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Occupational Exposure to Formaldehyde: GenotoxicRisk Evaluation By Comet Assay And Micronucleus TestUsing Human Peripheral LymphocytesSolange Costa a b , Carolina Pina a , Patrícia Coelho a b , Carla Costa a b , Susana Silva a ,Beatriz Porto c , Blanca Laffon d & João Paulo Teixeira a ba National Institute of Health, Environmental Health Department, Porto, Portugalb ICETA, Institute of Agricultural and Agri-foodstuffs Sciences and Technologies, University ofPorto, Porto, Portugalc ICBAS, Biomedical Sciences Institute of Abel Salazar, University of Porto, Porto, Portugald Toxicology Unit, Department Psychobiology, University of A Coruña, A Coruña, Spain

Available online: 27 Jun 2011

To cite this article: Solange Costa, Carolina Pina, Patrícia Coelho, Carla Costa, Susana Silva, Beatriz Porto, Blanca Laffon& João Paulo Teixeira (2011): Occupational Exposure to Formaldehyde: Genotoxic Risk Evaluation By Comet Assay AndMicronucleus Test Using Human Peripheral Lymphocytes, Journal of Toxicology and Environmental Health, Part A, 74:15-16,1040-1051

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Journal of Toxicology and Environmental Health, Part A, 74:1040–1051, 2011Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287394.2011.582293

OCCUPATIONAL EXPOSURE TO FORMALDEHYDE: GENOTOXIC RISK EVALUATIONBY COMET ASSAY AND MICRONUCLEUS TEST USING HUMAN PERIPHERALLYMPHOCYTES

Solange Costa1,2, Carolina Pina1, Patrícia Coelho1,2, Carla Costa1,2, Susana Silva1,Beatriz Porto3, Blanca Laffon4, João Paulo Teixeira1,2

1National Institute of Health, Environmental Health Department, Porto, Portugal2ICETA, Institute of Agricultural and Agri-foodstuffs Sciences and Technologies, University ofPorto, Porto, Portugal3ICBAS, Biomedical Sciences Institute of Abel Salazar, University of Porto, Porto, Portugal4Toxicology Unit, Department Psychobiology, University of A Coruña, A Coruña, Spain

Formaldehyde (FA) is a world high-production compound with numerous applications rang-ing from production of resins to medicines. Due to its sensitizing properties, irritating effectsand potential cancer hazard FA is of great environmental health concern. Numerous studiesin humans and experimental animals demonstrated that inhaled FA produced toxicity, geno-toxicity, and cancer at distal sites. IARC, based on sufficient data, reclassified FA as a humancarcinogen. The highest level of human exposure to this aldehyde occurs in occupational set-tings, namely, in pathology and anatomy laboratories, where FA is commonly used as a fixativeand tissue preservative. Several studies consistently showed that the levels of airborne FA inanatomy laboratories exceeded recommended exposure criteria. In order to assess the geno-toxic effects of chronic occupational exposure to FA, a group of pathology/anatomy workerswas assessed using a micronucleus (MN) test and comet assay. The level of exposure to FAwas also determined and the time-weighted average (TWA) of exposure was calculated foreach subject. The TWA mean value for FA exposed workers was 0.43 ± 0.06 ppm, exceedingnational and international recommended limit levels of 0.3 ppm. Both MN frequency andcomet assay parameters were significantly higher in exposed subjects. Data obtained confirma correlation between genetic damage and occupational exposure to FA. These data, alongwith recent implications of human carcinogenicity, point out the need for close monitoringof occupational exposure to FA. Implementation of security and hygiene measures as well asgood practices campaigns may be crucial to decrease risk.

Formaldehyde (FA) (CAS number 50-00-0) is an environmental health concernbecause this compound produces sensitiza-tion and irritation and is potentially a can-cer hazard. Given the economic importanceand widespread use, many individuals areexposed to FA environmentally and/or occu-pationally. Nonoccupational exposure includes

This work is supported by Fundação para a Ciência e a Tecnologia (FCT) under grants SFRH/BD/46929/2008 and PTDC/SAU-ESA/102367/2008.

Address correspondence to Solange Costa, National Institute of Health, Environmental Health Department; Rua AlexandreHerculano, 321, 4000-055 Porto, Portugal. E-mail: solange.costa2@gmail.com

vehicles emissions, tobacco smoke, and house-hold products, namely, furniture, plywoodand detergents.

Based on sufficient epidemiological andexperimental evidence the InternationalAgency for Research on Cancer (IARC) reclassi-fied FA as a human carcinogen (Group 1) (IARC2006). Increased incidences of nasopharyngeal

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OCCUPATIONAL EXPOSURE TO FA ON GENOTOXICITY 1041

cancer were found in populations occupation-ally exposed to FA (Hayes et al. 1990; Hansenand Olson 1995; Hauptmann et al. 2003).Animal studies demonstrated that high con-centrations of FA produce irreversible damageto the nasal epithelium of rats and that in somecases rats exposed to these concentrationsdeveloped neoplasia (Merck and Speit 1998).The postulated mode of action for nasal tumorsin rats was considered biologically plausibleand was considered likely to be relevant tohumans (Merck and Speit 1998).

More recently, due to IARC reclassification,the U.S. National Institute of EnvironmentalHealth Sciences (NIEHS) recommended thereview of FA carcinogen status and its reclas-sification as a known carcinogen in its 12threport on carcinogens (RoC). Formaldehydewas listed as reasonably anticipated humancarcinogen, since 2nd RoC’s in 1981, and itsstatus remained unchanged since then. IARCworking group also concluded, in their review,that there is strong but not sufficient evidencefor a causal association between leukemia andoccupational exposure to FA. A number of epi-demiological studies found a potential causalassociation between occupational exposure toFA and excess mortality from leukemia, espe-cially myeloid leukemia (Zhang et al. 2009;IARC 2006). However due to the mixed resultsand lack of an accepted biological model theevidence for FA leukemogenicity in humansremains controversial.

Formaldehyde-induced genotoxicity wasconfirmed in a variety of experimental sys-tems ranging from bacteria to rodents. Severalstudies demonstrated that both DNA damageand chromosome changes were induced byFA in cell culture experiments, and in vivo inhumans and experimental animals at the sitesof FA exposure (Liu et al. 2009; IARC 2006).Increased number of chromosomal aberrations(CA) (Jakab et al. 2010), elevated micronucleus(MN) frequency (He et al. 1998; Orsiére et al.2006; Costa et al. 2008) and enhanced sisterchromatid exchange (SCE) frequency (Shahamet al. 2002; Costa et al. 2008) were reportedin peripheral blood lymphocytes from individ-uals occupationally exposed to FA. However,

these results are more variable, with increasesin damage being reported in some studiesbut not in others (Ying et al. 1999; Heckand Casanova 2004; Schmid and Speit 2007).Biological evidence of cellular toxicity distantfrom the respiratory tract such as peripherallymphocytes is still inadequate and conflictingand thus requires further study.

The highest level of human exposureto FA occurs in occupational settings.Occupational exposure involves not onlyindividuals employed in the direct manu-facture of FA and products containing thischemical, but also those subjects using theseproducts, namely, in pathology and anatomylaboratories where FA is commonly used asa fixative and tissue preservative. Indoor airanalyses showed that the levels of airborneFA in pathology and anatomy laboratoriesexceeded the recommended exposure criterialevels (Paustenbach et al. 1997; Shaham et al.2002; Akbar-Khanzadeh and Pulido 2003). Inthese settings, absorption of FA occurs mainlythrough inhalation. Inhaled FA primarily affectsthe upper airways; the severity and extent ofphysiological response depend on chemicalconcentration in the air. The known humanoccupational exposure to FA, along with theimplications of human carcinogenicity, empha-sizes the need for close monitoring of exposureto FA.

Methods over a wide range are currentlyused for the detection of early biologicaleffects of DNA-damaging agents in occupa-tional settings. The micronucleus test, SCE,and CA in peripheral blood lymphocytes arewell-established cytogenetic techniques thatare used extensively to evaluate the pres-ence and extent of chromosome damage inhuman biomonitoring studies. High quantitiesof CA and MN are associated with increasedcancer risks in otherwise healthy individuals(Bonassi et al. 2008; Murgia et al. 2008). TheMN test provides a reliable measure of chro-mosomal breakage and loss at less expenseand is less time-consuming than CA (Bonassiet al. 2007).

The comet assay has been found to bea sensitive method for human biomonitoring

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1042 S. COSTA ET AL.

for detection of DNA damage, and a usefultool for detection of genetic damage at theindividual cell level (Collins 2004). The cometcombines the simplicity of biochemical tech-niques for detecting DNA single-strand breaks(strand breaks and incomplete excision repairsites), alkali-labile sites, and cross-linking, withthe single-cell approach typical of cytogeneticassays. The main advantages of the comet assayinclude: (a) collection of data at the level ofthe individual cell, allowing more robust sta-tistical analysis; (b) need for a small numberof cells per sample (<10,000); (c) sensitivityfor detecting DNA damage; and (d) use ofany eukaryote (and some prokaryote) single-cell population. The length of the comet “tail”(TL) and the intensity of DNA staining withinit (percent tail DNA) are proportional to theextent of DNA damage. Thus, in image-analysissystems these two parameters, TL and percenttail DNA, are usually the selected parame-ters used to assess DNA damage in humanbiomonitoring studies. However, TL tends toreach a maximum at a low level of dam-age and Collins (2008) recommended usingpercent tail DNA.

The simultaneous use of cytogenetic testsand comet assay in occupational studiesenables a comparison of the presence of(1) DNA strand breaks, due to both acute andchronic exposure, and (2) chromosome dam-age, due to clastogenic and aneugenic events.In the case of chronic exposure, comparing thelevels of damage from a cytogenetic techniquewith the comet assay may provide informa-tion regarding concurrent versus past exposure,contributing to a more comprehensive riskassessment (Albertini et al. 2000).

The aim of the present study was toassess both DNA and cytogenetic damage inperipheral lymphocytes of FA-exposed work-ers in pathology/anatomy laboratories. Geneticdamage was studied by means of the MNtest and comet assay (percent tail DNA andTL). For the environmental exposure assess-ment, air sampling was performed in worker’sbreathing zone for representative working peri-ods, and an 8-h time-weighted average (TWA)was determined.

TABLE 1. Characteristics of the Study Population

Control Exposed

Number ofsubjects

50 48

Gender 36 females 36 females14 males 12 males

Age (yr)a 37.4 ± 10.3 (24–61) 40.3 ± 9.9 (23–60)Years of

employmenta— 13.6 ± 8.7 (1–31)

Smoking statusNonsmokers 43 (86%) 38 (79%)Smokers 7 (14%) 10 (21%)Number of

cigarettes/daya13 ± 8 (6–30) 12 ± 5 (5–20)

Number ofyears

smokinga

19 ± 15 (4–51) 18 ± 10 (3–36)

aMean ± SD.

METHODS

Sample SelectionThe study population consisted of 48 sub-

jects occupationally exposed to FA, work-ing for at least 1 yr, from 5 hospitalpathology/anatomy laboratories, located inPortugal, and 50 nonexposed control employ-ees, matched by age, gender, and smokinghabits, working in the same area in administra-tive offices but without occupational exposurehistory to FA. The characteristics of both groupsare described in Table 1. Health conditions,medical history, medication, diagnostic tests (x-rays etc.), and lifestyle factors were assessedby means of questionnaires. Subjects of theexposed group also gave information relatedto working practices, such as use of protec-tive measures, years of employment, specificsymptoms related to FA exposure, and chronicrespiratory diseases such as asthma and oth-ers. All subjects were fully informed aboutthe procedures and objectives of this study,and each subject prior to the study signedan informed consent form. Ethical approvalfor this study was obtained from the institu-tional Ethical Board of the National Instituteof Health.

Environmental MonitoringAir sampling was performed in the work-

ers breathing zone for representative working

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OCCUPATIONAL EXPOSURE TO FA ON GENOTOXICITY 1043

periods; analysis of the samples allowed thecalculation of the 8-h TWA level of exposureto FA for each subject. Air sampling and FAanalysis were performed according to NIOSHmethod 3500 (NIOSH 1994).

Collection of Blood Samples forGenotoxic EvaluationHeparinized venous blood samples were

collected between 10 and 11 a.m. from eachdonor, and were immediately processed for thedifferent methodologies used in this study. Allsamples were coded and analyzed under blindconditions.

Micronucleus (MN) TestAliquots of 0.5 ml heparinized whole blood

were used to establish duplicate lymphocytecultures for cytokinesis-blocked MN test, asdescribed by Teixeira et al. (2004). Cultureswere incubated at 37◦C in the dark for a totalof 72 h; cytochalasin B (6 µg/ml) was added at44 h to prevent cytokinesis. Cells were col-lected by centrifugation and treated twice witha mixture of RPMI (pH 7.2) supplementedwith 2% fetal bovine serum (FBS). The cellswere centrifuged again and submitted to a mildhypotonic treatment in a mixture of RPMI (pH7.2) supplemented with 2% FBS–deionizedwater (1:4, v/v). Then the centrifuged cellswere placed on dry slides and smears were per-formed. After air-drying, slides were fixed withcold methanol–acetic acid (3:1, v/v). Air-driedslides were stained with 4% Giemsa in pH 6.8phosphate buffer. Microscopic analyses wereperformed using a Nikon Eclipse E400 lightmicroscope. To determine the total number ofMN in binucleated cells, in total 1000 binucle-ated cells with well-preserved cytoplasm (500per replicate) were scored for each subject.Micronuclei were scored blindly by the samereader and identified according to the criteriadefined by Fenech (2007).

Comet AssayLymphocytes were isolated, in dupli-

cate, from heparinized whole blood, by

centrifugation on a Ficoll density gradient. Thelymphocyte layer (buffy coat) was removedand washed thrice with ice-cold phosphatebuffer solution (PBS), pH 7.4, at approximately270 × g for 10 min. Cell viability, determinedby trypan blue exclusion, was higher than 80%in all cases.

The alkaline version of the comet assaywas performed as described by Singh et al.(1988) with minor modifications described byCosta et al. (2008). Two slides were pre-pared for each donor, and a “blind” scorerexamined 50 randomly selected cells fromeach slide (100 cells/donor) using a magni-fication of 400×. Image capture and anal-ysis were performed with Comet Assay IVsoftware (Perceptive Instruments). Both per-cent tail DNA and TL were determined.Percent DNA in tail represents tail inten-sity and is linearly related to DNA breakfrequency up to approximately 80% in tail,which defines the useful range of the assay(Collins 2004). Tail length indicates theextent of migration of genetic material inthe direction of the anode (Singh et al.1988) and is expected to be proportional tothe level of single-strand breaks and alkali-labile sites.

Statistical AnalysisThe distribution of variables was com-

pared with normal distribution by meansof the Kolmogorov–Smirnov goodness-of-fittest. The effects of occupational exposure,gender, age, and tobacco smoking wereassessed. Micronucleus was the only parame-ter that departed significantly from normalityand therefore the Mann–Whitney U-test andKruskal–Wallis test, both nonparametric, wereapplied to the data. For comet assay paramet-ric tests, one-way analysis of variance (ANOVA)and Student’s t-test were applied. The asso-ciations between two variables were analyzedby Pearson’s (parametric) and Spearman’s (non-parametric) correlation tests. The level of sig-nificance considered was p < .05. All analyseswere conducted using the SPSS for Windowsstatistical package 16.0.

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1044 S. COSTA ET AL.

RESULTS AND DISCUSSION

Environmental MonitoringIn order to assess the FA level of exposure

of the five hospital pathology/anatomy labora-tories, air samples were collected and analyzed.The FA concentrations varied depending on thetasks; the main FA vapor emissions occurredduring the macroscopic examination of FA-preserved specimens and during the disposalof specimens and waste solutions. The meanlevel of FA exposure of the 48 workers stud-ied was 0.43 ± 0.06 ppm (0.04–1.58 ppm).The current Portuguese occupational exposurestandard is 0.3 ppm (ceiling level), indicat-ing the maximal safe FA airborne concentra-tion that should not be exceeded during anylength of time in a worker’s breathing zone.The American Conference of GovernmentalIndustrial Hygienists (ACGIH) has also set forFA a ceiling limit at 0.3 ppm (ACGIH 2008).Hence, the results obtained in this study showthat workers in pathology/anatomy laborato-ries are exposed to levels of airborne FAthat exceed both the national guideline limitvalue and the ACGIH recommended expo-sure criteria.

Several studies on environmental monitor-ing of pathology/anatomy laboratories (Akbar-Khanzadeh and Pulido 2003; Dufresne et al.2002; Shaham et al. 1997) also found levelsof FA near or higher than recommended limitvalues. Shaham et al. (2002) reported for 14pathology departments mean levels of FA expo-sure of 0.4–2.24 ppm. Ohmichi et al. (2006)measured FA indoor concentrations and per-sonal exposure levels in a gross anatomy labduring 3 sessions out of a total 20 sessionsover 10 wk; the mean level of exposure toFA obtained was 0.44 ppm, while the aver-age personal FA exposure levels for instruc-tors and students ranged between 0.45 and

1.08 ppm. Other investigators reported similarresults; Akbar-Khanzadeh et al. (1994) reportedfor 34 workers of a gross anatomy labora-tory a mean FA exposure level of 1.24 ppm(0.07–2.94 ppm). Orsière et al. (2006) mea-sured the occupational exposure to FA inpathology and anatomy laboratories by per-sonal air sampling and reported a mean con-centration for an 8-h period of 0.1 ppm (0.1–0.7 ppm). Overall, our study, in agreement withother studies, shows pathology/anatomy lab-oratories as one of the occupational settingswhere workers are frequently exposed to lev-els of FA near or higher than recommendedlimit values, which indicates a potential risk toworker’s health.

GenotoxicityEffects of exposure on genotoxic endpoints

studied, MN frequency, and DNA damage(comet assay) are shown in Table 2. For the vari-ables studied, significant increases were foundin the FA-exposed workers compared with thecontrols.

The distribution of MN frequency andcomet assay parameters in the study populationcan be seen in Figure 1. For the endpoints stud-ied, the distribution in the exposed populationdisplays higher levels of damage. Moreover,the distributions of comet assay parameters,percent tail DNA and TL, are similar in bothgroups. Accordingly, a significant positive cor-relation was found between these two param-eters (r = .789). Between MN frequency andTL the correlation was close to significance(r = .199); with respect to MN frequency andpercent tail DNA no significant association wasfound. In addition, a positive significant corre-lation was noted between FA exposure levelsand MN frequency (r = .342).

Previous studies also found a signifi-cant increase of MN frequency in peripheral

TABLE 2. MN Frequency, Percent Tail DNA, and TL in Populations Examined

Mean MN (‰) ± SE, (range) Mean percent tail DNA ± SE, (range) Mean TL (µm) ± SE, (range)

Control 3.66 ± 0.51 (0–11) 8.01 ± 0.64 (2.83–24.40) 42. ± 1.6 (17.14–74.62)Exposed 6.19 ± 0.62∗ (1–18) 11.76 ± 0.74∗ (4.72–29.67) 54.55 ± 2.02∗ (33.14–99.09)

Note. SE, mean standard error. Asterisk indicates significant compared to control, p < .05.

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OCCUPATIONAL EXPOSURE TO FA ON GENOTOXICITY 1045

ExposedControls

0

10

20

30

4050

60

70

0–4 5–8 9–12 >13

% I

ndiv

idua

ls

Mean MN frequency

(A)

0

10

20

30

40

50

60

<40 41–55 56–70 >71

Mean TL (µm)

% I

ndiv

idua

ls

(C)

0

10

20

30

40

50

60

<6 7–10 11–14 >15

Mean % tail DNA

% I

ndiv

idua

ls

(B)

FIGURE 1. Distribution of MN frequency (A), percent tail DNA (B), and comet TL (C) in control and exposed populations.

lymphocytes of FA exposed workers and inepithelial cells (nasal and buccal). Suruda et al.(1993), during an embalming course, exam-ined the effect of inhaled FA (0.33 ppm) onoral, nasal, and lymphocyte cells of a groupof 29 mortician students. Results showed thatepithelial cells from the buccal mucosa had a12-fold elevation in MN frequency, micronu-cleated lymphocytes frequency rose 26%, andnasal epithelial MN increased 22%. A dose-response relationship was observed with cumu-lative exposure to FA. He et al. (1998) appliedMN assay to detect abnormalities in humanperipheral lymphocytes of 13 students exposedto FA (2.37 ppm) during a 12-wk anatomyclass. Micronuclei frequencies were signifi-cantly elevated compared with controls, andthe FA-exposed group also showed a signifi-cant rise in CA. Orsière et al. (2006) examinedthe genotoxic effect of occupational expo-sure to FA on 59 pathology and anatomylaboratory workers. Assessment of chromoso-mal damage was carried out by MN assayin peripheral lymphocytes; the frequency ofbinucleated micronucleated cells was signifi-cantly higher in pathologists/anatomists thanin controls. Overall these findings along withour results demonstrate the capacity of FA toinduce chromosomal damage on cells locateddistant from the exposure site, such as periph-eral lymphocytes of exposed workers.

However, some of these studies have beencritically reviewed and the reported humangenotoxic effects in lymphocytes were con-sidered to be unlikely due to the implausi-bility of systemic distribution of inhaled FA

(Schmid and Speit 2007). The main reasonspresented were the high potential of FA toreact and be metabolized, and the absence ofincrease of FA endogenous blood concentra-tions in experimental animals after FA inhala-tion (Casanova et al. 1988; Heck and Casanova2004). Nevertheless, recent studies showedsome evidence of FA systemic distribution inhumans. Pala et al. (2008) found significantlyhigher levels of FA–albumin adducts in labo-ratory workers exposed to high levels of FAcompared to low-level-exposed workers. Inaddition, significantly higher levels of FA–DNAadducts were noted in leukocytes of smokerscompared to nonsmokers. Inhalation of FA incigarette smoke was suggested as the proba-ble source for the elevated FA–DNA adducts insmokers (Wang et al. 2009).

Regarding the comet assay, in a differ-ent FA occupational exposure scenario (ply-wood factories), a significant increase of TLand olive tail moment (OTM) was describedby Yu et al. (2005) in peripheral blood lym-phocytes of 151 workers. Recently, Jiang et al.(2010) reported significantly greater damage asassessed by MN test and comet assay (OTM),in workers in the same type of factories (mean8-h TWA concentration 0.83 ppm) comparedwith controls. These results obtained for FA-exposed workers in an industrial occupationalscenario are in agreement with our results forpathology/anatomy workers.

The MN test represents a measure of bothchromosome breakage and chromosome loss,and therefore reflects exposure to agents withclastogenic and aneugenic modes of action.

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1046 S. COSTA ET AL.

Although in this study the presence of cen-tromeric signals in the MN test was notassessed, the combined significant increasesin MN frequency and comet assay parame-ters suggest a clastogenic effect for FA as theprimary mechanism underlying MN formation.Accordingly, a significantly higher fraction ofacentromeric MN in buccal and nasal cells col-lected from mortuary science students exposedto FA before and after a 90-d embalmingcourse was found by Titenko-Holland et al.(1996). In addition, Schmid and Speit (2007)using human blood cultures found the majorityof FA-induced MN were centromere-negative,indicating a clastogenic mode of action forFA. The observed clastogenic action of FA onhuman blood cultures is also in agreementwith results obtained in other in vitro studies(IPCS 1989).

However, Orsière et al. (2006), combiningthe MN test with fluorescent in situ hybridiza-tion (FISH) with a pan-centromeric DNAprobe, demonstrated a significantly higherfrequency of monocentromeric MN in FA-exposed pathologists/anatomists, whereas foracentromeric MN frequency no marked dif-ferences were obtained between exposed andcontrol groups, which may indicate that chro-mosomal damage leading to micronucleatedlymphocytes is due to chromosome loss and topotential FA-induced defects in mitotic appa-ratus. Hence, in order to define the primarymechanism leading to MN formation in FA-exposed subjects, more studies are necessarycombining the MN test with assays that enablediscrimination between clastogenic and aneu-genic events.

In human biomonitoring studies it is impor-tant to assess the influence of major confound-ing factors such as gender, age, and smok-ing habits. The effect of gender on MN fre-quency and comet assay is shown in Table 3.MN frequency was numerically higher inexposed females compared to males. However,among controls this difference reached sig-nificance; females showed a significant risein MN (4.36 ± 0.65) compared to males(1.86 ± 0.44). This is in agreement with currentknowledge on the effect of gender on geneticdamage that determines a 1.5-fold greater MNfrequency in females than males (Fenech et al.1999). This finding may be attributed to prefer-ential aneugenic events involving the X chro-mosome (Barale et al. 1998; Surralés et al.1996). No significant difference was foundbetween males and females from both groupsfor percent tail DNA and TL, which is in agree-ment with studies of Angerer et al. (2007) andMøller (2006).

Smoking habits did not influence MN fre-quency and comet assay parameters. Thereare some conflicting results regarding the effectof smoking on MN frequencies and cometassay parameters (Laffon et al. 2002; Bhalliet al. 2006; Ergene et al. 2007). In twometa-analysis studies no marked associationbetween smoking and these two genotoxicendpoints was noted (Hoffmann and Speit2005; Bonassi et al. 2003). Moreover, Bonassiet al. (2003) recommended that quantita-tive data about smoking habit should alwaysbe collected, and whenever the collectionis large enough to satisfy statistical require-ments the subgroup of heavy smokers (≥30

TABLE 3. Effect of Gender on MN Frequency, Percent Tail DNA, and TL

N MN (‰) ± SE, (range) Percent tail DNA ± SE, (range) TL (µm) ± SE, (range)

GenderControl

Females 36 4.36 ± 0.65∗ (0–11) 7.76 ± 0.81 (3.13–24.40) 40.99 ± 2.02 (17.14–74.62)Males 14 1.86 ± 0.44 (0–5) 8.66 ± 0.96 (2.83–15.92) 44.57 ± 2.29 (28.85–58.75)

ExposedFemales 36 6.61 ± 0.70 (1–18) 12.03 ± 0.92 (4.72–29.67) 55.59 ± 2.49 (33.14–99.09)Males 12 4.92 ± 1.32 (1–17) 10.96 ± 1.17 (6.29–20.43) 51.41 ± 3.08 (33.76–76.18)

Note. SE, mean standard error. Asterisk indicates significant difference between control females and control males, p < .05.

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OCCUPATIONAL EXPOSURE TO FA ON GENOTOXICITY 1047

TABLE 4. Effect of Age on MN Frequency, Percent Tail DNA, and TL

Group age N Group MN (‰) ± SE, (range) Percent tail DNA ± SE, (range) TL (µm) ± SE, (range)

Control≤30 yr 15 1 1.87 ± 0.45 (0–7) 9.16 ± 1.35 (4.53–24,40) 43.77 ± 3.05 (29.87–66.52)31–36 yr 17 2 2.59 ± 0.57 (0–9) 7.83 ± 0.98 (2.83–17.75) 41.16 ± 2.30 (17.14–59.05)37–47 yr 8 3 4.00 ± 1.10 (0–10) 7.51 ± 2.17 (3.13–20.97) 38.51 ± 4.23 (36.63–66.33)≥48 yr 10 4 7.90 ± 1.44a (2–11) 7.00 ± 0.84 (3.78–10.24) 43.55 ± 4.31 (28.85–74.62)

Exposed≤30 yr 10 1 3.40 ± 0.98 (1–10) 11.76 ± 1.54 (6.77–24.35) 57.70 ± 3.52 (42.37–80.79)31–36 yr 11 2 5.00 ± 0.56 (2–8) 10.76 ± 1.54 (5.99–20.43) 52.52 ± 4.14 (35.71–76.18)37–47 yr 15 3 6.60 ± 1.28 (1–17) 12.82 ± 1.49 (4.72–29.67) 58.07 ± 4.34 (33.76–99.09)≥48 yr 12 4 9.08 ± 1.27b (3–18) 11.36 ± 1.62 (5.20–24.88) 49.36 ± 3.50 (33.14–71.48)

Note. SE, mean standard error.aSignificant difference with regard to groups 1, 2, and 3, p < .05.bSignificant difference with regard to groups 1 and 2, p < .05.

cigarettes per day) should be specifically eval-uated, because in the absence of such data,the simple comparison of smokers versus non-smokers might be misleading. As shown inTable 1, no heavy smokers were found instudy population. However it is importantto note that the low number of smokingsubjects included in this study may affectour results.

In order to examine the influence of age(Table 4), exposed and nonexposed individ-uals were subdivided into 4 groups: ≤30,31–36, 37–47, and ≥48 yr. Micronuclei fre-quency increased with age in both populations.A significant rise of MN rate was found forcontrol subjects comparing the elder (≥48 yr)with the younger groups. In the exposed groupa significant elevation of MN frequency wasnoted between the elder and two youngergroups (≤30 yr, 31–36 yr). Moreover, a sig-nificant correlation was found between thesetwo parameters.

The correlation of increased chromosomaldamage with age was reported by Ramseyet al. (1995). The aging process is relatedto a progressive rise in spontaneous chromo-some instability and loss of efficiency in DNArepair mechanisms, resulting in accumulationof genetic lesions (Wojda et al. 2007; Orsièreet al. 2006), which consequently may influencethe MN frequency and provide a basis for sig-nificant elevation in MN frequency in the eldercompared with younger groups. In contrast,no significant effect of age was found in DNA

damage endpoints, in agreement with previousstudies (Laffon et al. 2006).

A positive correlation was found betweenthe duration of exposure (years of employment)and MN frequency (r = .41). No significantassociation was noted between duration ofexposure and comet assay parameters. Thebasis for the absence of association might bethe fact that the comet assay reflects recentexposures and detects a type of damage thatmight be easily repaired. Similar results wererecently observed in groups of workers exposedto petroleum fumes during an average of 7.3 yr(Singh et al. 2010), and exposed to pesticidesduring an average of 25.69 yr of employment(Remor et al. 2009).

CONCLUSIONS

Occupational exposure to FA in five hos-pital pathology/anatomy laboratories showedthat workers are exposed to high levels ofFA (0.43 ppm), exceeding national and inter-national recommended threshold limit values(0.3 ppm). Our results also showed that DNAand chromosomal damage occurred in periph-eral lymphocytes of pathology/anatomy work-ers exposed to FA. Moreover, a tendency foran accumulation of chromosomal damage withthe duration of FA exposure was found withsignificant positive correlation between MNfrequency and years of employment. For FAexposure assessment, the MN test and cometassay seem to be useful biomarkers in the

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evaluation of genotoxic effects in human pop-ulations occupationally exposed to this alde-hyde. In addition, the positive correlationobtained between comet assay parameters,percent tail DNA and comet tail length, indi-cated that either one may be used to reliablyassess DNA damage in FA-exposed subjects.

Overall, data obtained in this study indi-cate that genotoxic risk due to FA occupationalexposure cannot be excluded, which, alongwith implications of human carcinogenicity,emphasizes the need for close monitoring ofFA exposure in this type of workplace. Thus,implementation of security and hygiene mea-sures in this sector, as well as good practicecampaigns, may be crucial to decrease the risk.

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