Mutation Research 583 (2005) 85–94

Comparison of genetic damage in brazilian footwear-workers

exposed to solvent-based or water-based adhesive
Vanina Dahlström Heuser a , Vanessa Moraes de Andrade a , Juliana da Silva b,∗ ,
Bernardo Erdtmann a,c
a Programa de Pós-Graduação em Genética e Biologia Molecular (PPGGBM), Universidade Federal do Rio Grande do Sul (UFRGS),
Porto Alegre-RS, Brazil
b Laboratório de Genética Toxicológica, Programa de Pós-Graduação em Ensino de Ciências e Matemática (PPGECIM), Universidade
Luterana 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 2005
Available online 1 April 2005

Abstract

Research has shown that workers employed in footwear manufacture are at increased risk of some cancers, the strongest
evidence being for nasal cancer and leukemia. Footwear-workers are routinely exposed to complex mixtures of solvents in
degreasers, cleaners, primers, and adhesives used in the production process as toluene, n-hexane, acetone, and possibly dust
particles, additives in shoe materials and degradation products of materials. The recognition of the potential health-hazards of
solvent-based adhesives (SBAs) has lead to the development of adhesives with no organic solvents, the water-based adhesives
(WBA). We investigated footwear-workers (all males) exposed to SBA (n = 29) (for 3.98 ± 4.13 years), and WBA (n = 16), which
had spent the six months previous to the study employed in an experimental section which used only water-based adhesives,
although they had previously worked in sections which used solvent-based adhesives (for 5.80 ± 4.03 years); 25 healthy subjects
were used as controls. The Comet assay and the micronucleus test were used as endpoints, while the traditional parameters for
assessing exposure to toluene in organic mixtures by measuring the concentration of urinary hippuric acid were also assessed. Our
results 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 damage index
for the SBA (P < 0.001) group in comparison to the WBA group and control (P < 0.05). For the micronucleus test in binucleated
lymphocytes and exfoliated buccal cell, the three groups were not statistically different. Our study demonstrated that water-based
adhesives are clearly a better option for safeguarding the health of footwear-workers, even with possibility of isocyanate presence,
while 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 glues

∗ Corresponding author at: Programa de Pós-Graduação em Ensino de Ciências e Matemática (PPGECIM), Universidade Luterana do Brasil

(ULBRA), Prédio 14, Sala 230, Rua Miguel Tostes 101, Bairro São Luı́s, 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 footwear
in the world, which annually produces more than 700
million pairs of footwear (25–30% for export) in about
4,000 companies which together directly employ more
than 210,000 workers. The Sinos River Valley, located
in the southernmost Brazilian State of Rio Grande
do Sul (RS), is home to about 40% of all Brazilian
footwear production and 75% of all Brazilian footwear
exports [1,2].
Most studies find that workers in footwear manu-
facture are routinely exposed to complex mixtures of
solvents, including acetone, n-hexane, methylethylke-
tone, and large amounts of toluene [3,4]. None of these
solvents is considered genotoxic or carcinogenic [5–7].
However, the health effects of organic solvent mixtures
are 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 in
footwear manufacture are at increased risk of some
cancers, 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-workers
exposed to benzene has been well documented [8].
Due to these data, many risk factors in shoe and
leather factories, as benzene and hexavalent chromium,
were substituted by similar but less toxic products,
as toluene and trivalent chromium, respectively. Al-
though the modification was introduced by recom-
mendation of World Health Organization, the evalu-
ation by short-term genotoxicity tests presented posi-
tive results in shoe manufacturing and leather indus-
try [9–12]. Toluene and leather dust (with chromium)
are frequently cited as responsible for these results,
but individually they are considered non or little geno-
toxic [13,14]. While the neurotoxicity of toluene is
an accepted fact [15,16], its genotoxicity in complex
mixtures is still under discussion; perhaps its effects
are increasing the host susceptibility to carcinogens
and/or turning other bio-available genotoxic products
[3,11,17–20].
The recognition of the potential health-hazards of
adhesives containing organic solvents (solvent-based
adhesives – SBAs) has led to the development of
adhesives with no organic solvents (solvent-free ad-
hesives) or 100% water-based adhesives, but po-
tentially hazardous solvent-based adhesives are still
the most widely used adhesives in the Brazilian
footwear industry, probably because water-based ad-
hesives are four times more expensive and take 3 h
longer to dry than their solvent-based counterparts
[21].
Hippuric acid is the main metabolite resulting from
toluene exposure and has been suggested as a marker
for 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 detect
the early biological effects of DNA-damaging agents.
During the last few years, the alkaline single-cell gel
electrophoresis assay, also known as the Comet assay,
has been used in human biomonitoring studies as a
rapid and sensitive tool for demonstrating chemically-
induced DNA damage, cells with damaged DNA dis-
playing increased migration of DNA fragments from
the nucleus and the formation of a Comet shape
[25,26].
Another cytogenetic test used to measure occupa-
tional exposure to toxic agents is the micronucleus
test, which assesses the micronuclei originating from
chromosome fragments or whole chromosomes that
are not included in the main daughter nuclei dur-
ing nuclear division. The micronucleus test provides
a measure of both chromosome breakage and chro-
mosome loss and has shown to be at least as sensi-
tive an indicator of chromosome damage as the classi-
cal metaphase chromosome analysis [27,28]. The mi-
cronucleus test also has the advantage that it can be
used with cells with high rate of divisions, such as
epithelial cells, without the need of cell culture in
vitro. The analysis of micronuclei in exfoliated buccal
cells has been demonstrated to be a sensitive method
for monitoring genetic damage in human populations
[29–32].
In the study described in this paper, we investigated
footwear-industry workers exposed to water-based and
solvent-based adhesives using the Comet assay and the
micronucleus test as sensitive cytogenetic endpoints
for the detection of genotoxic effects and compared
the data produced to the traditional parameters for as-
sessing exposure to toluene (concentration of urinary
hippuric acid).
 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94 87

2. Materials and methods All the individuals examined in the study lived in
or near the city of Novo Hamburgo-RS and were re-
2.1. Study population and sample collection quired to answer a Portuguese version of a question-
naire from the International Commission for Protection
This study was approved by the Brazilian National against Environmental Mutagens and Carcinogens [33]
Ethical Committee on Research (Comissão Nacional and participate in a face-to-face questionnaire which
de Ética em Pesquisa – CONEP) and informed written included standard demographic data (age, gender, etc.)
consent was obtained from each individual prior to the as well as questions relating to medical issues (expo-
start of the study. sure to X-rays, vaccinations, medication, etc.), life style
The study involved 45 male footwear-industry (smoking, coffee, alcohol, diet, etc.) and their occupa-
workers, 29 of whom (the solvent-based adhesive tion (number of hours worked per day, time exposed
(SBA) group) were employed at two factories in the to organic solvents, use of protective measures, etc.).
sectors where they were occupationally exposed to Individuals were selected for the three groups (control,
glues, adhesives and solutions containing organic sol- SBA and WBA) in such a manner so as to ensure that
vent mixtures (mainly toluene and low concentrations except for occupational exposure to organic solvents,
of hexane, acetone and methylethylketone). The other there were no marked differences between the mem-
16 (the water-based adhesive (WBA) group) spent the bers of the groups. In all the groups, individuals who
6 months previous to the study employed in an experi- smoked more than five cigarettes per day for at least
mental section which used only water-based adhesives one year were considered smokers. The characteristics
containing little (<0.16% (v/v) acetone) or no organic of the three groups are presented in Table 1.
solvent, although they had previously worked in sec- Blood and urine samples were obtained from in-
tions which used solvent-based adhesives. Besides the dividuals in the SBA and WBA groups on the same
difference in solvent content in adhesives, water-based day during a normal shift during the workers periodi-
adhesives contained polyurethane, while solvent-based cal medical examinations by the nurses from a regional
adhesives contained polychroprene in their formula- Health and Safety at Work Medical Center (Centro
tion. The control group consisted of 25 healthy males de Saúde e Segurança do Trabalhador das Indústrias
with no occupational exposure. Calçadistas da Região de Parobé, Parobé-RS, Brasil
Table 1
Some characteristics of unexposed control individuals and footwear-industry workers exposed to water-based adhesives (WBA) and solvent-based
adhesives (SBA)
Control WBA SBA
No. of subjects 25 16 29
Mean age ± S.D. (years) 30.64 ± 8.76 25.38 ± 4.40 27.10 ± 7.07
Range (min–max) (19–44) (18–34) (19–44)
Time of exposure (mean ± S.D., years)a – 5.80 ± 4.03 3.98 ± 4.13
Range (min–max) – (0.42–14.00) (0.50–19.00)
Smoking status
No. of non-smokers 20 (76.00%) 13 (81.25%) 22 (75.86%)
No. of ex-smokers 2 (8.00%) 2 (12.50%) 6 (20.68%)
No. of current smokers 3 (12.00%) 1 (6.25%) 1 (3.45%)
Cigarettes/day ± S.D. 20 ± 10 10 ± 0 10 ± 0
Alcohol drinking status
No. of never drinkers 5 (20.00%) 10 (62.50%) 10 (34.48%)
No. of non-habitual drinkers (0–3 times/week) 19 (76.00%) 6 (37.50%) 19 (65.52%)
No. of habitual drinkers (4–7 times/week) 1 (4.00%) 0 0
Drinks/week ± S.D. 1.92 ± 2.97 1.23 ± 0.58 1.38 ± 0.57
a These data refer to exposure up to 6 months before blood samples were taken, the WBA group (who had previously been working with SBA)

having started working with WBA 6 months before the blood samples were taken; S.D. = standard deviation.
88 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94
(CESSTIC) – The Footwear Industry Health and Safety
at Work Center for the Parobé Region, Parobé-RS,
Brazil). For the control group, blood and urine sam-
ples were taken at the same region. All blood samples
were collected using venipuncture and heparinized va-
cutainers and processed as quickly as possible to avoid
the damage associated with storage [34], the blood cell
samples being transported to the laboratories at or be-
low 8 ◦ C and processed within 8 h of collection in order
to minimize loss of DNA and damage due to DNA re-
pair processes.
2.2. Hippuric acid assay
Analysis of urinary hippuric acid was carried out
using gas chromatography at a commercial laboratory
(Toxilab, Toxicological Laboratory, Porto Alegre-RS,
Brazil) using a Perkin–Elmer XL Auto System gas
chromatograph (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 agarose
and when the agarose had solidified the slides, they
were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA
and 10 mM Tris; pH 10.0–10.5) containing freshly
added 1% (v/v) Triton X-100 and 10% (v/v) dimethyl
sulfoxide (DMSO) for a minimum of 1 h and a maxi-
mum of 2 weeks. After treatment with lysis buffer, the
slides were incubated in freshly-made alkaline buffer
(300 mM NaOH and 1 mM EDTA; pH > 13) for 20 min
and the DNA was electrophoresed for 20 min at 25 V
(0.90 V/cm) and 300 mA after which the buffer was
neutralized with 0.4 M Tris (pH 7.5) and the DNA
stained with ethidium bromide (2 ␮g/ml). The elec-
trophoresis procedure and efficiency for each elec-
trophoresis run was checked using negative and posi-
tive internal controls consisting of whole human blood
collected in the laboratory, the negative control be-
ing unmodified blood and the positive control 50 ␮l
of blood mixed with 13 ␮l (8 × 10−5 M) of methyl
methanesulfonate (CAS No 66-27-3; Sigma, St. Louis,
MO, USA) and incubated for 2 h at 37 ◦ C. Each elec-
trophoresis run was considered valid only if the nega-
tive and positive controls yielded the expected results.
Images of 200 randomly selected cells (100 cells
from each of two replicate slides) were analyzed for
each 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 ␮m
at 200×) and a fluorescence microscope equipped
with a 12 nm BP546 excitation filter and a 590 nm
barrier filter. Cells were also visually allocated to
one of the five classes depending on tail size (class
0 = no tails, class 4 = longest tails) to give a single-
DNA damage score for each subject and hence for
each group studied; the group damage index (DI)
ranging from 0 (no tails on any cells, i.e. 200
cells × 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 altered
migration) was calculated based on the number of
cells with tails versus the number of cells without
tails. All the slides were scored blindly on the same
day on which they were subjected to electrophore-
sis.
2.4. Micronucleus test
2.4.1. Cytokinesis-blocked human lymphocyte MN
For each blood sample, duplicate lymphocytes cul-
tures were set up in culture flasks by adding 0.3 ml of
whole blood to 5 ml of RPMI 1640 medium (Nutricell,
Campinas-SP, Brazil) containing 20% fetal calf serum
and 1% (v/v) phytohemaglutinin. The flasks incubated
at 37 ◦ C for 44 h before adding 5 ␮g/ml of cytochalasin
B (Sigma) [27] and continuing incubation until the to-
tal incubation time reached was 72 h. After incubation,
the lymphocytes were harvested by centrifugation at
800 revs/min for 8 min, recentrifuged, fixed in 3:1 (v/v)
methanol/acetic acid, placed onto a clean microscope
slide and stained with 5% (v/v) Giemsa. For each blood
sample, 2000 binucleated lymphocytes (i.e. 1000 from
each of the two slides prepared from the duplicate cul-
tures) were scored for both micronuclei presence and
nucleoplasmic bridges (NPB) between daughter nuclei,
assessment being made using bright-field optical mi-
croscopy at a magnification of 200–1000 ×. All sides
were coded to blind analysis.
2.4.2. Buccal cells
Buccal cells were collected by swabbing the in-
ner cheek of the individuals with a moistened wooden
 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94
tongue depressor, the tip of which was immersed
in 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 saline
under the same centrifugation conditions to remove
bacteria 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 1–2
weeks and then feulgen-stained (hydrolysis in 1N
HCl at 60 ◦ C for 10 min followed by immersion in
Schiff’s reagent for 3–4 h) and the cytoplasm coun-
terstained by immersion in 0.5% (w/v) fast-green for
30 s.
The criteria used for micronuclei analysis were
those 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 diameter
of 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 a
smooth oval or round shape; and (e) be clearly sep-
arated from the main nucleus. Only cells that were
not smeared, clumped or overlapped and those who
contained intact nuclei were included in the analysis.
According to Tolbert et al. [39] and Gómez–Arroyo
et al. [41], exfoliated buccal cells undergo degenera-
tive processes which can produce anomalies that are
difficult to distinguish from micronuclei, examples of
such anomalies being binucleate cells, condensed chro-
matin, pinched nuclei with a feulgen-negative band
(so called ‘broken egg’ nuclei), pycnosis (shrunken
nuclei), karyorrhexis (disintegrated nuclei) and kary-
olysis in which nuclear dissolution occurs producing
a feulgen-negative ghost-like image of the nucleus.
In our study, cells which exhibited these character-
istics were excluded from the micronucleus analysis
and all the slides were coded to blind analysis. The
quality of the slides were determined at 200× mag-
nification using a bright-field Zeiss microscope and
the micronuclei frequency was estimated based on
the number of normal exfoliated buccal cells counted
using bright-field optical microscopy at a magnifica-
tion of 1000 ×. For each volunteer, 2000 buccal cells
(i.e. 1000 from each of the duplicate slides) were
scored.
89
2.5. Statistical analysis
The normality of the variables was evaluated using
the Kolmogorov–Smirnov test and the statistical dif-
ferences between the three groups (control, WBA and
WSBA) were analyzed using the non-parametric two-
tailed Kruskal–Wallis test with the Dunn correction
for multiple comparisons to perform a non-parametric
analysis of variances. The ␹2 and t-test were used to
compare 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 a P value of 5%.
3. Results
The main characteristics of the three groups stud-
ied are presented in Table 1, from which it can be
seen that there were no significant differences between
the 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 no
significant difference in the mean time of exposure to
organic 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 SBA
group used silicone gloves to prevent skin contact with
organic solvents. We also observed that all the factories
had ventilation in the work areas.
Box-plots of the urinary hippuric acid levels for
the three groups are shown in Fig. 1, where the line
within a box indicates the mean values and the bars
show the standard deviation (S.D.). The two-tailed
Kruskal–Wallis test showed that there was a signifi-
cantly lower mean concentration of hippuric acid (g/g
creatinine ± S.D.) in the control group (0.41 ± 0.31)
than there was in the WBA (0.69 ± 0.29; P < 0.05) and
SBA (0.99 ± 0.62; P < 0.001) groups.
The cytogenetic data for the three groups (Table 2)
was analyzed using the Kruskal–Wallis test. For the mi-
cronucleus test in lymphocytes and buccal exfoliated
cells, no significant difference were detected among
the groups. The Comet assay results showed that the
mean damage index (DI) for the SBA group was sig-
nificantly greater than that for the control (P < 0.05) and
90 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94

Fig. 2. Correlation between hippuric acid concentration (g/g cre-

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 the
standard deviation (S.D.). The significance of the WBA and SBA
values were compared using the two-tailed Kruskal–Wallis test with
the 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 WBA
group, while the mean damage frequency (DF) for the
SBA group was significantly higher as compared to the
WBA group (P < 0.001). No differences in DI and DF
values were observed between the means of the con-
trol and WBA groups. As expected, the electrophoresis
negative internal control Comet assay blood cells gave
a DI of 0–4 which indicated that these cells were not
damaged while the positive internal control blood had
a DI of 615–800 which indicates highly damaged cells.
With respect to the significant results observed, we
used the Spearman rank test to investigate possible cor-
atinine) and age for unexposed control individuals (n = 25) and
footwear-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 age
and DNA damage index and found that there was a pos-
itive correlation between the urinary concentration of
hippuric acid and age for the control group (P < 0.02)
and the SBA group (P < 0.001) (Fig. 2). There were no
correlations between Comet assay results and hippuric
acid concentration.
4. Discussion
Footwear-workers are routinely exposed to complex
mixtures of solvents in degreasers, cleaners, primers
and adhesives used in the production process, the main
solvent used being toluene, with lesser amounts of n-
hexane, acetone, methyethylketone [3], and possibly
dust particles (leather, polymers, and finishing materi-
als) additives in shoe materials and degradation prod-

Table 2
Cytogenetic 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 ± 2.34
Nucleoplasmatic bridges present in binucleated lymphocytes 3.00 ± 1.97 2.56 ± 2.53 3.69 ± 2.49
Micronuclei present in exfoliated buccal cells 0.62 ± 0.73 0.69 ± 0.87 1.15 ± 1.45
a Significant in relation to the control group at P < 0.05.
b Significant in relation to the WBA group at P < 0.001.
c Significant in relation to the WBA group at P < 0.05. All significances by the two-tailed Kruskal–Wallis, Dunn test. S.D. = standard deviation.
 V.D. Heuser et al. / Mutation Research 583 (2005) 85–94
ucts of materials (i.e. polyurethanes/isocyanates) and
additives [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 the
urine of the WBA and SBA groups than that of the con-
trol group. This finding agrees with previous studies of
footwear-workers that also used the hippuric acid as
a parameter to evaluate exposure to solvent mixtures
[3,23,24], in which it was found that the concentration
of urinary hippuric acid was higher in workers exposed
to toluene and other solvents than in unexposed indi-
viduals. The metabolite of toluene and hippuric acid
provided a good estimation of workplace exposure of
workers with SBG (Fig. 1), but it is necessary to take
into consideration the other factors which increase hip-
puric acid, such as age (Fig. 2). Similar results for
hippuric acid in Brazilian population were shown by
Siqueira 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 the
WBA and control groups (Table 2). According to Tice
et al. [36], DF values (based on the proportion of cells
with altered migration) are less informative (especially
for damaged cells) than measures related to the extent
of DNA damage, DI values (based on the length of
migration and the amount of DNA in the Comet tail)
considered as being a more sensitive measure of DNA
damage. There was a higher frequency of micronuclei
in buccal epithelial cells from the SBG group, although
no statistical difference was observed (Table 2).
Toluene alone gives negative results in most geno-
toxicity tests with different organisms [13,43–46].
Other results were obtained in a multitude of studies
with biological monitoring of various genotoxic effects
in peripheral blood lymphocytes from workers exposed
to toluene in the occupational environment [46–52], as
well as glue sniffers [53]. Some positive results have
been obtained in shoe workers, but these significant
responses might also be due to benzene contamina-
tion [11,54,55]. Pitarque et al. [3] obtained negative
results by Comet test in shoe-workers exposed mainly
to toluene, but found increased values for MN in periph-
eral lymphocytes in the same group [11]. In most cases,
91
confusion due to co-exposure to ink, other solvents and
various 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 spread
in aerosolized or gaseous form when heating or when
vapors escape to the workplace air from open vessels at
room temperature [58,59]. Respiratory disorders asso-
ciated with isocyanate exposure [59–61], and toxicity
and/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 carcinogen
in humans [69]. In our study, isocyanate was present
only in water-based adhesive composition to which
WBA workers were exposed; this group revealed no
positive results in our genotoxicity tests (Comet assay,
BNLMN, and EBCMN). However, it is possible that
the 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 in
SBG-exposed workers. Positive mutagenicity results of
chloroprene were reported [71,72], but at the exposure
concentrations used in the cancer inhalation studies,
chloroprene did not induce SCE or CA in mouse bone
marrow cells, nor did it increase the MN frequency in
peripheral blood erythrocytes [73,74]. Several studies
also reported an increased risk of cancer among shoe-
workers exposed to chloroprene and organic solvents
present in glues and adhesives [75–79]. Chloroprene is
classified as possibly carcinogenic to humans [78].
In conclusion, our study demonstrates the absence
of genotoxic effects in footwear-workers exposed to
water-based adhesives and although the workers ex-
posed to solvent-based adhesives did not show a
marked difference in relation to the control group, it
is clear that water-based adhesives are a better option
for safeguarding the health of footwear-workers. In the
genotoxicity evaluations made in this study, the Comet
assay was more sensitive than the micronucleus test,
both performed on blood cells which was also observed
in other studies [38,80]. The micronucleus test in buc-
cal epithelial cells showed a non-significant increase
in SBA group. Considering the less invasive sampling
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 acid
were higher in the SBA workers in relation to WBA
and controls, but the exposure responsible for the DNA
damage could not be identified with certainty, like in
many other studies about solvent mixtures exposure.
The genotoxicity, although low, detected in this study
in footwear-workers also reported by other studies [11],
and SBA glue sniffers [53] suggests that the use of glue
can present health risks, and the footwear-workers need
more evaluations to assess the risk associated to these
working conditions. The use of WBA glue seems to be
a lesser toxic option than the SBA glue, although more
costly.
Acknowledgments
The authors express their gratitude to all the indi-
viduals who volunteered to participate in this study.
We also thank Verônica R.S. de Moraes, Silvia Brito,
Eduardo Rissi from CESSTIC (Centro de Saúde e
Segurança do Trabalhador das Indústrias Calçadistas
da Região de Parobé, RS) and especially to Susi N. da
Silva for her valuable help during the sampling. We
also thank Luciana R. Somonet and Diolanda Barros
(Bom Pastor Laboratory) and Roseli O. de Cândido.
This work was financially supported by the Brazil-
ian agency Conselho Nacional para o Desenvolvimento
Cientı́fico e Tecnológico (CNPq).
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