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Evaluation of Primary DNA Damage, Cytogenetic Biomarkers and GeneticPolymorphisms for CYP1A1 and GSTM1 in Road Tunnel ConstructionWorkersM. Villarinia; M. Morettia; C. Fatigonia; E. Ageab; L. Dominicia; A. Mattiolic; R. Volpid; R. Pasquinia

a Dipartimento di Specialità Medico-Chirurgiche e Sanità Pubblica, Università degli Studi di Perugia,Perugia b U.O. Servizio S.I.M.T., ASL n. 1 Umbria, Città di Castello c Servizio Prevenzione e SicurezzaAmbienti di Lavoro, ASL n. 3 Umbria, Foligno d Agenzia S.E.D.E.S., Regione dell'Umbria, Perugia, Italy

To cite this Article Villarini, M. , Moretti, M. , Fatigoni, C. , Agea, E. , Dominici, L. , Mattioli, A. , Volpi, R. and Pasquini,R.(2008) 'Evaluation of Primary DNA Damage, Cytogenetic Biomarkers and Genetic Polymorphisms for CYP1A1 andGSTM1 in Road Tunnel Construction Workers', Journal of Toxicology and Environmental Health, Part A, 71: 21, 1430 —1439To link to this Article: DOI: 10.1080/15287390802328580URL: http://dx.doi.org/10.1080/15287390802328580

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Journal of Toxicology and Environmental Health, Part A, 71: 1430–1439, 2008Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287390802328580

UTEHEvaluation of Primary DNA Damage, Cytogenetic Biomarkers and Genetic Polymorphisms for CYP1A1 and GSTM1 in Road Tunnel Construction Workers

Genotoxic Risk in Tunnel Construction WorkersM. Villarini1, M. Moretti1, C. Fatigoni1, E. Agea2, L. Dominici1, A. Mattioli3, R. Volpi4, and R. Pasquini11Dipartimento di Specialità Medico-Chirurgiche e Sanità Pubblica, Università degli Studi di Perugia, Perugia, 2U.O. Servizio S.I.M.T., ASL n. 1 Umbria, Città di Castello, 3Servizio Prevenzione e Sicurezza Ambienti di Lavoro, ASL n. 3 Umbria, Foligno, and 4Agenzia S.E.D.E.S., Regione dell’Umbria, Perugia, Italy

In tunnel construction workers, occupational exposure to dust(a-quartz and other particles from blasting), gases (nitrogen dioxide,NO2), diesel exhausts, and oil mist has been associated with lungfunction decline, induction of inflammatory reactions in the lungswith release of mediators that may influence blood coagulation,and increased risk of chronic obstructive pulmonary disease. Thepresent molecular epidemiology study was designed to evaluatewhether occupational exposure to indoor pollutants during roadtunnel construction might result in genotoxic effects. A studygroup of 39 underground workers and a reference group of 34unexposed subjects were examined. Primary and oxidative DNAdamage, sister-chromatid exchanges (SCE), and micronuclei(MN) were measured in peripheral blood cells. The possible influ-ences of polymorphisms in gene encoding for CYP1A1 andGSTM1 xenobiotic-metabolizing enzymes were also investigated.Exposure assessment was performed with detailed interviews andquestionnaires. There were no significant differences in the levelof primary and oxidative DNA damage and frequency of SCEbetween the tunnel workers and controls, whereas the frequencyof MN showed a significant increase in exposed subjects com-pared to controls. No effects of CYP1A1 or GSTM1 variants wereobserved for the analyzed biomarkers. Since MN in peripheralblood lymphocytes are recognized as a predictive biomarker ofcancer risk within a population of healthy subjects, the genotoxic

risk of occupational exposure to various indoor environmentalpollutants during road tunnel construction cannot be excluded bythis biomonitoring study.

Tunnel construction involves heavy work with the use of hugemachines and explosives in a limited space, which pollutes theindoor atmosphere deep within the mountain. Tunnel constructionworkers are exposed to a variety of toxic substances, includingdust, asbestos, silica, concrete, and diesel fumes. Large amounts ofdust and gases are liberated when rock is blasted. Dust is also gen-erated by rock drilling, the spraying of concrete, and transportoperations. Oil mist is produced when mineral oil is sprayed ontomachinery to protect the surface against concrete spills and ontoconcrete forms to prevent concrete from sticking.

Exposure during underground construction exerts severaleffects on health. Decreased lung function, increased incidenceof airway inflammation, and bronchial hyperresponsivenesswere shown in tunnel workers (Bakke et al., 2001, 2004;Massin et al., 1996; Ulfvarson et al., 1991; Ulvestad et al.,2000, 2001; Vallyathan et al., 1995). Silicosis, a progressiveand sometimes fatal lung disease, is well known in the rock-drilling work force (NIOSH, 1992). A recent study showed thattunnel workers who work long hours, round-the-clock, andlong weeks have a mortality rate that is as high as that of otherconstruction workers. On the other hand, they are treated moreoften in hospitals for illness associated with construction work,such as cancer and other occupational diseases (Tuchsen et al.,2005). The diesel machinery that is used in most heavyconstruction work produces various known and suspected car-cinogens (IARC, 1989), and some epidemiological studies sug-gested an increased risk of lung cancer in subjects who havebeen exposed to heavy concentrations of these carcinogensthrough their work (Bhatia et al., 1998; Boffetta et al., 2001;Steenland et al., 1990), although these findings are not consistent.

Received 7 February 2008; accepted 12 May 2008.This work was partially supported by a grant from ISPESL—

Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro(Research No. B10/DOC/02). The authors thank the constructiontunnel workers for participating in the study and express theirgratitude to the blood donors (AVIS Umbertide) who served as thecontrols. We are indebted to the nursing staff of the TransfusionCenter at the Umbertide Hospital for their valuable contribution. Wealso thank Prof. Giuseppina Scassellati-Sforzolini, Emeritus Professorof Hygiene, University of Perugia, for her assistance during thisresearch and her constructive comments on the article.

Address correspondence to Dr. M. Moretti, Dipartimento diSpecialità Medico-Chirurgiche e Sanità Pubblica, Università degliStudi di Perugia, Via del Giochetto 06122, Perugia, Italy. E-mail:massimo.moretti@unipg.it

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GENOTOXIC RISK IN TUNNEL CONSTRUCTION WORKERS 1431

Biological monitoring of exposure to chemical substancesin the workplace is essential for evaluating risks to humanhealth as an integral part of developing strategies to improveoccupational health and safety conditions. The aim of biomon-itoring studies in human populations exposed to potentialmutagens is to assess the risk of genetic disease or cancer byanalyzing the relationship between internal exposure and thebiological effects in target cells also taking into account con-founding factors.

Human biomonitoring can be performed using differentgenetic markers. Sister chromatid exchanges (SCE) and micronu-clei (MN) in peripheral blood lymphocytes (PBL) have been usedfor the surveillance of work environments with low-dose expo-sures to mutagens or carcinogens. SCE are interchanges of DNAreplication products between two sister chromatids of a duplicat-ing chromosome at apparently homologous loci. SCE are pre-sumed to be a consequence of errors in the replication process,perhaps at sites of stalled replication complexes. For this reason,it has been suggested a correlation between this cytogenetic end-point and DNA adducts (Kriek et al., 1998). MN are additionalsmall nuclei derived from acentric chromosomal fragments orwhole chromosomes that have been excluded from either of thedaughter nuclei in cell division. The formation of MN in dividingcells is the result of chromosome breakage or chromosomemalsegregation due to spindle dysfunction produced by eitherclastogen or aneugen agents, respectively (Norppa & Falck2003). The methodologies for SCE and MN are well establishedand the criteria to be used in such approaches are well docu-mented (Natarajan et al., 1996; Albertini et al., 2000).

Primary DNA damage is considered to be an importantinitial event in carcinogenesis. The single-cell microgel elec-trophoresis (comet) assay has become the preferred test for thequalitative and quantitative assessment of DNA damage insingle cells (Singh et al., 1988; Rojas et al., 1999; Tice et al.,2000; Collins, 2004).

Attention has been recently focused on genetic polymorphismsthat seem appear to modulate human response to genotoxicinsults (Norppa, 2004). Genetic susceptibility might be due tovariations in genes encoding for carcinogen-metabolizingenzymes, such as cytochrome P-450 (CYP450) and glutathioneS-transferases (GST) (Wormhoudt et al., 1999). The membersof subfamily 1 of the CYP gene superfamily play a major rolein the catalysis of such metabolic activation (Nebert, 1991).Among several polymorphisms identified in the CYP1A1 gene,the T→C mutation (m1) in the 3′-flanking region of the genewas extensively studied in relation to cancer risk. The presenceof this mutation was associated with increased catalytic activity(Bartsch et al., 2000). Moreover, a positive associationbetween the presence of the variant alleles and increased pol-yaromatic hydrocarbon (PAH)–DNA adducts was reported(Pastorelli et al., 1998; Rojas et al., 1998; Teixeira et al., 2002).The CYP1A1 polymorphisms were examined extensively toevaluate the possible role they play in DNA damage and cancerpromotion (Grzybowska et al., 2000; Agundez, 2004).

The GSTM1 gene, encoding for the cytosolic enzymeglutathione S-transferase μ 1 (GSTM1) that detoxifies acti-vated forms of chemical carcinogens such as polyaromatichydrocarbons (PAH) and epoxides, is deleted in about 50% ofCaucasians, with a reported variation of 38 to 65% (Boardet al., 1990). The inherited absence of the GSTM1 gene (theGSTM1 null genotype) is therefore theoretically associatedwith a higher risk to the adverse effects of chemicals and itsinfluence on various exposure biomarkers was widely studied(Binkova et al., 1998; Pastorelli et al., 1998; Alexandrie et al.,2000; Sram & Binkova 2000).

Biomonitoring procedures have been used in many occupa-tional exposure studies (Pavanello & Clonfero, 2000; Sram &Binkova, 2000; Norppa, 2004), but never for road tunnel con-struction workers. The aim of the present molecular epidemiol-ogy (cross-sectional) study was to investigate the associationbetween occupational exposure and the occurrence of genotoxicoutcomes in peripheral leucocytes of road tunnel workers. Totest the biologically effective dose, the comet assay associatedwith specific repair enzymes (i.e., endonuclease III and forma-midopyrimidine DNA glycosylase) was applied to measurebaseline DNA damage and the presence of oxidized purines/pyrimidines. Early biological effects were determined by evalu-ating SCE and MN. To evaluate individual susceptibility, studysubjects were genotyped for phase I enzymes involved in theactivation and biotransformation of PAH (i.e., CYP1A1) andfor phase II enzymes involved in the inactivation of a variety ofreactive chemical species (i.e., glutathione S-transferase M1).

MATERIALS AND METHODS

Chemicals, Media, and EnzymesAll reagents used were of analytical grade. Acetic acid,

hydrochloric acid (HCl), dimethyl sulfoxide (DMSO), ethanol,ethylenediamine tetraacetic acid disodium salt (Na2EDTA),Giemsa stain solution, methanol, potassium chloride (KCl),sodium chloride (NaCl), and sodium hydroxide (NaOH) werepurchased from Carlo Erba Reagenti Srl, Milan, Italy.5-Bromodeoxyuridine (BrdU), cytochalasin-B, demecolcine,ethidium bromide, low-melting-point agarose, phytohemaggluti-nin (PHA), tris(hydroxymethyl)aminomethane (Tris base), andTriton X-100 were obtained from Sigma-Aldrich Srl, Milan, Italy.Vacutainer blood collection tubes were from Becton DickinsonItalia SpA, Milan, Italy. Precoated two-well slides (CometSlide),endonuclease enzymes (EndoIII and FPG), and enzyme buffersolutions were purchased from Trevigen, Inc., Gaithersburg,MD, USA. Gibco cell culture media, fetal calf serum, antibiot-ics, and Dulbecco’s phosphate-buffered saline, pH 7.4 (PBS),were purchased from Invitrogen Srl, Milan, Italy. DNA extrac-tion kit was supplied by Qiagen SpA, Milan, Italy. Primers weresynthesized by MWG-Biotech AG, Ebersberg, Germany. DNApolymerase, 2′-deoxynucleoside 5′-triphosphates (dNTPs), andmagnesium chloride (MgCl2) were obtained from Stratagene,

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1432 M. VILLARINI ET AL.

La Jolla, CA, USA. Agarose HR 3:1 was of EuroClone SpA,Milan, Italy. MspI restriction enzyme was from New EnglandBiolabs, Ipswich, MA, USA. Conventional microscope slidesand coverslips were supplied by Knittel-Glaser, Braunschweig,Germany. Distilled water was used throughout the experiments.

SubjectsWorkers from three different tunnel construction sites in Cen-

tral Italy (total of 39 males) were studied and compared to 34male nonexposed controls. The reference group comprised out-door workers (i.e., construction workers performing iron fixingand concrete work, truck drivers, and surveyors) from the samelocalities as the workers to minimize the influence of other envi-ronmental factors on DNA damage and cytogenetic biomarkers.The tunnel workers were employed in three different tunnelingconstruction sites in the Apennine Mountains (Umbrian Apen-nine), and the tunnels were between 500 and 6000 m long. Theworkers in tunnel I (total length 1200 m) were involved in finish-ing works (i.e., cement-based grouting and road paving),biomonitoring was performed when the work was situated in themiddle of the tunnel. The workers in tunnels II (final length 900 m)and III (final length 5950 m) were tunnel face workers engagedin tunnel excavation (i.e.. rock blasting, drilling, and transportoperations); the tunnel faces were at about 400 and 2500 m,respectively. In tunnels II and III fresh air was supplied to thetunnel face by the means of flexible ventilation tubes.

The workers and control subjects were informed about theaim and the experimental details of the study. Informed consentwas obtained from all of the participants subjects. A standardquestionnaire was used to interview each person about theirage, nature of occupation, years of service, personal habits(e.g., smoking habits, consumption of alcohol, etc.), and healthstatus. Subjects who had been exposed to radiations for thera-peutic or diagnostic purposes were not included in the study.

Blood Sample CollectionBlood sampling and processing was carried out simulta-

neously for the tunnel workers and control subjects. Bloodsamples were taken from the tunnel workers at the end of thelast day of their work week. Peripheral blood samples weretaken by venipuncture and collected in heparinized or lithiumEDTA vacuum tubes, for genotoxicity testing or genotyping,respectively. All blood samples were coded, cooled (4°C), pro-tected from light, and processed as quickly as possible (usuallywithin 4 h following the blood sampling).

Comet AssayThe comet assay is a technically simple and fast method that

allows DNA single- or double-strand breaks to be detected insingle cells without requiring cell culture (Moller, 2006). Forthe comet assay, a cell suspension is embedded in agarose on amicroscope slide and lysed by detergents at high salt concentration

to liberate the DNA. The slides are then treated under alkalineconditions to unwind the DNA from the strand breakage sites.Electrophoresis at high pH results in structures that resemblecomets when observed by fluorescence microscopy. The fluo-rescence intensity of the comet tail in relation to that of thehead reflects the number of DNA breaks. The sensitivity of thecomet assay is greatly influenced by the pH of lysis and elec-trophoresis buffers. The standard alkaline procedure (lysis andelectrophoresis steps at pH 10 and pH >13, respectively)allows single-strand DNA breaks to be detected as well asalkali labile lesions (i.e., apurinic/apirimidinic sites) that areconverted to strand breaks under alkaline conditions. A modi-fied enzymatic approach, based on DNA digestion after thelysis step, is a sensitive method for detecting oxidative DNAdamage. Lesion-specific enzymes remove the oxidized bases toform apurinic/apyrimidinic sites that are converted into strandbreaks by the activity of lyases. These breaks are detected bythe comet assay (Collins et al., 1993).

In this study, the standard alkaline comet assay procedure(Singh et al., 1988) was performed with minor modification(Villarini et al., 2000) to evaluate primary DNA damage. Thepresence of oxidized purines or pyrimidines was evaluatedusing the protocol proposed by Collins and coworkers (1993,1996), with slight modifications. Endo III (which recognizes oxi-dized pyrimidines) and FPG (which recognizes altered purines,including the major purine oxidation product 8-oxoguanine)were used. To prevent additional DNA damage, all steps wereperformed under yellow light.

Briefly, blood cells (30 μl whole blood) were mixed with3 ml of 0.75% low-melting-point agarose (in PBS) at 37°C.Aliquots (75 μl) of the cell suspensions were pipetted ontoeach of the two areas of the precoated two-well slides. Theslides were placed in the dark at 4°C for 10 min to accelerategelling of the agarose disc and then transferred in the lysisbuffer (2.5 M NaCl, 100 mM Na2EDTA and 10 mM Tris base;pH 10) containing freshly added 1% (v/v) Triton X-100 and10% (v/v) DMSO. The lysis was allowed to proceed for a min-imum of 1 h and a maximum of 18 h. After lysis, the slideswere washed with immersion in the enzyme buffer; the bufferwas changed three times over a 15-min period.

To prepare the slides for the analysis of the oxidative DNAdamage, 100 μl enzyme solution (1 U/sample) or 100 μlenzyme buffer was added to the microgels in each well (in eachslide an area served as buffer control and the other was sub-jected to the activity of FPG or Endo III). The slides were thenincubated at 37°C for 60 min in humidified atmosphere. Theslides prepared for the primary DNA damage analysis were notsubjected to this step.

To relax supercoiled DNA, the slides (for both primary andoxidative DNA damage) were incubated in freshly made alkalineelectrophoresis buffer (300 mM NaOH and 1 mM Na2EDTA;pH >13) at 4°C for 30 min. The electrophoresis was performedat 4°C for 20 min at 25 V (1 V/cm) and 300 mA. After electro-phoresis, the slides were washed three times with a neutralization

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GENOTOXIC RISK IN TUNNEL CONSTRUCTION WORKERS 1433

buffer (0.4 M Tris/HCl; pH 7.5) to remove excess alkali anddetergents. To preserve the slides, they were dehydrated in70% ethanol and allowed to dry at air temperature until staining.

For the microscope analysis, the slides were stained with60 μl ethidium bromide (2 μg/ml) and examined at 200 × mag-nification under a fluorescence microscope (BX41, Olympus,Tokyo, Japan), equipped with an excitation filter of 515–560 nmand a barrier filter of 590 nm. The microscope was equippedwith a black-and-white CCD camera connected to a computer-based image analysis system (Comet Assay III, PerceptiveInstruments Ltd., Haverhill, Suffolk, UK). The system acquiresimages, computes the integrated intensity profiles for each cell,estimates the comet cell components (i.e., head and tail) and thenevaluates a range of derived parameters. In this study, tail inten-sity (percent of DNA in the tail) was the chosen as the parame-ter to measure the DNA damage. In total, 150 comets permicrogel treatment (i.e., buffer, FPG or Endo III) were scoredfor each subject from replicated slides. The net amount of dam-age represented by the FPG- or Endo III-sensitive sites wasdetermined for each subject by subtracting the extent of DNAstrand breakage measured in cells incubated with the bufferalone from that observed in cells incubated with the enzymes.

Sister-Chromatid ExchangesSCE are analyzed in second division metaphases by BrdU

labeling using a staining method based on sister chromatiddifferentiation. BrdU closely resembles thymidine and isefficiently incorporated into the elongating DNA strands dur-ing replication. After two cell cycles in BrdUrd medium, thetwo sister chromatids differ in the amount of BrdUrd presentand the chromatid with more BrdUrd is lighter in appearance(“bleaching” effect). After treating the cells with a spindleinhibitor (e.g., colchicine, demecolcine) to accumulate cells ina metaphase-like stage of mitosis (c-metaphase), cells areharvested and chromosome preparations are made.

In this study, the SCE assay was performed following theoriginal method (Moorhead et al., 1960), with minor modifica-tions. Briefly, 4 parallel lymphocyte cultures/subject were setup by adding 0.3 ml of venous blood to 4.7 ml RPMI-1640medium supplemented with 20% fetal calf serum, 2% PHA,antibiotics (100 IU/ml penicillin, and 100 μg/ml streptomycin),and 10 μg/ml BrdU. The cultures were incubated in the dark for72 h, under 5% CO2 in air in a humidified atmosphere at 37°C,and demecolcine (0.1 μg/ml) was added 2 h prior to harvestingto arrest the cells at metaphase. The cells were then collected bycentrifugation, resuspended in a prewarmed hypotonic solution(75 mM KCl) for 15 min at 37°C, fixed for 1 h in Carnoy (3:1methanol/acetic acid; v/v), and finally dropped onto cleanslides. The slides were stained with fluorescence plus Giemsaprocedure (Perry & Wolff, 1974). Microscopic analyses wereperformed at 1000 × magnification on a light microscope(CX40, Olympus, Tokyo, Japan). In total, 30 well-spreadsecond-division metaphases containing 46 (±1) chromosomes

were examined for each subject to determine the number ofSCE/cell and the dispersion coefficient (H) of SCE frequencywas calculated according to the formula proposed by Margolinand Shelby (1985): H = variance/mean.

The same slides were also scored in order to detect theproportion of cells that undergo one (M1), two (M2) and threeor more (M3) divisions. The proliferative rate index (PRI) wascalculated according to the formula PRI = (M1+2M2+3M3)/Nwhere N indicates to the total number (i.e. 100) of metaphasesscored (Lamberti et al., 1983).

Cytokinesis-Block Micronucleus AssayThe conventional approach used in human biomonitoring

studies involves ex vivo culturing of peripheral blood lympho-cytes and the scoring of cells for MN after most cells havedivided. In the cytokinesis-block (CB) technique, in whichcytochalasin-B is used as a cytokinesis inhibitor, dividing cellsare accumulated in the binucleated stage (Fenech & Morley,1985). This approach allows reliable data scoring because onlythe MN of those cells that have completed one nuclear divisionare analyzed.

In this approach, the cytokinesis-block micronucleus(CBMN) assay was performed following the original method(Fenech 1993), with minor modifications. Briefly, lymphocytecultures were established in duplicate by adding 0.3 ml ofwhole blood to 4.7 ml of RPMI-1640 medium supplementedwith 20% fetal calf serum, 2% PHA, and antibiotics (100 IU/mlpenicillin, and 100 μg/ml streptomycin). The cultures wereincubated in the dark for 72 h at 37°C, under 5% CO2 in air in ahumidified atmosphere. After 44 h of incubation, cytochalasin-B was added to the cultures at a concentration of 6 μg/ml toblock cytokinesis. The cells were collected by centrifugationand treated for 3 min with a prewarmed mild hypotonic solution(75 mM KCl). After centrifugation and removal of the superna-tant, the cells were fixed with a fresh mixture of methanol/aceticacid (3:1 v/v). Centrifugation and resuspension were carriedout 3 times and the cells were then dropped onto clean slides todetect the MN by conventional staining with 5% Giemsa.Microscope analyses were performed at 1000× magnificationon a light microscope (CX40, Olympus, Tokyo, Japan). For eachsubject 1000 binucleated lymphocytes with well-preservedcytoplasm were scored following the established criteria forMN evaluation (Fenech et al., 2003). The effects of the expo-sure on cell proliferation were estimated by calculating themitotic index (MI), which corresponds to the ratio of binucle-ated and polynucleated cells to 1000 stimulated lymphocytes.

DNA Extraction and GenotypingBlood samples of all subjects were stored at −20°C until

used. Genomic DNA was obtained from 200 μl of whole bloodusing a commercially available kit (QIAamp DNA extractionkit) according to the manufacturer’s instructions. Each DNAsample was stored at −20°C until analysis.

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1434 M. VILLARINI ET AL.

CYP1A1m1 (MspI) PolymorphismThe CYP1A1 m1 polymorphism was detected by PCR

followed by restriction fragment length polymorphism (RFLP)analysis using the restriction enzyme MspI (Hayashi et al.,1991). The primers for the CYP1A1 m1 mutation were 5′-AAGAGG TGT AGC CGC TGC ACT-3′ and 5′-TAG GAG TCTCTC ATG CCT-3′, which amplified a 335-bp fragment.Approximately 300 ng of genomic DNA was amplified in a totalvolume of 50 μl mixture containing 2 mM MgCl2, 250 μMdNTPs, 1.5 U PfuTurbo DNA polymerase, and 1 μM of eachprimer. The PCR was conducted with a PCRSprint thermocycler(Hybaid Ltd., Ashford, Middlesex, UK). Initial denaturation wascarried out at 95°C for 5 min, followed by 35 cycles of denatur-ation for 30 s, annealing at 63°C for 30 s, extension at 72°C for30 s, and a final cycle of 72°C for 5 min. The PCR products weredigested with 40 units of Msp1 restriction enzyme at 37°C for 5 h.DNA fragment were subsequently electrophoresed in a 3%agarose HR 3:1 gel stained with ethidium bromide (250 ng/ml).

The wild-type genotype w1/w1 (lacking the MspI site) formedan uncleaved 340-bp band, while the variant genotype m1/m1(homozygous for the allele carrying the mutation with the MspIsite) generated 2 bands of 200 and 140 bp. The heterozygousgenotype m1/w1 showed 3 bands of 340, 200, and 140 bp.

GSTM1 PolymorphismGSTM1 genotyping for gene deletions was carried out by

detecting the presence or absence of the intact gene (Bell et al.,1993). The primers used for GSTM1 amplification were 5′-GAACTC CCT GAA AAG CTA AAG C-3′ and 5′-GTT GGG CTCAAA TAT ACG GTG G-3′. β-Globin gene primers were used asan internal positive control: 5′-CAA CTT CAT CCA CGT TCACC-3′ and 5′-GAA GAG CCA AGG ACA GGT AC-3'. PCRreaction was carried out in a 50 μl volume containing 100 ng ofgenomic DNA, 2 mM MgCl2, 250 μM concentration of eachdNTP, 0.1 μM of both GSTM1 and β-globin primers, and 1.5 Uof PfuTurbo DNA polymerase. In the thermocycling procedure,initial denaturation at 95°C for 5 min was followed by 35 cyclesof denaturation at 94°C for 45 s, annealing at 58°C for 45 s, andextension at 72°C for 45 s before a final extension at 72°C for 5 min.PCR products were separated by electrophoresis on a 3% agaroseHR 3:1 gel stained with ethidium bromide (250 ng/ml).

The GSTM1 positive genotype was defined by the presenceof a specific band (215 bp) present in wild-type homozygotesand heterozygotes for the deletion (not differentiated in theanalysis and both expressing GSTM1 enzyme), whereas theabsence of the GSTM1-specific PCR product indicated the cor-responding null genotype (homozygous deletion of the GSTM1gene). A β-globin-specific fragment (268 bps) confirmed theproper function of the reaction (positive genotype).

Statistical AnalysisStatistical analyses were carried out using SPSS 10.0.7

package (SPSS, Inc., Chicago, IL, USA). The presence of

possible significant differences between exposed subjects andcontrols were tested with the nonparametric Kolmogorov–Smirnov Z-test (a test based on the largest difference betweenthe two cumulative distribution), which is sensitive to any typeof differences in both the locations and the shapes of the ana-lyzed distributions. The Hardy–Weinberg equilibrium test forCYP1A1 genotype distribution was performed using a χ2 testwith 1 degree of freedom; the GSTM1 genotype was coded aspositive (wild-type homozygotes and heterozygotes for thedeletion) or null (homozygous deletion), making direct calculationof Hardy–Weinberg equilibrium impossible. The Pearson’s χ2

test was used to evaluate differences in the distributions ofallele frequencies between exposed subjects and controls. Thelevel of statistical significance was set at p < .05.

RESULTSIn this study, primary DNA damage and two cytogenetic

biomarkers (SCE and MN) were evaluated in 39 road tunnelconstruction workers and 34 control subjects; the two groupswere comparable in terms of age, smoking habits, and socio-economic status. Table 1 shows the main characteristics of the

TABLE 1 Main Characteristics and Genotype Distribution of the Study

Population Grouped According to Exposure Status

Exposed workers Controls

Subjects (n) 39 34

Demographic characteristicsa

Ageb 42.28 ± 10.14 38.59 ± 11.17≤ 40 yearsc 15 (38.5%) 19 (55.9%)> 40 yearsc 24 (61.5%) 15 (44.1%)

Occupational featuresYears employedb 13.76 ± 11. 18 —

Smoking habitsNon-smokers 20 (51.3%) 20 (58.2%)Smokers 19 (48.7%) 14 (41.2%)Cigarettes/dayd 18.68 ± 8.37 11.93 ± 6.93

GenotypesCYP1A1 wt/wt 32 (82.1%) 31 (91.2%)CYP1A1 wt/m1 + m1/m1 7 (17.9%) 3 (8.8)GSTM1 positive 28 (71.8%) 24 (70.6%)GSTM1 null 11 (28.2%) 10 (29.4%)

Data are reported as the number of subjects (% between brackets).aAll subjects were males.bAge and years employed are expressed in years and reported

as the group mean ± standard deviation.cCut-off defined according to the mean value (i.e. 40.56 years)

of the observed age distribution in the whole population.dThe number of cigarettes smoked per day is reported as the mean ±

standard deviation.

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GENOTOXIC RISK IN TUNNEL CONSTRUCTION WORKERS 1435

experimental and control groups. No differences wereobserved between the exposed and control subjects withrespect to age, smoking habits and cigarettes smoked daily.Table 1 also shows the CYP1A1 and GSTM1 gene expressionof the studied populations. The distribution of the CYP1A1wild-type genotype was 82.1% in the exposed population and91.2% in the controls. The observed CYP1A1 m1 genotypesamong the exposed and control subjects were in Hardy–Weinberg equilibrium (data not shown). The prevalence of theGSTM1 null genotype was 71.8% in the tunnel workers and70.6% among the controls.

The comet tail intensity in peripheral blood leukocytes fromexposed workers and controls and the extent of oxidative DNAdamage (leukocytes treated ex vivo with FPG or Endo III) aresummarized in Table 2. To analyze age effects, the road tunnelworkers and the controls were divided into two subgroups: age≤ 40 or > 40 yr. Statistical analysis indicated that there were nosignificant differences in the level of primary and oxidativeDNA damage between the tunnel workers and the controls.Smoking did not significantly influence the levels of DNA

damage in either group. There was no effect of age or geno-types on comet tail intensity.

The frequencies of SCE and MN in lymphocytes of all thestudy subjects, grouped by age, smoking habits, and genotypes,are shown in Table 3 and Table 4. No statistically significantdifferences were observed between the road tunnel workersand control subjects for SCE, whereas the results relative to thefrequency of MN showed significantly higher genotoxiceffects in exposed subjects. No effects of age were noted onSCE or MN frequencies, neither in the exposed workers nor inthe controls. SCE frequency was quantitatively higher insmokers than in nonsmokers, but the difference was non signif-icant. No effects of CYP1A1 or GSTM1 variants were observedfor the cytogenetic biomarkers analyzed.

Among the exposed group, the subjects worked in 3 differenttunnel projects: 12 subjects worked in tunnel I, 12 in tunnel II,and 15 in tunnel III. When exposed workers were divided intothree subgroups on the basis of the work place, differences interms of genotoxic risk were observed. The highest extent ofprimary DNA damage and MN frequency were observed in

TABLE 2 Primary and Oxidative DNA Damagea in Peripheral Blood Leukocytes of Exposed Workers and Control Subjects,

also in Relation to Age, Smoking Habits and Genotype

Exposed Controls

PrimaryDNA damage

Oxidized basesb

PrimaryDNA damage

Oxidized basesb

n FPG sites Endo III sites n FPG sites Endo III sites

Total 39 3.08 ± 0.29 1.00 ± 0.38 0.41 ± 0.29 34 2.85 ± 0.18 1.16 ± 0.33 0.82 ± 0.24Tunnel

I 12 2.14 ± 0.30 0.30 ± 0.33 0.47 ± 0.38 — — —II 12 5.28 ± 0.40§ 1.53 ± 1.12 −0.27 ± 0.73 — — —III 15 2.08 ± 0.21 1.13 ± 0.33 0.90 ± 0.34 — — —

Agec

≤ 40 years 15 3.19 ± 0.47 0.98 ± 0.34 0.65 ± 0.55 19 2.96 ± 0.23 0.88 ± 0.50 0.68 ± 0.35> 40 years 24 3.02 ± 0.38 1.01 ± 0.58 0.26 ± 0.33 15 2.71 ± 0.31 1.52 ± 0.39 0.99 ± 0.31

Smoking habitsNon-smokers 20 3.18 ± 0.44 1.47 ± 0.65 0.06 ± 0.46 20 3.04 ± 0.25 1.13 ± 0.48 0.78 ± 0.34Smokers 19 2.98 ± 0.39 0.51 ± 0.36 0.91 ± 0.31 14 2.58 ± 0.26 1.21 ± 0.41 0.87 ± 0.31

GenotypesCYP1A1 wt/wt 32 3.13 ± 0.34 1.06 ± 0.46 0.42 ± 0.35 31 2.88 ± 0.20 1.22 ± 0.35 0.88 ± 0.25CYP1A1 wt/m1 + m1/m1 7 2.86 ± 0.47 0.71 ± 0.37 0.37 ± 0.26 3 2.55 ± 0.42 0.59 ± 0.64 0.18 ± 0.44GSTM1 positive 28 3.28 ± 0.36 1.22 ± 0.50 1.18 ± 0.37 24 2.91 ± 0.25 0.95 ± 0.41 0.81 ± 0.26GSTM1 null 11 2.58 ± 0.43 0.44 ± 0.43 0.99 ± 0.34 10 2.69 ± 0.19 1.67 ± 0.51 0.84 ± 0.50

Extent of DNA strand breakage reported as the group mean (± SEM) of the average individual data; 150 cells were scored for each individual.aThe percentage of DNA in the comet tail (i.e. tail intensity) was taken as a measure of DNA damage.bOxidative DNA damage was recognized by DNA repair enzymes formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo

III); the results obtained for FPG or Endo III were normalized by subtracting the level of DNA damage observed for the enzyme buffer only.cAge, considered as categorical variable in this descriptive data set, classified according to the mean value (i.e. 40.56 years) of the observed

age distribution in the whole population.§= p < 0.05, vs. whole control group: Kolmogorov-Smirnov Z test.

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1436 M. VILLARINI ET AL.

tunnel II workers, whereas the highest number of SCE wasobserved in tunnel III subgroup.

Tunnel workers were also gathered into three groups basedon the type of work. The first group consisted of drillers andtunnel boring machine operators (22 subjects), the secondgroup consisted of shotcreters and tunnel concrete workers(7 subjects), and the last group consisted of support workers(10 subjects). The drillers perform conventional drill and blastoperations and used pneumatic handheld equipment for rockdrilling. The tunnel boring machine crew operates tunnel-boringmachines that drill the entire cross section of the tunnel orshaft. The shotcreters spray concrete on the tunnel walls and thetunnel concrete workers do iron and carpentry work after the tun-nel has been excavated. The support workers are responsible forinstalling and maintaining ventilation ducting, compressed air,cables and pipes, and for transporting materials. Types of workfeatures did not affect the biomarkers evaluated (data not shown).

DISCUSSIONIn tunnel construction workers, the occupational exposure

to dust (α-quartz and other particles from blasting), gases(NO2), diesel exhaust, and oil mist has been associatedwith lung function decline (Bakke et al., 2004), induction of

inflammatory reactions in the lungs with release of mediatorsthat may influence blood coagulation such as interleukin-6)(Hilt et al., 2002), and an increased risk of chronic obstructivepulmonary disease (Ulvestad et al., 2000; Oliver & Miracle-McMahill, 2006). To our knowledge only one study investi-gated the relationship between occupational exposure duringroad tunnel construction and genotoxic endpoints includingchromosome aberrations: Kjuus et al. (2005), who reported nostatistically significant differences between exposed workersand control subjects for cells with chromosome aberrations orfor chromatid breaks, chromosome breaks, or chromosomegaps. Lewne et al. (2007) recently investigated the exposure ofpersonnel to diesel, petrol exhaust fumes, particles, elemental car-bon, and nitrogen dioxide in various occupational environments,including tunnel construction workers. Lewne et al. (2007)showed that the tunnel construction workers were exposed tosignificantly higher levels of all the indicator substances thanother occupational groups.

This molecular epidemiology approach was used to investigatewhether occupational exposure to pollutants during road tunnelconstruction induced genotoxic damage. In particular, theprimary and oxidative DNA damage was evaluated by usingthe alkaline comet assay (i.e., biomarker of biologically effec-tive dose) and cytogenetic damage was evaluated on the basis

TABLE 3 Frequencies of SCE Peripheral Blood Lymphocytes of Exposed Workers and Control Subjects, also in Relation

to Age, Smoking Habits and Genotype

Exposed Controls

n SCE/cell SCE H PRI n SCE/cell SCE H PRI

Total 39 5.07 ± 0.11 0.29 ± 0.03 2.29 ± 0.03* 34 4.88 ± 0.08 0.31 ± 0.02 2.43 ± 0.01Tunnel

I 12 4.71 ± 0.25 0.39 ± 0.05 2.31 ± 0.04 — — —II 12 5.09 ± 0.14 0.20 ± 0.01§ 2.36 ± 0.26 — — —III 15 5.34 ± 0.16§ 0.28 ± 0.02 2.19 ± 0.60§ — — —

Agea

≤ 40 years 15 4.93 ± 0.19 0.31 ± 0.05 2.37 ± 0.04 19 4.75 ± 0.11 0.29 ± 0.02 2.45 ± 0.01> 40 years 24 5.16 ± 0.14 0.28 ± 0.02 2.24 ± 0.04 15 5.03 ± 0.13 0.34 ± 0.03 2.41 ± 0.03

Smoking habitsNon-smokers 20 5.05 ± 0.18 0.30 ± 0.03 2.26 ± 0.04* 20 4.73 ± 0.10 0.31 ± 0.03 2.44 ± 0.02Smokers 19 5.09 ± 0.14 0.28 ± 0.02 2.32 ± 0.04 14 5.08 ± 0.13 0.31 ± 0.03 2.43 ± 0.02

GenotypesCYP1A1 wt/wt 32 5.13 ± 0.13 0.29 ± 0.02 2.29 ± 0.03* 31 4.86 ± 0.09 0.31 ± 0.02 2.44 ± 0.02CYP1A1 wt/m1 + m1/m1 7 4.78 ± 0.20 0.31 ± 0.03 2.28 ± 0.08 3 5.09 ± 0.39 0.30 ± 0.03 2.42 ± 0.03GSTM1 positive 28 4.95 ± 0.12 0.29 ± 0.03 2.32 ± 0.03* 24 4.86 ± 0.10 0.31 ± 0.02 2.44 ± 0.01GSTM1 null 11 5.38 ± 0.23 0.28 ± 0.03 2.19 ± 0.06* 10 4.91 ± 0.14 0.32 ± 0.04 2.42 ± 0.03

Results are reported as the group mean (± SEM) of average individual SCE counts; 30 cells were scored for each individual for SCE.aAge, considered as categorical variable in this descriptive data set, classified according to the mean value (i.e. 40.56 years) of the observed

age distribution in the whole population.*= p < 0.05, vs. the corresponding control group; §= p < 0.05, vs. whole control group: Kolmogorov-Smirnov Z test.

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GENOTOXIC RISK IN TUNNEL CONSTRUCTION WORKERS 1437

of SCE and MN (i.e., biomarkers of early biological effects). Inaddition, the study subjects were genotyped for polymorphismsin the genes encoding for CYP1A1 and GSTM1 xenobiotic-metabolizing enzymes, which are biomarkers of individualsusceptibility.

The present study is characterized by both positive (strengths)and negative (weaknesses) aspects. An advantage of thisapproach is that the study design allows for the simultaneousevaluation of several genotoxicity endpoints associated withsubjects genotyping for xenobiotic metabolizing enzymes. Aweakness of the study could be the limited number of subjectsenrolled, but this was the consequence of the size of the work-force in this occupational branch. The genotoxic endpoints eval-uated, in particular DNA strand breakage (comet assay) andMN, have well-defined roles as biomarkers of biologically effec-tive dose (Mussali-Galante et al., 2005) and early biologicaleffects (Bonassi et al., 2007), respectively.

In this biomonitoring study there was a significant increase inMN frequency among tunnel construction workers in compari-son to controls. On the contrary, occupational exposure to vari-ous indoor pollutants during road tunnel construction did notaffect primary or oxidative DNA damage (comet assay), orinduce higher frequencies of SCE in peripheral bloodlymphocytes of exposed workers. Decreased cell kinetics wereobserved extensively for SCE and MN in the exposed subjects.

Stratification of exposed subjects according to the tunnelsite revealed differences in terms of genotoxic risk. The highestextent of primary DNA damage and MN frequency wereobserved in tunnel II workers, whereas the highest number ofSCE was observed in tunnel III subgroup. At tunnel II and IIIsites similar numbers and types of diesel-powered machineswere used, and the tunnels were excavated using the sametechnology. The observed differences in term of genotoxic out-comes were not thus totally as expected.

The effect of smoking habits was evaluated as a potentialconfounding factor in a large number of human biomonitoringstudies. As in other studies, no association was observed in thiswork between smoking and biomarkers of genotoxic damagesuch as primary DNA damage, SCE, and MN. For thecomet assay, conflicting results were recently evaluated in ameta-analysis study (Hoffmann et al., 2005) and the authorsconcluded that an effect of smoking could not be formallydemonstrated when the evaluation of DNA damage was basedon image analysis. For MN, a few reports showed positiveresults, whereas the preponderance of studies did not find anyassociation between MN and smoking (data reviewed inBonassi et al., 2003). As a hypothesis, these observations maybe explained in term of adverse effects of smoking wheresmoke components destroy cells in culture or delay the cellcycle, thus, making it not possible to carry out the MN test. For

TABLE 4 Frequencies of MN in Peripheral Blood Lymphocytes of Exposed Workers and Control Subjects,

also in Relation to Age, Smoking Habits and Genotype

Exposed Controls

n MN MI n MN MI

Total 39 6.31 ± 0.61* 0.68 ± 0.01* 34 4.71 ± 0.28 075 ± 0.01Tunnel

I 12 5.17 ± 0.49 0.73 ± 0.02 — —II 12 10.88 ± 0.97§ 0.63 ± 0.02§ — —III 15 3.57 ± 0.25 0.69 ± 0.02 — —

Agea

≤ 40 years 15 6.40 ± 0.88 0.67 ± 0.02* 19 4.79 ± 0.36 0.75 ± 0.02> 40 years 24 6.25 ± 0.84 0.69 ± 0.02 15 4.60 ± 0.46 0.75 ± 0.02

Smoking habitsNon-smokers 20 6.28 ± 0.75 0.70 ± 0.02 20 4.80 ± 0.40 0.74 ± 0.02Smokers 19 6.34 ± 0.99 0.67 ± 0.02* 14 4.57 ± 0.37 0.77 ± 0.02

GenotypesCYP1A1 wt/wt 32 6.63 ± 0.71 0.68 ± 0.01* 31 4.71 ± 0.30 0.75 ± 0.01CYP1A1 wt/m1 + m1/m1 7 4.86 ± 0.88 0.73 ± 0.03 3 4.67 ± 0.88 0.78 ± 0.03GSTM1 positive 28 6.55 ± 0.76 0.68 ± 0.01* 24 5.00 ± 0.35 0.77 ± 0.02GSTM1 null 11 5.68 ± 0.98 0.70 ± 0.03 10 4.00 ± 0.36 0.71 ± 0.02

Results are reported as the group mean (± SEM) of individual MN number; 100 cells were scored for each individual for MN.aAge, considered as categorical variable in this descriptive data set, classified according to the mean value (i.e. 40.56 years)

of the observed age distribution in the whole population.*= p < 0.05, vs. the corresponding control group; §= p < 0.05, vs. whole control group: Kolmogorov-Smirnov Z test.

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1438 M. VILLARINI ET AL.

SCE, smoking was often associated with an increase in SCEfrequency in human biomonitoring studies; however, a com-parative study with 200 healthy subjects failed to demonstratean effect of smoking on SCE (Betti et al., 1995).

The observed negative results (comet assay and SCE) could,at least in part, be due to the appropriate use of protectiveequipment (e.g., helmet, safety glasses, leather gloves, and,in particular, dust mask) by trained workers. However, thedata obtained in this study (MN) indicate that the genotoxicrisk of occupational exposure associated with the tunnelconstruction work cannot be excluded. Moreover, it shouldbe take into consideration a possible effect of exposure onthe lymphocyte proliferation rates. In fact, exposure togenotoxic xenobiotics could generate false negative resultsif damaged cells are delayed in their cell cycle. Geneticpolymorphisms of CYP1A1 and GSTM1 did not affect theconsidered biomarkers of exposure/effect. Nevertheless,these results must be cautiously interpreted, due to the rela-tively small number of exposed and control individualsincluded in this study.

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