Mutation Research 583 (2005) 85–94

Comparison of genetic damage in brazilian footwear-workersexposed to solvent-based or water-based adhesive

Vanina Dahlstrom Heusera, Vanessa Moraes de Andradea, Juliana da Silvab,∗,Bernardo Erdtmanna,c

a Programa de P´os-Gradua¸cao em Gen´etica e Biologia Molecular (PPGGBM), Universidade Federal do Rio Grande do Sul (UFRGS),Porto Alegre-RS, Brazil

b Laboratorio de Genetica Toxicologica, Programa de P´os-Gradua¸cao em Ensino de Ciˆencias e Matem´atica (PPGECIM), UniversidadeLuterana do Brasil (ULBRA), Canoas-RS, Brazil

c Instituto de Biotecnologia, Universidade de Caxias do Sul (UCS), Caxias do Sul-RS, Brazil

Received 4 September 2004; received in revised form 3 March 2005; accepted 4 March 2005Available online 1 April 2005

Abstract

Research has shown that workers employed in footwear manufacture are at increased risk of some cancers, the strongestevidence being for nasal cancer and leukemia. Footwear-workers are routinely exposed to complex mixtures of solvents indegreasers, cleaners, primers, and adhesives used in the production process as toluene,n-hexane, acetone, and possibly dust

azards ofd adhesives

adhesives,tsameters foressed. Our

age indextedter-basedpresence,

s

sil

particles, additives in shoe materials and degradation products of materials. The recognition of the potential health-hsolvent-based adhesives (SBAs) has lead to the development of adhesives with no organic solvents, the water-base(WBA). We investigated footwear-workers (all males) exposed to SBA (n= 29) (for 3.98± 4.13 years), and WBA (n= 16), whichhad spent the six months previous to the study employed in an experimental section which used only water-basedalthough they had previously worked in sections which used solvent-based adhesives (for 5.80± 4.03 years); 25 healthy subjecwere used as controls. The Comet assay and the micronucleus test were used as endpoints, while the traditional parassessing exposure to toluene in organic mixtures by measuring the concentration of urinary hippuric acid were also assresults showed a significantly lower mean concentration of hippuric acid in the control group than found in the SBA (P< 0.001)and WBA (P< 0.05) groups. The Comet assay results showed that there was a significant increase in the mean damfor the SBA (P< 0.001) group in comparison to the WBA group and control (P< 0.05). For the micronucleus test in binuclealymphocytes and exfoliated buccal cell, the three groups were not statistically different. Our study demonstrated that waadhesives are clearly a better option for safeguarding the health of footwear-workers, even with possibility of isocyanatewhile the positive results observed in SBA group might be explained by chloroprene presence in the adhesive.© 2005 Published by Elsevier B.V.

Keywords:Comet assay; Micronucleus test; Exfoliated buccal cells; Footwear-workers; Hippuric acid; Occupational exposure to glue

∗ Corresponding author at: Programa de Pos-Graduac¸ao em Ensino de Ciencias e Matematica (PPGECIM), Universidade Luterana do Bra(ULBRA), Predio 14, Sala 230, Rua Miguel Tostes 101, Bairro Sao Luıs, CEP 92420-280, Canoas-RS, Brazil.

E-mail address:juliana.silva@ulbra.br (J. da Silva).

1383-5718/$ – see front matter © 2005 Published by Elsevier B.V.doi:10.1016/j.mrgentox.2005.03.002

86 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94

1. Introduction

Brazil is one of the largest producers of footwearin the world, which annually produces more than 700million pairs of footwear (25–30% for export) in about4,000 companies which together directly employ morethan 210,000 workers. The Sinos River Valley, locatedin the southernmost Brazilian State of Rio Grandedo Sul (RS), is home to about 40% of all Brazilianfootwear production and 75% of all Brazilian footwearexports[1,2].

Most studies find that workers in footwear manu-facture are routinely exposed to complex mixtures ofsolvents, including acetone,n-hexane, methylethylke-tone, and large amounts of toluene[3,4]. None of thesesolvents is considered genotoxic or carcinogenic[5–7].However, the health effects of organic solvent mixturesare not well known, but the elevated risk was consid-ered to be a consequence of the exposure to this com-plex mixture[4].

Research has shown that workers employed infootwear manufacture are at increased risk of somecancers, the strongest evidence being for nasal can-cer and leukemia. The increased risk of nasal can-cer was associated with exposure to leather dust.The occurrence of leukemia among footwear-workersexposed to benzene has been well documented[8].Due to these data, many risk factors in shoe andleather factories, as benzene and hexavalent chromium,were substituted by similar but less toxic products,a Al-t m-m lu-a osi-t dus-t m)a ults,b no-t isa xm ectsa ensa cts[

s ofa aseda t ofa ad-

hesives) or 100% water-based adhesives, but po-tentially hazardous solvent-based adhesives are stillthe most widely used adhesives in the Brazilianfootwear industry, probably because water-based ad-hesives are four times more expensive and take 3 hlonger to dry than their solvent-based counterparts[21].

Hippuric acid is the main metabolite resulting fromtoluene exposure and has been suggested as a markerfor estimating exposure to both high and low con-centrations of toluene[22], even in solvent mixtures[3,23,24]. Blood cells have also been used to mon-itor biomarkers in human populations, with cytoge-netic markers having been extensively used to detectthe early biological effects of DNA-damaging agents.During the last few years, the alkaline single-cell gelelectrophoresis assay, also known as the Comet assay,has been used in human biomonitoring studies as arapid and sensitive tool for demonstrating chemically-induced DNA damage, cells with damaged DNA dis-playing increased migration of DNA fragments fromthe nucleus and the formation of a Comet shape[25,26].

Another cytogenetic test used to measure occupa-tional exposure to toxic agents is the micronucleustest, which assesses the micronuclei originating fromchromosome fragments or whole chromosomes thatare not included in the main daughter nuclei dur-ing nuclear division. The micronucleus test provides

hro-ensi-ssi-

n beasinalthod

ons

tedandd theintsredr as-ary

s toluene and trivalent chromium, respectively.hough the modification was introduced by recoendation of World Health Organization, the evation by short-term genotoxicity tests presented p

ive results in shoe manufacturing and leather inry [9–12]. Toluene and leather dust (with chromiure frequently cited as responsible for these resut individually they are considered non or little geoxic [13,14]. While the neurotoxicity of toluenen accepted fact[15,16], its genotoxicity in compleixtures is still under discussion; perhaps its effre increasing the host susceptibility to carcinognd/or turning other bio-available genotoxic produ

3,11,17–20].The recognition of the potential health-hazard

dhesives containing organic solvents (solvent-bdhesives – SBAs) has led to the developmendhesives with no organic solvents (solvent-free

a measure of both chromosome breakage and cmosome loss and has shown to be at least as stive an indicator of chromosome damage as the clacal metaphase chromosome analysis[27,28]. The mi-cronucleus test also has the advantage that it caused with cells with high rate of divisions, suchepithelial cells, without the need of cell culturevitro. The analysis of micronuclei in exfoliated bucccells has been demonstrated to be a sensitive mefor monitoring genetic damage in human populati[29–32].

In the study described in this paper, we investigafootwear-industry workers exposed to water-basedsolvent-based adhesives using the Comet assay anmicronucleus test as sensitive cytogenetic endpofor the detection of genotoxic effects and compathe data produced to the traditional parameters fosessing exposure to toluene (concentration of urinhippuric acid).

V.D. Heuser et al. / Mutation Research 583 (2005) 85–94 87

2. Materials and methods

2.1. Study population and sample collection

This study was approved by the Brazilian NationalEthical Committee on Research (Comissao NacionaldeEtica em Pesquisa – CONEP) and informed writtenconsent was obtained from each individual prior to thestart of the study.

The study involved 45 male footwear-industryworkers, 29 of whom (the solvent-based adhesive(SBA) group) were employed at two factories in thesectors where they were occupationally exposed toglues, adhesives and solutions containing organic sol-vent mixtures (mainly toluene and low concentrationsof hexane, acetone and methylethylketone). The other16 (the water-based adhesive (WBA) group) spent the6 months previous to the study employed in an experi-mental section which used only water-based adhesivescontaining little (<0.16% (v/v) acetone) or no organicsolvent, although they had previously worked in sec-tions which used solvent-based adhesives. Besides thedifference in solvent content in adhesives, water-basedadhesives contained polyurethane, while solvent-basedadhesives contained polychroprene in their formula-tion. The control group consisted of 25 healthy maleswith no occupational exposure.

All the individuals examined in the study lived inor near the city of Novo Hamburgo-RS and were re-quired to answer a Portuguese version of a question-naire from the International Commission for Protectionagainst Environmental Mutagens and Carcinogens[33]and participate in a face-to-face questionnaire whichincluded standard demographic data (age, gender, etc.)as well as questions relating to medical issues (expo-sure to X-rays, vaccinations, medication, etc.), life style(smoking, coffee, alcohol, diet, etc.) and their occupa-tion (number of hours worked per day, time exposedto organic solvents, use of protective measures, etc.).Individuals were selected for the three groups (control,SBA and WBA) in such a manner so as to ensure thatexcept for occupational exposure to organic solvents,there were no marked differences between the mem-bers of the groups. In all the groups, individuals whosmoked more than five cigarettes per day for at leastone year were considered smokers. The characteristicsof the three groups are presented inTable 1.

Blood and urine samples were obtained from in-dividuals in the SBA and WBA groups on the sameday during a normal shift during the workers periodi-cal medical examinations by the nurses from a regionalHealth and Safety at Work Medical Center (Centrode Saude e Seguranc¸a do Trabalhador das IndustriasCalcadistas da Regiao de Parobe, Parobe-RS, Brasil

Table 1Some characteristics of unexposed control individuals and footwear-industry workers exposed to water-based adhesives (WBA) and solvent-basedadhesives (SBA)

ontrol

N 25

M 30.64±(19–4

T– .00)

S20 (76 6%)2 (8.0 )3 (12. )

20± 10

A5 (20. %)

19 ( 52%)1 (4.

1.92± 2

amples with SBA)h mples

C

o. of subjects

ean age± S.D. (years)Range (min–max)

ime of exposure (mean± S.D., years)a –Range (min–max)

moking statusNo. of non-smokersNo. of ex-smokersNo. of current smokersCigarettes/day± S.D.

lcohol drinking statusNo. of never drinkersNo. of non-habitual drinkers (0–3 times/week)No. of habitual drinkers (4–7 times/week)Drinks/week± S.D.

a These data refer to exposure up to 6 months before blood saving started working with WBA 6 months before the blood sa

WBA SBA

16 29

8.76 25.38± 4.40 27.10± 7.074) (18–34) (19–44)

5.80± 4.03 3.98± 4.13(0.42–14.00) (0.50–19

.00%) 13 (81.25%) 22 (75.80%) 2 (12.50%) 6 (20.68%00%) 1 (6.25%) 1 (3.45%

10± 0 10± 0

00%) 10 (62.50%) 10 (34.4876.00%) 6 (37.50%) 19 (65.00%) 0 0.97 1.23± 0.58 1.38± 0.57

were taken, the WBA group (who had previously been workingwere taken; S.D. = standard deviation.

88 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94

(CESSTIC) – The Footwear Industry Health and Safetyat Work Center for the Parobe Region, Parobe-RS,Brazil). For the control group, blood and urine sam-ples were taken at the same region. All blood sampleswere collected using venipuncture and heparinized va-cutainers and processed as quickly as possible to avoidthe damage associated with storage[34], the blood cellsamples being transported to the laboratories at or be-low 8◦C and processed within 8 h of collection in orderto minimize loss of DNA and damage due to DNA re-pair processes.

2.2. Hippuric acid assay

Analysis of urinary hippuric acid was carried outusing gas chromatography at a commercial laboratory(Toxilab, Toxicological Laboratory, Porto Alegre-RS,Brazil) using a Perkin–Elmer XL Auto System gaschromatograph (Perkin–Elmer, USA).

2.3. Comet assay

The alkaline Comet assay was performed as de-scribed by Singh et al.[35] with the modifications sug-gested by Tice et al.[36]. Blood cells (5�l) were em-bedded in 95�l of 0.75% low-melting point agaroseand when the agarose had solidified the slides, theywere placed in lysis buffer (2.5 M NaCl, 100 mM EDTAand 10 mM Tris; pH 10.0–10.5) containing freshlyadded 1% (v/v) Triton X-100 and 10% (v/v) dimethyls xi-m thes ffer( ina 5 V( asn As -t lec-t posi-t odc be-iom uis,M -t ga-t ults.

Images of 200 randomly selected cells (100 cellsfrom each of two replicate slides) were analyzed foreach person. Comet image lengths (IL) (i.e., the nu-clear region + tail) were measured in arbitrary units us-ing a calibrated eyepiece micrometer (1 unit =≈5�mat 200×) and a fluorescence microscope equippedwith a 12 nm BP546 excitation filter and a 590 nmbarrier filter. Cells were also visually allocated toone of the five classes depending on tail size (class0 = no tails, class 4 = longest tails) to give a single-DNA damage score for each subject and hence foreach group studied; the group damage index (DI)ranging from 0 (no tails on any cells, i.e. 200cells× 0) to 800 (all cells with maximally long tails,i.e. 200 cells× 4) [34,37,38]. The damage frequency(DF (%), i.e. the proportion of cells with alteredmigration) was calculated based on the number ofcells with tails versus the number of cells withouttails. All the slides were scored blindly on the sameday on which they were subjected to electrophore-sis.

2.4. Micronucleus test

2.4.1. Cytokinesis-blocked human lymphocyte MNFor each blood sample, duplicate lymphocytes cul-

tures were set up in culture flasks by adding 0.3 ml ofwhole blood to 5 ml of RPMI 1640 medium (Nutricell,Campinas-SP, Brazil) containing 20% fetal calf serumand 1% (v/v) phytohemaglutinin. The flasks incubatedaB o-t ion,t n at8 /v)m copes loods rome cul-t andn clei,a l mi-cw

2in-

n den

ulfoxide (DMSO) for a minimum of 1 h and a maum of 2 weeks. After treatment with lysis buffer,

lides were incubated in freshly-made alkaline bu300 mM NaOH and 1 mM EDTA; pH > 13) for 20 mnd the DNA was electrophoresed for 20 min at 20.90 V/cm) and 300 mA after which the buffer weutralized with 0.4 M Tris (pH 7.5) and the DNtained with ethidium bromide (2�g/ml). The elecrophoresis procedure and efficiency for each erophoresis run was checked using negative andive internal controls consisting of whole human bloollected in the laboratory, the negative controlng unmodified blood and the positive control 50�lf blood mixed with 13�l (8 × 10−5 M) of methylethanesulfonate (CAS No 66-27-3; Sigma, St. LoO, USA) and incubated for 2 h at 37◦C. Each elec

rophoresis run was considered valid only if the neive and positive controls yielded the expected res

t 37◦C for 44 h before adding 5�g/ml of cytochalasin(Sigma)[27] and continuing incubation until the t

al incubation time reached was 72 h. After incubathe lymphocytes were harvested by centrifugatio00 revs/min for 8 min, recentrifuged, fixed in 3:1 (vethanol/acetic acid, placed onto a clean micros

lide and stained with 5% (v/v) Giemsa. For each bample, 2000 binucleated lymphocytes (i.e. 1000 fach of the two slides prepared from the duplicate

ures) were scored for both micronuclei presenceucleoplasmic bridges (NPB) between daughter nussessment being made using bright-field opticaroscopy at a magnification of 200–1000×. All sidesere coded to blind analysis.

.4.2. Buccal cellsBuccal cells were collected by swabbing the

er cheek of the individuals with a moistened woo

V.D. Heuser et al. / Mutation Research 583 (2005) 85–94 89

tongue depressor, the tip of which was immersedin 5 ml of cold saline (0.9% (w/v) aqueous NaCl)in a conical tube and transported under refrigera-tion to the laboratory where the saline was cen-trifuged at 1500 revs/min for 8 min and the sedi-mented buccal cells washedtwice more with salineunder the same centrifugation conditions to removebacteria and cell debris which would have compli-cated scoring. After washing, a drop of cell suspen-sion was placed onto each of two duplicate micro-scope slides and dried at room temperature for 1–2weeks and then feulgen-stained (hydrolysis in 1NHCl at 60◦C for 10 min followed by immersion inSchiff’s reagent for 3–4 h) and the cytoplasm coun-terstained by immersion in 0.5% (w/v) fast-green for30 s.

The criteria used for micronuclei analysis werethose of Tolbert et al.[39] and Titenko–Holland et al.[40], i.e. for a structure to be considered as a micronu-cleus, it must be: (a) less than one-third of the diameterof the main nucleus; (b) be in the same plane of fo-cus as the main nucleus e; (c) have the same color,texture and refraction as the main nucleus; (d) have asmooth oval or round shape; and (e) be clearly sep-arated from the main nucleus. Only cells that werenot smeared, clumped or overlapped and those whocontained intact nuclei were included in the analysis.According to Tolbert et al.[39] and Gomez–Arroyoet al. [41], exfoliated buccal cells undergo degenera-tive processes which can produce anomalies that ared ofs chro-m and( kenn ry-o inga eus.I ter-i ysisa Theqn ndt ont tedu ca-t lls( eres

2.5. Statistical analysis

The normality of the variables was evaluated usingthe Kolmogorov–Smirnov test and the statistical dif-ferences between the three groups (control, WBA andWSBA) were analyzed using the non-parametric two-tailed Kruskal–Wallis test with the Dunn correctionfor multiple comparisons to perform a non-parametricanalysis of variances. The�2 and t-test were used tocompare the basic characteristic of study populations.The Spearman’s rank test was used to analyze the cor-relation. The critical level for rejection of the null hy-pothesis was considered to be aP value of 5%.

3. Results

The main characteristics of the three groups stud-ied are presented inTable 1, from which it can beseen that there were no significant differences betweenthe mean age of individuals in the different groups(Kruskal–Wallis test), nor in their smoking or drink-ing habits (�2 and Kruskal–Wallis test). There was nosignificant difference in the mean time of exposure toorganic solvents prior to the start of this trail of individ-uals in the water-based adhesive (WBA) and solvent-based adhesive (SBA) groups (t-test).

The replies to the questionnaire showed that 94%of workers in the WBA group and 86% in the SBAgroup used silicone gloves to prevent skin contact witho oriesh

ortw barss iledK nifi-c g/gctS

w mi-c atedc ongt t them sig-n

ifficult to distinguish from micronuclei, examplesuch anomalies being binucleate cells, condensedatin, pinched nuclei with a feulgen-negative b

so called ‘broken egg’ nuclei), pycnosis (shrunuclei), karyorrhexis (disintegrated nuclei) and kalysis in which nuclear dissolution occurs producfeulgen-negative ghost-like image of the nucl

n our study, cells which exhibited these characstics were excluded from the micronucleus analnd all the slides were coded to blind analysis.uality of the slides were determined at 200× mag-ification using a bright-field Zeiss microscope a

he micronuclei frequency was estimated basedhe number of normal exfoliated buccal cells counsing bright-field optical microscopy at a magnifi

ion of 1000×. For each volunteer, 2000 buccal cei.e. 1000 from each of the duplicate slides) wcored.

rganic solvents. We also observed that all the factad ventilation in the work areas.

Box-plots of the urinary hippuric acid levels fhe three groups are shown inFig. 1, where the lineithin a box indicates the mean values and thehow the standard deviation (S.D.). The two-taruskal–Wallis test showed that there was a sigantly lower mean concentration of hippuric acid (reatinine± S.D.) in the control group (0.41± 0.31)han there was in the WBA (0.69± 0.29;P< 0.05) andBA (0.99± 0.62;P< 0.001) groups.The cytogenetic data for the three groups (Table 2)

as analyzed using the Kruskal–Wallis test. For theronucleus test in lymphocytes and buccal exfoliells, no significant difference were detected amhe groups. The Comet assay results showed thaean damage index (DI) for the SBA group wasificantly greater than that for the control (P< 0.05) and

90 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94

Fig. 1. Box-plot of the urinary hippuric acid levels (g/g creatinine)of unexposed control individuals and footwear-industry workers ex-posed to water-based (WBA) and solvent-based (SBA) adhesives.Line within a box indicates the mean values and the bars show thestandard deviation (S.D.). The significance of the WBA and SBAvalues were compared using the two-tailed Kruskal–Wallis test withthe Dunn correction (*P< 0.05; ** P< 0.001).

WBA (P< 0.001) groups and that there was a signifi-cant increase (P< 0.05) in the mean image length (IL)of the SBA and control groups in relation to the WBAgroup, while the mean damage frequency (DF) for theSBA group was significantly higher as compared to theWBA group (P< 0.001). No differences in DI and DFvalues were observed between the means of the con-trol and WBA groups. As expected, the electrophoresisnegative internal control Comet assay blood cells gavea DI of 0–4 which indicated that these cells were notdamaged while the positive internal control blood hada DI of 615–800 which indicates highly damaged cells.

With respect to the significant results observed, weused the Spearman rank test to investigate possible cor-

Fig. 2. Correlation between hippuric acid concentration (g/g cre-atinine) and age for unexposed control individuals (n= 25) andfootwear-industry workers exposed to water-based (WBA,n= 16)and solvent-based (SBA,n= 29) adhesives (Spearman’s rank test).

relations between hippuric acid concentration and ageand DNA damage index and found that there was a pos-itive correlation between the urinary concentration ofhippuric acid and age for the control group (P< 0.02)and the SBA group (P< 0.001) (Fig. 2). There were nocorrelations between Comet assay results and hippuricacid concentration.

4. Discussion

Footwear-workers are routinely exposed to complexmixtures of solvents in degreasers, cleaners, primersand adhesives used in the production process, the mainsolvent used being toluene, with lesser amounts ofn-hexane, acetone, methyethylketone[3], and possiblydust particles (leather, polymers, and finishing materi-als) additives in shoe materials and degradation prod-

Table 2Cytogenetic parameters (mean± S.D.) of unexposed control individuals and footwear-industry workers exposed to water-based adhesives (WBA)and solvent-based adhesives (SBA)

Cytogenetic parameters Control (n= 25) WBA (n= 16) SBA (n= 29)

Comet assay (200 leukocytes/subject)Damage index (index range = 0–800) 3.44± 3.24 2.13± 2.45 8.35± 7.85a,b

Image length (�m) 20.57± 0.93c 19.89± 0.60 20.68± 1.05c

Damage frequency (%) 1.52± 1.31 0.78± 0.91 2.76± 1.99b

Micronucleus tests (2000 cells/subject)Micronuclei present in binucleated lymphocytes 5.20± 2.33 3.88± 1.93 4.90± 2.34Nucleoplasmatic bridges present in binucleated lymphocytes 3.00± 1.97 2.56± 2.53 3.69± 2.49Micronuclei present in exfoliated buccal cells

a Significant in relation to the control group atP< 0.05.b Significant in relation to the WBA group atP< 0.001.c Significant in relation to the WBA group atP< 0.05. All significances b iation.

0.62± 0.73 0.69± 0.87 1.15± 1.45

y the two-tailed Kruskal–Wallis, Dunn test. S.D. = standard dev

V.D. Heuser et al. / Mutation Research 583 (2005) 85–94 91

ucts of materials (i.e. polyurethanes/isocyanates) andadditives[4].

Our data on urinary hippuric acid data (Fig. 1) indi-cate a relationship between urinary hippuric acid con-centration and exposure to toluene present in the mix-tures of organic solvents used, with a higher mean con-centration of this toluene metabolite appearing in theurine of the WBA and SBA groups than that of the con-trol group. This finding agrees with previous studies offootwear-workers that also used the hippuric acid asa parameter to evaluate exposure to solvent mixtures[3,23,24], in which it was found that the concentrationof urinary hippuric acid was higher in workers exposedto toluene and other solvents than in unexposed indi-viduals. The metabolite of toluene and hippuric acidprovided a good estimation of workplace exposure ofworkers with SBG (Fig. 1), but it is necessary to takeinto consideration the other factors which increase hip-puric acid, such as age (Fig. 2). Similar results forhippuric acid in Brazilian population were shown bySiqueira and Paiva[42].

The Comet assay values for the SBA group were sig-nificantly higher than the values for the control group,although there were no differences in damage index(DI) and damage frequency (DF) values between theWBA and control groups (Table 2). According to Ticeet al.[36], DF values (based on the proportion of cellswith altered migration) are less informative (especiallyfor damaged cells) than measures related to the extentof DNA damage, DI values (based on the length ofm il)c DNAd cleii ghn

eno-tO diesw tsi sedtw veb cantr ina-t er ainlyt iph-e ,

confusion due to co-exposure to ink, other solvents andvarious genotoxic substances in the environment can-not be excluded.

Some authors suggest that the presence of iso-cyanates in glues can explain positive findings[11,56].Several studies describe the isocyanate emission po-tential of polyurethane adhesives[57] that may spreadin aerosolized or gaseous form when heating or whenvapors escape to the workplace air from open vessels atroom temperature[58,59]. Respiratory disorders asso-ciated with isocyanate exposure[59–61], and toxicityand/or genotoxicity, even in polymerized form, are de-scribed in a number of assays[56,58,61–65]and occu-pational exposures[66]. The results pertaining to iso-cyanate induction of MN formation were inconsistent[67,68]. Isocyanate has been classified as a carcino-gen in animals[69,70] and is a suspected carcinogenin humans[69]. In our study, isocyanate was presentonly in water-based adhesive composition to whichWBA workers were exposed; this group revealed nopositive results in our genotoxicity tests (Comet assay,BNLMN, and EBCMN). However, it is possible thatthe use of chloroprene as a monomer in the produc-tion of the polychloroprene adhesive by SBG work-ers can explain the increase in Comet assay values inSBG-exposed workers. Positive mutagenicity results ofchloroprene were reported[71,72], but at the exposureconcentrations used in the cancer inhalation studies,chloroprene did not induce SCE or CA in mouse bonemarrow cells, nor did it increase the MN frequency inp sa hoe-w entsp sc

enceo tow s ex-p w am , iti ptionf theg meta test,b rvedi c-c asei ling

igration and the amount of DNA in the Comet taonsidered as being a more sensitive measure ofamage. There was a higher frequency of micronu

n buccal epithelial cells from the SBG group, althouo statistical difference was observed (Table 2).

Toluene alone gives negative results in most goxicity tests with different organisms[13,43–46].ther results were obtained in a multitude of stuith biological monitoring of various genotoxic effec

n peripheral blood lymphocytes from workers expoo toluene in the occupational environment[46–52], asell as glue sniffers[53]. Some positive results haeen obtained in shoe workers, but these signifiesponses might also be due to benzene contamion [11,54,55]. Pitarque et al.[3] obtained negativesults by Comet test in shoe-workers exposed mo toluene, but found increased values for MN in perral lymphocytes in the same group[11]. In most cases

eripheral blood erythrocytes[73,74]. Several studielso reported an increased risk of cancer among sorkers exposed to chloroprene and organic solvresent in glues and adhesives[75–79]. Chloroprene ilassified as possibly carcinogenic to humans[78].

In conclusion, our study demonstrates the absf genotoxic effects in footwear-workers exposedater-based adhesives and although the workerosed to solvent-based adhesives did not shoarked difference in relation to the control group

s clear that water-based adhesives are a better oor safeguarding the health of footwear-workers. Inenotoxicity evaluations made in this study, the Cossay was more sensitive than the micronucleusoth performed on blood cells which was also obse

n other studies[38,80]. The micronucleus test in bual epithelial cells showed a non-significant incren SBA group. Considering the less invasive samp

92 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94

of such types of cells in a proper design of the test,it can present advantages to monitor human popula-tions exposed to inhaled/ingested genotoxins. The ex-posure to solvents estimated by urinary hippuric acidwere higher in the SBA workers in relation to WBAand controls, but the exposure responsible for the DNAdamage could not be identified with certainty, like inmany other studies about solvent mixtures exposure.The genotoxicity, although low, detected in this studyin footwear-workers also reported by other studies[11],and SBA glue sniffers[53] suggests that the use of gluecan present health risks, and the footwear-workers needmore evaluations to assess the risk associated to theseworking conditions. The use of WBA glue seems to bea lesser toxic option than the SBA glue, although morecostly.

Acknowledgments

The authors express their gratitude to all the indi-viduals who volunteered to participate in this study.We also thank Veronica R.S. de Moraes, Silvia Brito,Eduardo Rissi from CESSTIC (Centro de Saude eSeguranc¸a do Trabalhador das Industrias Calc¸adistasda Regiao de Parobe, RS) and especially to Susi N. daSilva for her valuable help during the sampling. Wealso thank Luciana R. Somonet and Diolanda Barros(Bom Pastor Laboratory) and Roseli O. de Candido.This work was financially supported by the Brazil-i entoC

R

-

a,metganic

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