Ecotoxicology and Environmental Safety 92 (2013) 265–270

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Ecotoxicology and Environmental Safety

0147-65

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journal homepage: www.elsevier.com/locate/ecoenv

Bio-monitoring for the genotoxic assessment in road construction workers asdetermined by the buccal micronucleus cytome assay

Ayla C- elik a,n, Seda Yildirim b, Seda Yaprak Ekinci b, Bahar Tas-delen c

a Mersin University, Faculty of Science and Letters, Department of Biology, 33343 Mersin, Turkeyb Mersin University, Graduate School of Natural and Applied Science, Mersin, Turkeyc Mersin University, Faculty of Medicine, Department of Biostatistics, Mersin, Turkey

a r t i c l e i n f o

Article history:

Received 30 November 2012

Received in revised form

24 January 2013

Accepted 25 January 2013Available online 6 March 2013

Keywords:

Cytogenetic monitoring

Asphalt exposure

Buccal micronucleus cytome assay

Repair index

13/$ - see front matter & 2013 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2013.01.030

esponding author. Fax: þ90 324 3610047.

ail address: aylace67@gmail.com (A. C- elik).

a b s t r a c t

Buccal micronucleus cytome (BMCyt) assay monitors genetic damage, cell proliferation and cell death

in humans exposed to occupational and environmental agents. BMCyt is used as an indicator of

genotoxic exposure, since it is associated with chromosomal instability. There is little research on the

occupational exposure among road construction workers for genotoxicity testing. In the present study,

we evaluated MN frequencies and other nuclear changes, karyorrhexis (KR), karyolysis (KL), broken egg

(BE), binucleate (BN), condensed chromatin cell (CCC), and picnotic cell (PC) in buccal mucosa cells of

40 road construction workers (twenty smokers and twenty non-smokers) and 40 control groups

consisting of healthy persons (twenty smokers and twenty non-smokers). Microscopic observation was

performed of 2000 cells per individual in both road construction workers and control group. In control

and worker groups, for each person repair index (RI) was calculated via formula KRþL/BEþMN. The

results showed a statistically significant increase in the frequency of MN in buccal epithelial cells of

exposed group compared with control group (po0.001). There is no significant difference between

smokers and non-smokers for incidence of MN or nuclear changes and value of RI in exposed group. In

road construction workers, RI is lower than the control group. There is a significant difference between

workers and control group (po0.001) for RI. Our data reveal that asphalt fumes during road paving

operations are absorbed by workers and that asphalt fume exposure is able to significantly induce

cytogenetic damage in buccal mucosa cells of workers after controlling some possible confounding

factors, such as age, sex and smoking habits.

In addition to determination of nuclear changes and the micronucleus, the determination of RI value

presents a new approach to genotoxic bio-monitoring assessment studies of occupationally exposed

population.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Asphalts (identified as bitumens) are complex hydrocarbonmixtures with molecular weights ranging from 500–2000, highboiling range (above 500 1C) (Schreiner, 2011). The workersengaged in such occupation are exposing themselves to coal-tar,bitumen and asphalt fumes by inhalation and dermal absorption(McClean et al., 2004). These fumes are petroleum derivativecompounds and contain complex types of polycyclic aromatichydrocarbons (PAHs) and heterocyclic compounds (King et al.,1984) and may be inhaled or deposited on skin or clothing. Mostof them such as Benzo (a) pyrene, phenanthrene, naphthaleneand chrysene are carcinogenic in nature (Machado et al., 1993).The Benzo (a) pyrene (B (a) P), a representative of polycyclicaromatic hydrocarbons (PAHs), is found as classic DNA-damaging

ll rights reserved.

agent (Gupta et al., 1988). Humans are exposed to PAHs byinhalation, ingestion, and skin contact. The main route of occupa-tional exposure is, in most industries, inhalation; in many cases,however, skin exposure represents an important route (McCleanet al., 2004).

Accumulating evidence suggests that petroleum derivativecompounds constitute a complex mixture of chemicals, some ofthem are well-known genotoxic agents (Pitarque et al., 1997). Thepotential risk for developing cancer has been investigated amongroad workers exposed to asphalt or bitumen fumes and it isdetermined that lung cancer frequency is over than 100.(Partanen and Boffetta, 1994; Partanen et al., 1995; Reinke et al.,2000). Therefore, risk assessment represents relevant data forprotecting these individuals against potential harm, such as cancerin different target tissues, especially because there are few data inthe literature as well as conflicting results so far (C- elik et al., 2003).

Biomarkers used in toxicology to assist in diagnosing, stagingdisease as well as evaluating the risk assessment are divided intothree groups: 1. to define the exposure to carcinogenic agents, 2.

Table 1General characteristics of exposed and control groups.

Study groups N Age

mean7SE

Years of smoking

cigarette

mean 7SE

Years of

employment

mean 7SE

Control

Smokers 20 27.870.39 9.5070.19 �

Non-smokers 20 27.070.57 � �

Total 40 27.470.34 9.5070.19 �

Workers

Smokers 20 39.071.89 21.972.16 8.2572.06

Non-smokers 20 43.371.06 � 9.6571.39

Total 40 34.370.97 21.972.16 8.9571.23

N, number of subjects; SE, standard error.

A. C- elik et al. / Ecotoxicology and Environmental Safety 92 (2013) 265–270266

to show biological effects on the target tissue and 3. to giveinformation about the individual susceptibility. A variety ofassays has been proposed to determine potential biomarkers inbio-monitoring studies, including those that assess metaphasechromosomal aberrations, sister chromatid exchanges and hostcell reactivation. However, these methods are typically laboriousand time-consuming or require highly trained technicians toaccurately read and interpret slides (Induslki and Lutz, 1997).

The BMCyt assay has been used to measure biomarkers of DNAdamage (micronuclei and/or nuclear buds, Broken egg), cytoki-netic defects (binucleated cells) and proliferative potential (basalcell frequency) and/or cell death (karyorrhexis, karyolytic, picno-tic cells and cells with condensed chromatin). The formation ofmicronuclei can be induced by substances that cause chromo-some breakage (clastogens) as well as by agents that affect thespindle apparatus (aneugens) (Belien et al., 1995). Buccal mucosais considered as a suitable target site for monitoring humanexposure to occupational and environmental genotoxins. Mucosalcells are not only in the direct route of exposure to ingestedpollutants but also capable of metabolizing chemical agents toreactive species, for example 7,12-dimethylbenz[a]-anthracenevia gamma-glutamyl transpeptidase (GGT) activity (Zhang andMock, 1989; Zhang, 1994).

In the present study, we studied cytogenetic and toxicitybiomarkers in oral mucosa cells obtained from road constructionworkers and control subjects using BMCyt. In order to monitorcytogenetic effects ‘‘micronucleus and broken egg’’, cytotoxiceffects ‘‘binucleate’’, apoptotic effects ‘‘karyolysis andkaryorrhexis, picnotic cell, condensed chromatin’’ were alsoevaluated in this study. Epidemiological studies have oftenreported abusive consumption of smoking habits to be associatedwith increased occurrence of different kinds of oral carcinoma(Boffetta et al., 1992; MacFarlane et al., 1995). Probably, theconstant action of mutagenic agents such as chemical exposureand tobacco increases the rate of cellular deaths, as indicated bythe significant increase of this anomaly in workers exposed andcancer patients. In fact, production of KL and KR anomalies shouldrepresent the repair process due to intercellular selection, occur-ring before cytogenetic damages, as indicated by BE and MNstructures. Repair index may reflect the dynamic of carcinogen-esis (Ramirez and Saldanha, 2002). A different way to evaluate thecarcinogenesis process in oral cancer based on metanucleatedanomalies was recently proposed by Bhattathiri (2001).

The aim of this study was to evaluate cytogenetic or cytotoxicdamage and cellular death induced by exposure to asphalt,petroleum product, using BMCyt. In this study, workers occupa-tionally exposed to asphalt (bitumen fumes) and control groupwere monitored for buccal MN and nuclear changes.

2. Materials and method

2.1. Study population

The study was carried out in a group of 40 male road construction workers

(twenty smokers and twenty non-smokers) working in asphalt work from the

province of Mersin (TURKEY) occupationally exposed to different mixtures of

chemicals.

Participants (control and exposed groups) were informed about the objectives

of the study. They were asked to sign an informed consent form and each

participant was personally interviewed by a standard questionnaire having

information related to confounding factors like age, sex, smoking, alcohol intake,

tobacco chewing, personal medical history and occupational history of exposure

to bitumen, asphalt and coal-tar fumes. Table 1 reports the main characteristics

age, smoking habit, and years of exposure of both groups. None of exposed and

control group had alcohol habit. This questionnaire is similar to that published by

the International Commission for Protection against Environmental Mutagens and

Carcinogens. The control group consisted of 40 healthy men (twenty smokers and

twenty nonsmokers) working (academic and administrative person) on the Mersin

University campus, without indication of any exposure to solvents or other

potential genotoxic substances. None of these study groups showed significant

differences with regard to lifestyle and personal factors. To evaluate lifestyle

factors, donors were stratified as smokers or nonsmokers. Smokers were current

smokers and they smoked 4100 cigarettes in last 3 months.

2.2. Cytome assay

Subjects were asked to rinse their mouths with water before sampling. Buccal

epithelial cell samples were collected with a wooden spatula at the end of the

work shift. The samples were then applied to clean microscope slides. Slides were

coded by one author (A.C- .). Smears were air dried and fixed in methanol: acetic

acid (3:1). Slides were stained by the Feulgen reaction technique according to

Stich and Rosin (1984). Microscopic analysis was performed by two authors (S.Y.E.

and S.Y.) blinded to sample type. Two slides were prepared for each subject, and

1000 cells were evaluated per slide to determine micronucleus (MN) frequencies

and nuclear changes. Nuclear changes were classified according to Thomas et al.

(2009) as follows:

�

Normal basal cells have a larger nucleus-to-cytoplasm ratio than the differ-

entiated buccal cell.

�

Normal differentiated cells have a uniformly stained nucleus and are distin-

guished from basal cells by their larger size and by a smaller nucleus-to-

cytoplasm ratio.

�

Cells with micronucleus are identified by the presence of both a main nucleus

and one or more smaller nuclear structures. A micronucleus should be less

than one-third of the diameter of the main nucleus; on the same plane of

focus; have the same colors, texture, and refraction as the main nucleus; have

a smooth, oval, or round shape and be clearly separated from the main

nucleus.

�

Cells with nuclear buds (broken egg) contain a nucleus with an apparent sharp

constriction at one end of the nucleus. The nucleus and nuclear buds are

generally in very close proximity and appear to be attached to each other.

Buds have similar staining intensity as the main nucleus, and these are called

‘‘broken egg.’’

�

Binucleated cells contain two main nuclei instead of one. The nuclei are usually

close and of similar size and staining intensity.

�

Buccal cells with condensed chromatin show a nucleus structure with aggre-

gated chromatin, and in these cells the nucleus exhibits a striated pattern.

�

Picnotic cells are defined by a small contracted nucleus with a high density of

nuclear material that is uniformly but intensely stained.

�

Karyorrhectic cells have a nucleus containing extensive aggregated chromatin.

These cells may be undergoing a late stage of apoptosis. Nuclear fragmenta-

tion would be evident.

�

Karyolytic cells are apparent as a ghost-like image. In these cells, the nucleus is

not stained by Feulgen. In these cells, nucleus is depleted of DNA.

RI was calculated with the following formula:

RI¼KRþKL/BEþMN (Ramirez and Saldanha, 2002).

2.3. Statistical evaluations

Statistical analysis was performed using SPSS 11.5 for Windows (SPSS,

Chicago, IL) by only one author (B.T.). po0.05 was considered as the level

A. C- elik et al. / Ecotoxicology and Environmental Safety 92 (2013) 265–270 267

significance. Mann–Whitney U test was used to evaluate the MN, BN, KR, and KL,

BE frequencies data and RI values. The relationship between MN, BN, KR, KL, BE,

RI, PC, CCC and age, work duration, smoking years were tested by Spearman rank

correlation analysis. Multiple comparisons were performed by Kruskal–Wallis

post hoc test.

Table 3Multiple comparisons of differences among and within groups.

Groups MN BE KL KR BN PC CCC RI

Control NS – Exposed NS *** ** *** ** *** *** *** ***

Control S – Exposed S ns ns * ** ns *** *** ***

Exposed S – Exposed NS ns ns ns ns ns *** *** ns

Control S – Control NS *** ** ** *** *** ** *** ns

MN, micronucleus; BE, binucleated cell; KL, karyolitic cell; KR, karyorrhetic cell;

BN, binucleated cell; PC, picnotic cell; CCC, condensed chromatin cell; RI, repair

Indexn po0.05.nn po0.01.nnn po0.001.

3. Results

The results for chromosome damage marker (cells with MNand BE), failed cytokinesis marker (binucleated cells), and celldeath marker (karyorrhectic, karyolitic, picnotic cells and cellswith condensed chromatin) are summarized in Table 2.

In Cytome assay, there is statistically significant differencebetween exposed group and control group for MN, CCC, PC, BNfrequency, RI values and BE frequency ( po0.001 and po0.01,respectively) (Table 2).

The percentage of binucleated cells was significantly(po0.001) higher in road construction workers as compared tocontrol individuals. Percentage of karyorrhetic cells in roadconstruction workers was higher than the control. Multiplecomparisons of differences among and within groups were pre-sented in Table 3. There is a significant difference between controlnon-smoker and exposed non-smoker for MN, BE, KL, KR, BN, PC,CCC frequency and RI value (po0.001, po0.01, po0.001, po0.01,

po0.001, po0.001, po0.001and po0.001, respectively). Thedifference between control smoker and exposed smoker groupdid not reach to a significant level for MN, BE, BN frequency, butnot for KL, KR, PC, CCC frequency and RI value (po0.05, po0.01,

po0.001, po0.001 and po0.001). There is a significant differenceonly between exposed smoker and exposed non-smoker group forPC and CCC frequency (po0.001). There is significant differencebetween control smoker and control non-smoker group for MN,BE, KL, KR, BN, PC, CCC frequency ( po0.001, po0.01, po0.01,

po0.001, po0.001, po0.01, po0.001). But there is no significantdifference for RI value.

As seen in Table 4, Spearman rank correlation analysis showsthat a strong negative correlation is found between MN-RI andBE-RI (r¼� 0.750, r¼�0.623 respectively) and there is nocorrelation between BN-other nuclear changes and RI valuesand there is a slight correlation between MN-BE (r¼0.414) andBE-KR (r¼0.486) in smoker workers. There is a strong negativecorrelation between MN-RI and BE-RI (r¼�0.697, r¼�0.622)and a slight positive correlation between BE-KR (r¼0.418) MN-BE(r¼543) and BE-CCC (r¼0.416) in non-smoker workers.

In smoker control group, spearman correlation analysis repre-sents that there is a strong negative correlation between MN-RI(r¼�0.760), and a slight negative correlation between BE-RI(r¼�0. 498) on the contrary, there is a strong positive correlationbetween MN-KR and BE-KR, BN-CCC (r¼0.568, r¼0.678, r¼0.570,respectively). In non-smoker control group, Spearman rank cor-relation analysis shows that there is a slight positive correlation

Table 2Repair index values and frequencies (%) of micronuclei and other nuclear changes in

Groups MN mean7SE BE mean7SE KL mean7SE KR m

Exposed Smokers 10.971.25 5.7671.10 6.1070.51 6.65

Exposed Non-Smokers 9.9070.67 4.3371.09 5.9570.61 9.30

Total 10.470.70*** 5.1770.79** 6.0370.39 7.97

Control Smokers 7.0070.39 3.1570.22 9.5570.39 9.40

Control-Non- Smokers 1.9570.18 1.0670.06 2.9070.19 3.15

Total 4.4870.45 2.2270.21 6.2370.57 6.28

MN, micronucleus; BE, broken egg; BN, binucleated cell; CCC, condensed chromatin cell

repair indexnn po0.01.nnn po0.001.

between MN-KR (r¼0.527), KR-KL (r¼0.574) and KL-RI(r¼0.420).

4. Discussion

BMCyt assay in exfoliated oral mucosa cells has been system-atically used in genetic biomonitoring of populations exposed toseveral genotoxic chemicals, such as tobacco products, pesticidesand alcohol consumption (Sarto et al., 1990; Bloching et al., 2000;Pastor et al., 2001; Nersesyan, 2006a, 2006b; C- elik et al., 2003).The key advantage of the BMCyt assay is the relative ease ofscoring, the limited costs and person–time required, and theprecision obtained from scoring larger numbers of cells (Burgazet al., 1998). C- elik et al. (2003) found that cytogenetic damage inpetrol station workers. The presence of carcinogenic PAH inasphalts has stimulated laboratory research employing studiesinhalation exposure in rats. Studies performed in 1960s did notemploy method in use in present day. No lung tumors werereported but it was observed significant levels of exposureindicating respiratory irritation. Heuper and Payne (1960)reported that there was extensive fibrosing pneumonitis andadenomatosis in 65 rats exposed to oxidized asphalt fumes(5 h/day, 4 days/weeks for 2 years). Simmers (1964) performed aset of inhalation studies with mice exposed to a mixture of sixpenetration grade and oxidized asphalt from six different places.Twenty mice were exposed 30 min/day, 5 days/weeks, for 17months to a mixture of asphalt and water droplet aerosol ofunstated concentration. Only one mouse developed a lung adenomathat was considered spontaneous by the investigators. Recently,Fuhst et al. (2007) reported the effects of inhalation on ratrespiratory epithelial cells and histopathology identified slightdose-related degenerative, inflammatory and proliferative lesionsin parenchymal cells of nasal cavity. Population studies investigat-ing cytogenetic damage induced by asphalt fumes are inconclusivedue to difficulty in interpreting confounding exposure. In Turkey,

exfoliated buccal cells of control and exposed subjects.

ean7SE BN mean7SE PC CCC RI mean7SE

70.67 30.873.10 5.6570.30 7.8570.20 1.0270.12

71.10 20.672.77 3.4070.16 5.7570.20 1.3870.16

70.67 25.772.21*** 4.5270.24*** 6.8070.22*** 1.2070.10***

70.39 10.170.57 1.4070.15 2.4070.13 1.9670.09

70.19 2.5570.19 0.6070.11 0.6570.10 2.3070.12

70.54 6.3570.67 1.0070.11 1.5270.16 2.1370.08

; PC, picnotic cell; KL, karyolytic cell; KR, karyorrhetic cell; BN, binucleated cell; RI,

Table 4Spearman correlation analysis between nuclear changes within groups.

Groups Nuclear Changes MN BE KR KL RI CCC

Smoker-workers MN 1 0,414b a a �0.750c a

BE 0.414b 1 0.486b a �0.623c a

BN a a a a a a

KR a 0.486b 1 a a a

KL a a a 1 a a

RI �0.750c�0.623c a a 1 a

Non-smoker workers MN 1 0.543b a a �0.697c a

BE 0.543b 1 0.418b a �0.622c 416*

BN a a a a a a

KR a 0.418b 1 a a a

KL a a a 1 a a

RI �0.697c�0.622c a a 1 a

Smoker-control MN 1 0.403b a 0.568c�0.760c a

BE 0.403b 1 a 0.678c�0.498b a

BN a a a a a 570c

KR a a 1 a a a

KL 0.568c 0.678c a 1 �0.439b a

RI �0.760c�0.498b a �0.439b 1 a

Non-smoker control MN 1 a 0.527b a a a

BE a 1 a a a a

BN � � � � � a

KR 0.527b a 1 0.574c a a

KL a a 0.574b 1 0.420b a

RI a a a 0.420b 1 a

aCorrelation is not significant.b Correlation is significant at the 0.05 level (two-tailed).c Correlation is significant at the 0.01 level (two-tailed).

A. C- elik et al. / Ecotoxicology and Environmental Safety 92 (2013) 265–270268

Burgaz et al. (1998) found that SCE and MN frequency increased inperipheral blood lymphocytes of asphalt workers. Partanen andBoffetta (1994) performed a meta analysis of twenty epidemiologystudies and reported that there is aggregate relative risk for lungcancer was 0.9 (95 percent CI 0.8–1.0) in highway maintenanceworkers and 1.8 (95 percent CI 1.5–2.1) in roofers. Thereforeauthors indicated that the factors such as smoking, coal tar,asbestos and other potential carcinogens could contribute to theincrease in lung cancer risk. Conclusions from epidemiology studieson the role of asphalt fume exposure in lung cancer or othersystemic cancer are complicated by co-exposure to coal tar,asbestos, diesel exhaust, smoking and other lifestyle factors.Tobacco is known to contain various genotoxic chemical andsmoking is a well-documented cause of cancer including the oralcavity (Speit et al., 2003). Watkins et al. (2002) suggested thatsmoking habits contributed strongly to lung cancer among workersexposed to asphalt. It was expected that the micronucleus frequen-cies were significantly different between control and exposedsubjects in this trial. Such findings are fully in line with otherauthors (C- elik et al., 2003; Benites et al., 2006; Hallare et al.,2009).

For this reason, smokers and nonsmokers are comparativelyinvestigated in this study because smoking is supposed to be arelevant exposure to genotoxins. Our results demonstrated thatthere is no difference in micronuclei frequency between smokersand non-smokers in the exposed group. On the other hand, someresearchers have postulated increased DNA damage in heavysmokers (Schwartz et al., 2003; C- elik et al., 2003; C- elik andKanık 2006). In addition, exposure to nicotine caused a statisti-cally significant increase of micronucleus frequency in humangingival fibroblasts in vitro (Argentin and Chicchetti, 2004).Smoking has been implicated in oral and lung cancer risk andalso induction of MN in the buccal mucosal cells (C- elik and Kanik,2006). However, others have shown lack of correlation betweensmoking and MN frequency (). In the present study a positivecorrelation between smoking and MN both exposed and controlgroups (po0.01). In the present study, in control group, the

presence of statistically significant difference between smokerand non-smoker groups indicates alone the high effect of smokingon the micronucleus frequency as well as nuclear changes such askaryorhexis, karyolysis etc. Other researches also supported thisresults of the present study (C- elik and Kanık, 2006; C- elik et al.,2003).

To monitor cytotoxic effects, the frequencies of KR, KL and PCwere evaluated into this experimental study. Results demon-strated that asphalt exposure was able to induce cellular deathas depicted by statistically significant differences (po0.05)between non-smoker exposed and non-smoker control groups.The present study shows that nuclear abnormalities, such asbinucleates, karyolysis, and karyorrhexis, pycnosis are also usefulindices of chemical exposure and toxic response. Similar resultswere described by other researchers (C- elik et al., 2003; Beniteset al., 2006). Taken as a whole, such results support the notionthat asphalt was also a cytotoxicant agent on the buccal cells. It isimportant to stress that cytotoxicity interferes with micronucleusinduction since some micronucleated cells are inevitably lost aftercytotoxic injury. It has been shown that repeated exposure tocytotoxicants can result in cell injury and ultimately tumordevelopment (Swenberg, 1993). Some investigations reportedthat observation of the increased rates of nuclear anomalitieswhich reflect cytotoxicity are indicative for adverse effects suchas inflammation. In this context, it is notable that increased riskrates of cancer in the various organs may be due to tumorpromoting effects that could be caused by chronic inflammationswhich were detected in earlier studies including exposed subjects(Jhonson et al., 2009).

This study shows that exposed group (smoker and non-smoker) is different from control group for BN frequency. BNfrequency was investigated by many researchers in occupationalstudies and there are data indicating that BN is cytotoxicitymarker (D’Agostini et al., in press). Lately, many researchers(Stavropoulou et al., 2005; Shi and King, 2005; Thomas et al.,2009) indicated that the presence of two or more nuclei must beconsidered as the result of a cytokinesis block after completion of

A. C- elik et al. / Ecotoxicology and Environmental Safety 92 (2013) 265–270 269

nuclear division, resulting in disruption of the cellular skeletonand formation of supernumerary centrosomes and multipolarmitotic spindles.

The combination of cell death parameters like karyorrhectic,and karyolytic cells, pycnosis as potential biomarkers within thebuccal mucosa, in addition to MN, may be useful as a possiblediagnostic to identify individuals with a high risk of tobaccorelated tissue damage The proportion of basal cells and cellsundergoing cell death in buccal mucosa is an indication of theregenerative capacity of this tissue. The regenerative capacity oftissues and organs in the body is fundamental to healthy ageing.Regeneration is dependent on the number and division rate of theproliferating (basal) cells, their genomic stability and their pro-pensity for cell death (Shojaei, 1998; Squier and Kremer, 2001).These events can be studied in the buccal mucosa which is aneasily accessible tissue for sampling cells in a minimally invasivemanner and does not cause undue stress to study subjects (Sartoet al., 1990; Burgaz et al., 1999; Holland et al., 2008). The use ofcells from the buccal mucosa provides a unique opportunity tostudy the regenerative capacity of the epithelial tissue, which is ofectodermal origin in humans. Considering that 490 percent of allhuman cancers are of epithelial origin (C- elik et al., 2003) and thatbuccal mucosa is a high risk site for oral cancer (Kujan et al.,2006), we assumed that asphalt or bitumen fumes are able toinduce genomic instability in oral cells, as a result of chromoso-mal breakage or loss. In this study, there is difference betweensmokers and non-smokers for incidence of MN frequency andnuclear changes and RI in control group. In road constructionworkers, no significant difference was found between smokersand non-smokers for MN and all the nuclear changes. In roadconstruction worker group, the increase in MN and BE frequencydecreased the RI values; therefore the decrease in RI valuesaffected negatively the regeneration capacity of oral cell regard-less of the smoking. On the contrary, in control groups, RI valuesdisplayed the dissimilarity related with smoking. There is someevidence indicating the increase in MN frequency and nuclearchanges in exfoliated buccal cells of smoking workers exposedoccupationally to various chemicals and substances and controlgroups (C- elik and Kanık, 2006; Ergene et al., 2007). The differ-ences among studies may be dependent on number of cigarettessmoked per day, age, exposure time and individual discrepancies,however, more studies including molecular cytogenetic methodsare required for a meaningful conclusion.

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