cytogenetic biomonitoring of primary school children exposed to air pollutants: micronuclei analysis...

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Environmental Science and PollutionResearch ISSN 0944-1344 Environ Sci Pollut ResDOI 10.1007/s11356-013-2001-6

Cytogenetic biomonitoring of primaryschool children exposed to air pollutants:micronuclei analysis of buccal epithelialcells

Gonca Çakmak Demircigil, Onur Erdem,Eftade O. Gaga, Hicran Altuğ, GülçinDemirel, Özlem Özden, Akif Arı, SerminÖrnektekin, et al.

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RESEARCH ARTICLE

Cytogenetic biomonitoring of primary school children exposedto air pollutants: micronuclei analysis of buccal epithelial cells

Gonca Çakmak Demircigil & Onur Erdem & Eftade O. Gaga & Hicran Altuğ &

Gülçin Demirel & Özlem Özden & Akif Arı & Sermin Örnektekin &

Tuncay Döğeroğlu & Wim van Doorn & Sema Burgaz

Received: 2 May 2013 /Accepted: 10 July 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract There is an increasing attempt in the world todetermine the exposures of children to environmentalchemicals. To analyze the genotoxic effect of air pollution,micronucleus (MN) assay was carried out in buccal epithelialcells (BECs) of children living in an urban city of Turkey.Children from two schools at urban-traffic and suburban siteswere investigated in summer and winter seasons for the de-termination of BEC-MN frequency (per mille) and frequencyof BEC with MN (per mille). The same children were alsorecruited for lung function measurements within a MATRA

project (“Together Towards Clean Air in Eskisehir andIskenderun”) Measured NO2 and SO2 concentrations did notexceed the European Union (EU) limit levels either in urban-traffic or suburban regions. Higher O3 concentrations weremeasured in the suburban site especially in the summer peri-od. Particulate matter (PM2.5 and PM10) levels which did notdiffer statistically between two regions were above the EUlimits in general. Although BEC-MN frequencies of childrenliving in the suburban sites were higher in general, the differ-ence between two regions was not significant either in the

Responsible editor: Philippe Garrigues

G. Ç. Demircigil : S. Burgaz (*)Department of Toxicology, Gazi University, 06330 Ankara, Turkeye-mail: [email protected]

G. Ç. Demircigile-mail: [email protected]

O. ErdemDepartment of Toxicology, Gulhane Military Medical Academy,06010 Ankara, Turkeye-mail: [email protected]

E. O. Gaga :H. Altuğ :G. Demirel :Ö. Özden :A. Arı :T. DöğeroğluDepartment of Environmental Engineering, Anadolu University,İki Eylül Campus, 26555 Eskişehir, Turkey

E. O. Gagae-mail: [email protected]

H. Altuğe-mail: [email protected]

G. Demirele-mail: [email protected]

Ö. Özdene-mail: [email protected]

A. Arıe-mail: [email protected]

T. Döğeroğlue-mail: [email protected]

S. ÖrnektekinDepartment of Chemistry, Art and Science Faculty, Mustafa KemalUniversity, 31040 Antakya, Turkeye-mail: [email protected]

W. van DoornDepartment of Clean Technology, Royal Haskoning, P.O. Box 151,6500 AD, Nijmegen, The Netherlandse-mail: [email protected]

Present Address:S. ÖrnektekinMolecular Biology and Genetics, Faculty of Science and Letters,İstanbul Kültür University, 34156 Istanbul, Turkey

Environ Sci Pollut ResDOI 10.1007/s11356-013-2001-6

Author's personal copy

summer or winter periods. BEC-MN frequencies of the urban-traffic children were found to be significantly higher in sum-mer period (mean ± SD, 2.68±1.99) when compared to winterperiod (1.64±1.59; p=0.004). On the other hand, no season-ality was observed for the suburban children. Similar resultshave been obtained in the BEC frequency with MN in ourstudy. In summer, BEC-MN frequencies were significantlyincreased with the decrease in pulmonary function levelsbased on forced expiratory flow between 25 and 75 % of vitalcapacity (FEF25–75 %) levels (p<0.05). As a conclusion, chil-dren living in urban-traffic and suburban areas in the city ofEskişehir exhibited similar genotoxicity. Seasonal variation ingenotoxicitymay be interpreted as relatively high ozone levelsand increasing time spent at outdoors in the summer.

Keywords Air pollution . Children .Personalbiomonitoring .

Buccal epithelial cells .Micronucleus assay

Introduction

It has been known that anthropogenic activities such as traffic,residential heating, and industry affect the air quality in urbanareas. High ozone (O3) concentrations during a warm and sunnyweather, oxides of nitrogen emitted from motor vehicles, andairborne particles with different size distribution (such as PM2.5,PM10) and composition were considered among the main com-ponents of air pollution (Braunekreef and Holgate 2002).Volatile organic compounds (VOCs) such as benzene, toluene,ethylbenzene, and isomers of xylene (BTEX) were related toadverse health effects (Han and Naeher 2006). The com-plex mixture of gaseous and particulate-bound pollutantssuch as polycyclic aromatic hydrocarbons (PAHs),nitrated PAHs, metals, and other toxic compounds hasbeen investigated extensively in urban locations due totheir adverse health effects (Perera et al. 1992;Kyrtopoulos et al. 2001; Sørensen et al. 2003a, b).

Epidemiological studies indicate that in adults, there is anassociation between high levels of urban air pollution andincreased risk of lung cancer. There were few studies on therisk of cancer among children living in urban sites with highlevels of hazardous pollutants (Reynolds et al. 2003;Whitworth et al. 2008). The biomarker-based molecular epi-demiology studies may help to understand relative contribu-tion of ambient air pollution as a risk factor of cancer andfacilitate health risk assessments especially under conditionsof moderate or low air pollution (Kyrtopoulos et al. 2001;Hrelia et al. 2004). Biomarkers such as DNA, protein adducts,and cytogenetic alterations (e.g., micronuclei, chromosomalaberrations) may help in identifying the exposure and reveal-ing its early effects (Sørensen et al. 2003a; Hrelia et al. 2004).

In the last 10 years, some studies have been carried out toevaluate the effect of environmental exposure to genotoxic

agents in children because of the two main concerns: chil-dren may be more sensitive than adults to genotoxic agentsand genetic damage appearing at younger ages may affect thelifetime risk of adverse health outcomes, e.g., cancer (Neriet al. 2006). These studies imply that air pollution may affectlevels of genomic stability and potentially cancer in children(Calderon-Garciduenas et al. 1997; Huen et al. 2006;Pedersen et al. 2006; Ruchirawat et al. 2007; Van Leeuwenet al. 2008).

Micronuclei frequencies represent both clastogenicity(chromosome breakage) and aneuploidy (chromosome loss)in cells studied, and it has been extensively used to identifypotential genotoxic exposures and also chromosomal instabil-ity (Fenech 2002). It was shown that there was an associationbetween micronuclei (MN) frequencies in lymphocytes andincreased risk of cancer in humans (Bonassi et al. 2007). Theusage of human exfoliated buccal cells supplies a complemen-tary method for measuring MN in an easily accessible tissuewithout tissue culture requirement for monitoring human ex-posure to occupational and environmental genotoxins (Burgazet al. 1999, 2002; Holland et al. 2008; Fenech et al. 2011).Since this tissue is on the direct route of airborne pollutantsother than nasal epithelial cells and it may metabolize proxi-mate carcinogens, it is an important target site for inhaledtoxicants and they may exemplify early genotoxic eventsinduced by carcinogenic agents entering the body (Autrupet al. 1985; Vondracek et al. 2001; Holland et al. 2008).

In this population-based study, we investigated how ex-posure to air pollutants affected cytogenetic damage bymeasuring MN frequencies in buccal epithelial cells(BECs) from primary schoolchildren living at a rapidly ur-banizing mid-northwestern city of Turkey, Eskişehir.

Materials and methods

Study design

The study presented here is a part of a Matra project(“Together Towards Clean Air in Eskisehir and Iskenderun”9S0635.01) carried out in Eskişehir to investigate air pollutionand its health effects on primary schoolchildren. Ambient airconcentrations of pollutants during study period were deter-mined by using reliable methods in the literature and details ofair quality measurements and health survey have been de-scribed elsewhere (Gaga et al. 2012; Altuğ et al. 2013).Briefly, spatial distribution of pollutants was investigated bya preliminary passive sampling campaign carried out at 65points. Based on the results of this campaign, schools for themeasurement of lung function parameters were selected (Gagaet al. 2012). Pulmonary function tests were performed for1,841 and 1,497 children from 16 schools in summer (May27–June 12, 2008) and winter (February 27–March 12, 2009)

Environ Sci Pollut Res


seasons, respectively, for health survey. Ambient air concen-trations of NO2, SO2, and O3 were measured at school gardensby passive samplers during the health survey. Concentrationsof PM2.5 and PM10 were measured at four monitoring stationsestablished at urban-traffic and suburban sites to provide PMdata for the health survey.

For MN assay, two primary schools, school A and schoolB, were selected from suburban and urban-traffic sites, respec-tively, to represent different levels of air pollution and person-al monitoring based on pollutant maps prepared by using aGeographic Information System software (ArcGIS ver. 9.0) inthe preliminary passive sampling campaign. School locationsand major pollutant sources were shown in Fig. 1 adaptedfrom Gaga and Ari (2011). Personal exposure concentrationsof BTEX, NO2, and O3 of those children were also measuredby personal passive samplers within the project only in wintercampaign (Demirel 2010; Demirel et al, Personal exposure ofTurkish primary school children to BTEX, NO2 and ozone:relationship with indoor/outdoor concentrations and risk as-sessment, Submitted).

Study area

Eskişehir (600,000 inhabitants living in the metropolitancenter) is located in the northwest of the Central Anatoliain Turkey. Its topographical structure consists of plainssurrounded by mountains, so daytime and nighttime temper-atures differ significantly especially in the winter time. The

dominant wind direction is from east to west in winter andnorthwest to southwest in summer. The major source fordomestic heating in winter season was coal before 1996and it has been gradually replaced with natural gas since1996. Currently, almost 50 % of the residences use coal forheating in winter time. Fossil fuel consumption for domesticheating and traffic is the main polluting source of air pollu-tion in Eskişehir City. Domestic heating (for SO2 and PM)and traffic (for NOx and VOCs), rather than the industry, areresponsible for the pollution in the city (Özden et al. 2008).

Study population

Buccal mucosa samples of primary schoolchildren fromschool A and school B were collected during 2 weeks ofsummer and winter ambient air sampling campaigns. Fifty(30 girls and 20 boys) and 51 (25 girls and 26 boys) childrenaged 9–13 from school A and school B, respectively, wereinvolved in buccal sampling in the summer campaign. Forty-six of the same children from school A and 47 from school Bparticipated in the winter sampling, too. The subjects of thispopulation were chosen based on the responses of theInternational Study of Asthma and Allergies in Childhood(ISAAC) questionnaire applied to whole population (ca1,667 students). The selection criteria were parental smokinghabits, gender, being in fourth and fifth grade of primaryschool, and closeness of their houses to the schools theyattend. All children were nonsmokers. Time activity data

Fig. 1 Locations of the schoolsstudied in Eskişehir



were available in winter period and children spent almost98 % of their times in indoor environments such as schooland home (Demirel 2010; Demirel et al, Personal exposureof Turkish primary school children to BTEX, NO2 andozone: relationship with indoor/outdoor concentrations andrisk assessment, Submitted).

Pulmonary function tests were carried out in both summerand winter campaigns. Forced expiratory volume in 1 s(FEV1), forced vital capacity (FVC), FEV1/FVC ratio, peakexpiratory flow (PEF), and forced expiratory flow between25 and 75 % of vital capacity (FEF25–75 %) levels of thosechildren were also measured by using a spirometer,according to the American Thoracic Society standards(American Thoracic Society 1995).

A consent form was signed by the parents who were alsorequested to complete a detailed questionnaire about person-al history, health history, routine lifestyle activities, and foodhabits of their children. The ISAAC protocol was used todetermine and compare the prevalence of asthma and relatedsymptoms among schoolchildren involved in our study. Inaddition to the ISAAC core questions, other issues on vari-ous environmental risk factors (parental smoking habits,coal/wood stove usage, having furry domestic pets, havingmolds at home, etc.) were also asked. The questionnaireswere distributed to the children at school to be completed bythe parents at home. This study was approved by the localethical committee of Mustafa Kemal University, Faculty ofMedicine in agreement with the Helsinki Declaration(April 9, 2008, no. 4/22).

MN assay in exfoliated buccal cells

Children were asked to rinse their mouths with water, and a pre-moistened wooden tongue depressor was used to sample cellsfrom the both sides of the inside of the cheeks. The spatula wasapplied to a pre-cleaned and pre-moistened microscope slide.For each individual, two slides were prepared by smearing thecells onto pre-cleaned slides. Smears were air dried and fixed in80%methanol. Slides were stained by the Feulgen reactive andthen counterstained with Fast Green. Criteria of scoring weredescribed by Tolbert et al. (1992). One thousand differentiatedand basal cells per subject for each season, i.e., summer andwinter in total 2,000 cells, were scored on coded slides by twoobservers blindly using a light microscope (Zeiss, Primo Star).Degenerated cells were observed but not scored. In this study,two measures of MN frequency were used, i.e., the meanfrequency of MN per thousand buccal epithelial cells (BEC-MN frequency) and the mean frequency of cells bearing at leastone micronucleus per thousand buccal epithelial cells (frequen-cy of BECwith MN). These two indexes have been consideredsimilar performances; BEC-MN frequency was suggested aspotentially more sensitive; however, the frequency of BECwithMN was suggested as more stable (Ceppi et al. 2010).

Statistical analysis

Data analysis was performed by using SPSS for Windows,version 11.5 (SPSS Inc., Chicago, IL, USA). Shapiro–Wilkinson test was used for the fitness of the variables tothe normal distribution. Continuous variables were repre-sented as mean ± standard deviation or median (minimum–maximum), where appropriate. Wilcoxon signed rank testwas used to test the seasonal differences of the dependentparameters. Mann–Whitney U test was applied for determin-ing the median differences. Degrees of associations betweencontinuous variables were analyzed by Spearman's correla-tion tests. A p value less than 0.05 was considered statisti-cally significant. Of all possible multiple comparison tests,Bonferroni correction was applied for controlling type Ierror.

Results

Ambient air concentrations of the pollutants during the studyperiod

As mentioned before, concentrations of NO2, SO2, and O3

were measured at school gardens by passive sampling duringthe buccal measurements in both seasons. Figure 2 shows theseasonal weekly average air pollutant concentrations (micro-grams per cubic meter) for the selected schools. Passivesamplers were deployed to sampling points and left for aweek, collected back, and analyzed to represent weekly pol-lutant average. Two weekly consecutive sampling was carriedout and the results of which are presented in Fig. 2. AmbientNO2 concentrations of the urban-traffic school (school B) inthe summer period were almost 2.2 times greater than thesuburban school (school A) (12 vs 5 μg/m3). The differenceis more pronounced in winter time having approximately fivetimes greater NO2 concentrations than the suburban school(39 vs 7 μg/m3). Considering all other passive samplingpoints, average concentrations of NO2 in urban-traffic siteswere higher than suburban sites both for the summer andwinter seasons. European Union (EU) set an annual limitvalue of 40 μg/m3 for NO2. Although summer and winteraverage NO2 values obtained in this study may not representthe whole year, neither summer nor winter season averagesexceeded the annual EU limit value.

An inverse trend was observed for the O3 concentrationshaving higher concentrations in suburban school in both sea-sons which is related with the chemical reactions between O3,NOx, and VOCs. There is a factor of 1.5 difference betweentwo the schools considering O3 concentrations in summerperiod (122 vs 79 μg/m3) and the difference was more pro-nounced in the winter period (90 vs 36 μg/m3). Again, a similartrend was observed in both seasons considering all sampling



points in urban-traffic and suburban sites. Comparison of week-ly average O3 concentrations with 8 h average EU limit value is

not appropriate. The EU limit value is 120 μg/m3, and it shouldnot be exceeded no more than 25 days per calendar yearaveraged over 3 years. However, we may note that measuredweekly average concentrations of O3 in school Awere close oreven slightly above this value in the summer period.

Regarding SO2 concentrations, it seemed that summer SO2

concentrations at the suburban school were measured higher(17 μg/m3) than the urban school (10 μg/m3). On the otherhand, in the winter period, SO2 average concentration of urbanschool was 64 μg/m3, whereas it was 28 μg/m3 for thesuburban school. The possible reason for elevated SO2 levelsin the urban-traffic school was widespread use of coal/woodstoves for residential heating in the winter season. In a previ-ous study, it was found that the main reason of elevated SO2

emissions in Eskişehir is the coal use in residential heating(Özden et al. 2008; Altuğ et al. 2013). However, any signif-icant difference was not observed considering all samplingpoints in urban-traffic and suburban sites in summer andwinter periods (Gaga et al. 2012; Altuğ et al. 2013). Dailyaverage concentration of 125 μg/m3 SO2 which should not beexceeded more than three times in a calendar year was set as astandard by EU. Weekly SO2 concentrations measured bypassive samplers in summer and winter periods did not exceedthis limit value. Actually, the average of summer and winterSO2 concentrations was far below 125 μg/m3.

Particulate matter data collected from urban and suburbanstations do not reveal any differences between urban andsuburban averages. In the summer period, daily averageconcentrations of PM10 measured at suburban stations(85.9±22.60 μg/m3) were not significantly higher than urbanstations (75.0±17.34 μg/m3). Similarly, daily average con-centrations of PM2.5 at urban stations (25.2±6.79 μg/m3)were also not found to be significantly higher than suburbanstations (22.6±7.13 μg/m3) in the summer period. SummerPM10 averages varied from 65 μg/m3 (urban stations) to102 μg/m3 (suburban stations).

A similar trend was also observed for the winter PM10

concentrations which varied from 55.9 μg/m3 (urban station1) to 120.7 μg/m3 (urban station 2). Considering averagewinter concentrations, there was no significant differencebetween urban and suburban stations having average dailyconcentrations of 45.7±17.2 and 47.6±17.1 μg/m3, respec-tively. In fact, considering all PM10 and PM2.5 concentra-tions, it can be said that there is not any sharp differencebetween urban and suburban stations in both seasons.

PM10 concentrations exceeded the daily EU limit val-ue of 50 μg/m3 (daily limit that should not to beexceeded more than 35 days in a year) in all fourstations almost everyday during summer and winter pe-riods. Annual limit value of PM2.5 proposed by EU to beattained by 2015 is 25 μg/m3 (Directive 2008/50 CE,Annex XI) and it was also exceeded during summer andwinter campaigns.

Fig. 2 Seasonal weekly average air pollutant concentrations (micro-grams per cubic meter) in schools A and B



Demographic characteristics, personal exposureconcentrations, and pulmonary function test results

As it can be seen from Table 1, the children from school A andschool B were not statistically different according to theirgender, parental smoking habits, and maximum parental edu-cation level (p>0.05). On the other side, the use of coal/woodstoves for domestic heating was significantly more wide-spread in the urban school (p<0.05). Data for parental educa-tion were used as proxy for socioeconomic status in this study.Age of children seemed to vary significantly between tworegions, although the distributions were narrow (p<0.05).Even the if age range of the children in the schools were 9–13, most of the children were in the age group of 10–11 years.Despite the significant difference in age, the height of childrenin school A and school B was not significantly different.

Regarding respiratory complaint data those recruited fromthe ISAAC questionnaire, 10% of the children were asthmaticin school A, whereas it was 3.9 % in school B. According tothe health survey held in the summer sampling campaign,children were found to complain of wheezing (during or afterexercise), dry cough at night (apart from a cough associated

with a cold or chest infection), and whistling in the chest in theprevious 12 months in school A (8, 44, and 12, respectively)and school B (2, 25.5, and 5.9 %, respectively). As shown inTable 1, the prevalence of asthma symptoms (ever diagnosedasthma or wheezing in previous 12 months or cough at nightin previous 12 months or wheezing/whistling ever orwheezing/whistling in the previous 12 months) was signifi-cantly higher in children from school A (p<0.05). FEF25–75 %

was significantly higher for summer, whereas FEV1/FVCvalue was significantly higher for winter sampling period inschool A vs school B (p<0.05, Table 1). PEF values ofchildren from school A were significantly higher both forsummer and winter periods (p<0.05, Table 1).

The personal BTEX, NO2, and O3 levels were shown inTable 2. Accordingly, BTEX and NO2 levels of children inschool Awere significantly lower than those in school B, whilethe O3 levels were significantly higher (p<0.05, Table 2).

Buccal epithelial cell micronucleus frequencies

To classify MN frequency, a total of 194,000 buccal epithe-lial cells was analyzed in the present study, giving an overall

Table 1 Characteristics and pulmonary function test measurements of the children in schools A and B

School A (suburban) School B (urban-traffic)

Summer Winter Summer Winter

Children (n) 50 46 51 47

Gender

Boys (n) 20 19 26 24

Girls (n) 30 27 25 23

Age* (years; mean ± SD) 10.32±0.59 10.00±0.28

Heighta (cm; mean ± SD) 138.3±8.4 138.9±9.3

Parental smoking habitsb 27 (54 %) 24 (47.1 %)

Coal/wood stove usage [n (%)] 2 (4 %) 9 (17.65 %)

Maximum parental education [n (%)]c 12 (24 %) 12 (23.53 %)

Asthma symptoms [n (%)]d 26 (52 %) 13 (25.49 %)

Children with PFT (n) 43 43 38 38

FVC (L; mean ± SD) 2.10±0.34 2.50±0.47 2.11±0.33 2.38±0.41

FEV1 (L; mean ± SD) 1.86±0.26 2.04±0.32 1.78±0.30 1.93±0.28

FEF25–75 % (L/s; mean ± SD) 2.39±0.58** 2.17±0.81 2.01±0.61 2.05±0.78

PEF (L/s; mean ± SD) 3.99±0.78** 4.49±1.02*** 3.72±0.87 4.22±0.96

FEV1/FVC 0.892±0.06 0.828±0.09*** 0.845±0.08 0.819±0.09

SD standard deviation, PFT lung function test, FVC forced vital capacity, FEV1 forced expiratory volume in 1 s, FEF25–75 % forced expiratory flowbetween 25 and 75 % expired volumes, PEF peak expiratory flow, FEV1/FVC values for both summer and winter seasons

*p<0.001 (school A vs school B), **p<0.05 (school A vs school B for summer), ***p<0.05 (school A vs school B for winter)aMeasured at initial (summer period) lung function testb At least one person smokes in the child's homec Education of 8 years or mored Prevalence of any of the asthma symptoms (ever had asthma or wheezing in previous 12 months or cough at night in previous 12 months orwheezing/whistling ever or wheezing/whistling in the previous 12 months)



MN frequency of 2.33±1.87 (mean ± SD) and cells withmicronuclei frequency of 1.98±1.48 in children living inEskişehir.

The MN frequencies of the children those were availablein both seasons were involved in the statistical analysis,namely the data of 46 children in school A (suburban) and47 children in school B (urban) as their own controls werepresented in Tables 3 and 4.

There were no statistical differences between summer andwinter MN frequencies of suburban children (school A)(p>0.05) (Table 3). On the other hand, MN frequencies ofurban-traffic children (school B) were significantly higher inthe summer than that of the winter values (p<0.05, Table 3).There were no significant differences between schools A and Bin both seasons regarding MN frequencies (p>0.05, Table 3).Similar findings were also observed with cell frequencies withMN (Table 3). Passive smoking and gender were found to benot related withMN frequencies. Although, themean age of thechildren between the schools was significantly different, therewas no relationship between the age of the children and MNfrequencies. Coal/wood stove usage, education level of parents,and prevalence of any of the asthma symptoms were not relatedwith MN frequencies for both seasons (p>0.05).

The MN frequencies of the children from both of theschools treated as one group were significantly increased insummer time compared to the winter time MN frequenciesand cell frequencies with MN (p<0.05, Table 4).

The relationship of the MN frequencies and the pulmo-nary function tests revealed negative correlation between theFEF25–75 % and MN frequency and cell frequency with MN insummer sampling period (r=−0.254, p=0.02 and r=−0.220,p=0.04, respectively). There was no significant associationbetween pulmonary function parameters and BEC-MN fre-quencies for the winter period. On the other side, the height ofchildren was significantly negatively correlated with bothMNfrequency and cell frequency with MN for the winter period(r=−0.310, p=0.006 and r=−0.278, p=0.01, respectively).Height and age are known to be the major determinants ofpulmonary function. According to the personal monitoring, o-xylene levels were negatively correlatedwith theMN frequen-cy and cell frequency with MN (r=−0.285, p=0.04 andr=−0.320, p=0.02, respectively).

Discussion

Air pollution is an established health hazard in many citiesaround the world because ambient air contains a complexmixture of gaseous and particulate-bound compounds withinmany of them are genotoxic and potentially carcinogenic.Long-term adverse effects of moderate or low levels ofpollutants are not always clearly shown in the epidemiolog-ical studies (Carere et al. 2002). Especially in small-scalestudies, inherent limitations were suggested to be less suit-able for showing the subtle effects of air pollution(Brunekreef and Holgate 2002). At that point, the need forbiomarker-based population studies is taking attention. Thegenotoxicity assays among molecular epidemiology studies

Table 2 Personal exposure concentrations (micrograms per cubicmeter; mean ± SD) of children (n), in suburban and urban sites ofEskişehir, to O3, NO2, and BTEX in the winter period

School A* School B

O3 50.58±14.32 (26) 26.67±6.46 (24)

NO2 31.66±10.20 (26) 51.17±17.44 (25)

Benzene 0.92±0.80 (26) 2.22±0.85 (26)

Toluene 17.18±31.20 (26) 28.43±13.41 (25)

m+p-Xylene 0.69±0.41 (26) 1.45±0.71 (26)

o-Xylene 0.49±0.33 (26) 1.09±0.58 (26)

Ethylbenzene 0.43±0.33 (26) 0.90±0.43 (26)

Compiled from Demirel (2010). Numbers in parenthesis represent thenumber of samples measured

*p<0.001, school A vs school B for BTEX, NO2, and O3

Table 3 Comparison of BEC-MN frequencies and BEC fre-quencies with MN of the chil-dren living in Eskişehir by loca-tion and season

a School A (suburban) vs schoolB (urban-traffic)b Summer vs winter

Season School A(suburban)

School B(urban-traffic)

p value* School A(suburban)

School B(urban-traffic)

p valuea

n=46 n=47 n=46 n=47

Summer period 2.78±2.00 2.68±1.99 >0.05 2.78±2.00 2.68±1.99 >0.05

Winter period 2.11±1.70 1.64±1.59 >0.05 1.87±1.47 1.38±1.15 >0.05

p valueb >0.05 0.004 >0.05 0.005

Table 4 Comparison of BEC-MN frequencies and BEC frequencieswith MN of children living at Eskişehir by season

Season Total population (school A + school B)

MN frequencies(‰) (mean ± SD)(n=93)

BEC frequency withMN (‰) (mean ± SD)(n=93)

Total cellnumbercounted

Summer period 2.73±1.98 2.28±1.57 93×103

Winter period 1.87±1.66 1.62±1.33 93×103

p valuea 0.001 0.003

a Summer vs winter



gained wide acceptance as to be sensitive to detect the effectsof environmental exposures to many pollutants (Carere et al.2002; Han and Naeher 2006).

In the present study, we evaluated the genotoxic effects ofambient air pollution by using MN assay in exfoliated buccalcells from the same children in summer and winter seasons.Since children have different patterns of breathing thanadults, deposition of inhaled pollutants may change. At rest,children are more commonly mouth breathers, while adultstypically breathe predominantly through the nose (Batesonand Schwartz 2008). Thus, the use of exfoliated buccal cellsseems to be a reasonable choice for studying the effects of airpollutants in children.

The study was carried out in Eskişehir which is a rapidlyurbanizing city in Turkey. The results of the emission inven-tory study of Özden et al. (2008) for Eskişehir indicated thatthe contribution of domestic heating to SO2 and PM pollu-tion were 70 and 84 %, respectively, while traffic was re-sponsible for 60 % of the total NOx emissions. Domesticheating (for SO2 and PM) and traffic (for NOx and VOC)were found to be responsible for the pollution in the city(Özden et al. 2008). Considering seasonal contributions, coalheating in winter prevailed on higher SO2 and PM concen-trations and high traffic densities on higher NO2 concentra-tions. Complex photochemical reactions were suggested tobe the reason of higher O3 concentrations in remote areasduring summer season (Özden et al. 2008). As compared tothe data of some cities in Turkey and in the world havingsimilar populations, the air quality in Eskişehir may beconsidered moderate (Özden et al. 2008).

In the present study, winter and summer campaigns forgenotoxicity assessment in the children were carried out intwo schools located at suburban and urban-traffic sites inEskişehir. In the winter sampling campaign of genotoxicityassessment, personal sampling of BTEX, NO2, and O3 wasalso carried out. Our data on genotoxicity indicated thatchildren living in the urban region of the city were notdifferent from those living in the suburban region. In theurban site, summer season MN frequencies were significant-ly higher compared to the winter season. Similarly, regard-less of discrimination for urban and suburban of the city, thetotal MN frequencies of the children were also significantlyhigher in summer season. Similar results have been obtainedwith two measures of MN frequency (MN frequency andBEC frequency with MN) in our study (Tables 3 and 4).

In order to investigate the health risks of children due toair pollution, co-exposures as well as interactions of the airpollutants should be considered (Buthbumrung et al. 2008).Personal exposures may be affected from different variablessuch as activity, location, and microenvironmental condi-tions (Chang et al. 2003; Ashmore and Dimitroulopoulou2009); therefore, it is also critical to not consider only ambi-ent air concentration measurements (Weisel 2002).

In our study, regarding PM monitoring data and school-based ambient passive sampling results of SO2, no significantdifferences were observed between urban and suburban regionswhich were in line with the genotoxicity data. Concentrationsof NO2 measured at urban locations were found to be signifi-cantly higher than suburban regions in both seasons and anopposing significant trend was observed for the O3. On theother hand, all the pollution components analyzed in the regionby personal sampling showed higher levels in the urban regionexcept for O3 having higher values in suburban region.Personal exposure levels of BTEX, NO2, and O3 were notfound to be associated with the MN findings except for thenegative association for o-xylene. It should be kept in mind thatchildren spent relatively less amount of time at the outdoors in avery cold winter in Eskişehir (Demirel 2010; Demirel et al,Personal exposure of Turkish primary school children toBTEX, NO2 and ozone: relationship with indoor/outdoor con-centrations and risk assessment, Submitted). Such informationwas not available for the same children in summer period.However, it is obvious that children spent more time at theoutdoors in the summer season (Gauderman et al. 2000). Theremight be strong indoor sources for NO2 such as cooking,tobacco smoke, woodstoves, and fireplaces which may resulthigher indoor concentrations (Cyrys et al. 2000; Chao, 2001;Willers et al. 2006). In fact, personal exposure concentrations ofNO2 were measured higher than ambient air concentration inthis study. On the other hand, there is no strong indoor source ofO3, and in some studies, it has been found that indoor concen-trations are affected from outdoor concentrations (Chao 2001;Blondeau et al. 2005). Considering low rates of home ventila-tion during winter period, one might expect to measure lowerindoor and personal O3 concentrations than ambient levelswhich are the case for this study.

The only pollutant with a parallel trend with the MN fre-quencies was O3 in Eskişehir. Personal O3 concentrations weremeasured significantly higher in suburban schoolchildren inwinter period, whereas similarly higher MN frequencies insuburban were found; however, it was not significant. Also,O3 levels were higher in the summer period when compared tothe winter period which is in line with the seasonal pattern ofBEC-MN frequencies.

The number of asthmatic children based on the question-naires from school A in suburban region was almost twofoldof the ones from the school B in urban region. Similarly, therespiratory symptoms such as cough or wheezing werehigher in the suburban region of Eskişehir. Higher preva-lence of asthma and related symptoms in suburban schoolmay be associated with ambient O3 pollution. Multivariablelogistic regression analysis results of the large dataset from1,667 children and 16 schools found a significant positiveassociation between summertime O3 concentrations andprevalence of the asthma symptoms (unpublished data),which is supporting our findings. Also, subacute effects of



air pollution on impaired lung function and current symp-toms in schoolchildren were estimated by multivariate logis-tic regression analyses in the large dataset from 16 schools.The results of this recent study showed that increasing ozoneconcentrations may cause short-term impairment in lungfunction of school-aged children (Altuğ et al. 2013).

Moreover, summer MN frequencies were significantlyincreased with the decrease in pulmonary function test.Considering the relationship identified between summertimeO3, which is significantly higher in the summer period (Gagaet al. 2012), with asthma and related symptoms, an increasein O3 may also be related with genotoxic effects.

The study of Clark et al. (2010) suggested that the higherlevels of air pollution exposure in the early years of lifeincreased the risk of asthma in the preschool period. Theyalso concluded that the burden of the disease had a significantincrease on a population level, not at an individual level,because the urban and suburban settings can show similartrend of air pollution levels derived from traffic (Clark et al.2010). Rossnerova et al. (2011) recently showed that smokingasthmatic children in the most polluted region of the CzechRepublic, Ostrava, were at higher risk of DNA damage mea-sured as the frequency of micronuclei in peripheral bloodlymphocytes when compared to the non-asthmatic children.Neri et al. (2006) reviewed genotoxicity studies on childrenexposed to environmental pollutants with the biomarkers ofchromosomal aberrations, micronuclei, DNA adducts, andDNA damage in peripheral lymphocytes and exfoliated epi-thelial cells. Following this review, genotoxicity studies withregards to the effects of air pollution on children were contin-ued (Huen et al. 2006; Mielzyńska et al. 2006; Pedersen et al.2006; Ruchirawat et al. 2007; Tuntawiroon et al. 2007;Buthbumrung et al. 2008).

Among the aforementioned studies, a limited number ofstudies analyzed genotoxicity in exfoliated epithelial cells ofchildren exposed to air pollution (Calderon-Garciduenaset al. 1997; Valverde et al. 1997; Lahiri et al. 2000; Huenet al. 2006), whereas the rest used peripheral blood lympho-cytes. Calderon-Garciduenas et al. (1997) and Valverde et al.(1997) used comet assay in their studies. Calderon-Garciduenas et al. (1997) found higher DNA damage in thenasal epithelial cells of children (n=148) living in an area ofhigh air pollution when compared with those in relativelylow pollution area (n=19) in Mexico. Atmospheric pollut-ants and meteorological conditions were monitored in thepolluted region (O3, SO2, and NO2) (Calderon-Garciduenaset al. 1997). Valverde et al. (1997) observed an increasedDNA damage in lymphocytes and nasal cells, but not in thebuccal cells of young adults (mean age 19 years) in thesouthern part of the Mexico City compared with those inthe northern part of the city. In the southern part of the city,average of the time at which O3 concentrations exceeded thehourly national standard value (110 ppb=235.4 μg/m3) was

1.46 h, while in the northern part, it was 0.93 h (Valverdeet al. 1997). Lahiri et al. (2000) and Huen et al. (2006)carried out buccal epithelial cell MN assay on children toinvestigate the genotoxicity of air pollutants. Lahiri et al.(2000) found that MN frequency in buccal epithelial cells ofurban children (n=153) was significantly higher than therural ones (n=116) (2.2 vs 1.7 ‰, respectively) living inCalcutta. The PM10 annual average concentrations (133–232 μg/m3) which were far above their national standard of60 μg/m3 were also reported in the same study. Compared toour study, it can be said that measured PM10 levels wereconsiderably higher in Calcutta. An increase in the preva-lence of respiratory symptoms such as cough and wheezingin the urban region of Calcutta has been also reported andsuggested as the indication of the air pollution.

Huen et al. (2006) analyzed the associations between geo-graphic information system-based air pollution levels as asurrogate of traffic-related pollutants and micronucleus fre-quencies of children and adults. Accordingly, they (Huen et al.2006) demonstrated associations between regional O3 levels(ranged between 0.01 and 0.03 ppm, 8 h average) and buccalMN frequencies of 65 children (0.67‰), which agreed with alongitudinal study of Chen et al. (2006). Huen et al. (2006)observedMN induction by summertime in buccal cells, whichwas in accordance with our study. Genotoxicity studies eval-uate the effects of ambient air pollution on children living inurban-traffic vs relatively suburban regions reflecting differ-ences due to the location of the study (geographical andmeteorological conditions, traffic density, atmospheric pollut-ants) and the characteristics of the study population (diet,exercise, lifestyle, population size) which are complicatingthe comparisons. In our study, since we could not control thebackground exposure conditions, it was difficult to identifythe major pollutant(s) responsible for the observed effects oncytogenetic damage as MN in buccal cells of children fromEskişehir. Soares et al. (2003) suggested that chronic exposureto urban air pollutants, e.g., PM10, CO, NO2, and S02, maycause MN induction in peripheral erythrocytes of rodentsin vivo. Coronas et al. (2009) showed the mutagenic activityof PM10 samples (9–62 μg/m3) due to presence of oil refineryand the increased primary DNA damage by the Comet assayin individuals living and/or working in this area. On the otherhand, these authors did not find significant differences in MNfrequencies in buccal mucosa cells. Recent findings ofKoehler et al. (2010) indicated that there was no DNA damageby the micronucleus assay in human nasal cells of ten patientsexposed to N02 ranging between 0.01 and 10 ppm for half anhour. However, those authors emphasized that further researchshould be needed to resolve the effects of long-term exposure.

As a conclusion, children from Eskişehir for both urban-traffic and suburban sites may be vulnerable to the genotoxicdamage especially in summer season probably due to rela-tively high ozone concentrations in our study.



Acknowledgments We would like to thank Kees Meliefste and Insti-tute for Risk Assessment Sciences for their support during field studies.We also thank very much to the school directors and teachers for theirvery valuable help during the sampling studies. The authors are gratefulto the children and their parents for their participation in this study. Wewould also like to thank Mr. Salih Ergocen for performing the statisticalanalysis.

This study was part of the MATRA project “Together Towards CleanAir in Eskişehir and İskenderun” financed by the Dutch Ministry ofForeign Affairs.

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