school is illustrated in Figure 1. For children in the four Bangkok schools, the predominant PAH in air samples from the breathing zone was BghiP (range 0.86–1.28 ng/m3, representing 24–37% of total PAHs burden). BghiP was also the predominant PAH in ambient air at the roadsides (2.67–21.25 ng/m3, 34–45%) as well as in the school areas (0.97–2.44 ng/m3, 29–41%). A similar 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%).


Figure 1
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  		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.

 
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 µmol/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 concentration on Day 0 did not correlate with total PAHs exposure in Bangkok schoolchildren (r = 0.145, P = 0.141); however, a weak, but significant correlation could be established between Day 1 concentration and total PAHs exposure (r = 0.244, P = 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 = –0.620, P = 0.000) or BaP equivalent exposure (rs = –0.442, P = 0.000) were observed; however, no correlation between DNA adduct level and total PAHs exposure was seen in Chonburi schoolchildren (r = 0.066, P = 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 schoolchildren 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 = –0.042, P = 0.654) or BaP equivalent exposure (r = –0.134, P = 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 = 0.288, P = 0.006; DNA adduct r = 0.443, P = 0.000) in these children.


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  		Table III Biomarkers of early biological effects in schoolchildren

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
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 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 schoolchildren. 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 x 10–5 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 excretion of 1-HOP may be a consequence of PAHs exposure from the previous day. A number of studies have been reported a relatively long half-life for urinary excretion of 1-HOP in adults. In workers exposed to coke oven, half-life for urinary excretion of 1-HOP is reported as ~18 h (41) or up to 35 h (42). No study has reported on the half-life of 1-HOP in children. The concentrations of urinary 1-HOP in Bangkok schoolchildren were lower than those reported in children living in urban areas of other Asian countries such as China (43) and in some European countries such as the Netherlands (36) and Poland (44).

The potential impact from PAHs exposure on children's health can be demonstrated by a significantly greater number of bulky carcinogen–DNA adduct observed in Bangkok schoolchildren when compared with that of the provincial schoolchildren which was 5-fold higher levels. The increase of this precarcinogenic lesion may suggest an increased health risk for the development of certain diseases such as cancer as a result of genotoxic PAHs exposure during childhood (10). An association between DNA adducts and 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 information 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 environmental 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 acknowledge 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 schoolchildren. 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 mechanism. 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 schoolchildren who attended schools located in proximity to high traffic 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.


   Acknowledgments
 
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.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 26, 2006; revised August 28, 2006; accepted September 2, 2006.


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