Individual sensitivity to DNA damage induced by styrene invitro: influence of cytochrome P450, epoxide hydrolase and
glutathione S-transferase genotypes
Blanca Laffon a,b, Beatriz Perez-Cadahıa a,b, Eduardo Pasaro a,Josefina Mendez a,*
a Departmento Biologıa Celular y Molecular, Facultad de Ciencias, Universidade da Coruna, Campus A Zapateira s/n, 15071, A Coruna,
Spainb Instituto de Ciencias de la Salud, Universidade da Coruna, 15071, A Coruna, Spain
Received 4 October 2002; received in revised form 2 December 2002; accepted 2 December 2002
Abstract
Styrene is a monomer of great commercial interest; its polymers and copolymers are used in a wide range of
applications. In humans, styrene metabolism involves oxidation by cytochrome P450 monooxygenases (CYPs) to
styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure and detoxification
by means of epoxide hydrolase (EH) and glutathione S -transferases (GSTs). The objective of this study was to
investigate if genetic polymorphisms of metabolic enzymes modulate styrene-induced DNA damage in human
leukocytes. CYP2E1, CYP1A1, EH, GSTP1, GSTM1 and GSTT1 polymorphisms were determined in 30 healthy
donors and alkaline comet assay was carried out in isolated leukocytes exposed to 5 and 10 mM styrene, using 1%
acetone as solvent control. The results obtained suggest that CYP1A1 m1, m2 and m4, CYP2E1 Dra I and GSTP1
(exons 5 and 6) polymorphisms may affect styrene induction of DNA damage in human leukocytes.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Styrene; Comet assay; Genetic polymorphisms; Cytochrome P450; Epoxide hydrolase; Glutathione S -transferase
1. Introduction
Styrene (CAS No. 100-42-5) is one of the most
important monomers worldwide; its polymers and
copolymers are used in a wide range of applica-
tions with great commercial interest. Styrene is
used at a level of :/40% as a reactive diluent in
unsaturated polyester resins, most of which are
reinforced with fiberglass (Pfaffli and Saamanen,
1993). The highest occupational exposures to
styrene occur during the manufacture of these
products, especially in large items, such as boats,
that involve manual lamination procedures (Miller
et al., 1994).
Styrene has been shown to induce sister-chro-
matid exchanges (SCE), micronuclei (MN), aneu-
* Corresponding author. Tel.: �/34-981-167000; fax: �/34-
981-167065.
E-mail address: [email protected] (J. Mendez).
Toxicology 186 (2003) 131�/141
www.elsevier.com/locate/toxicol
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 7 2 9 - 1
ploidy, single-strand breaks and chromosomebreaks in several cell systems in vitro, primarily
after metabolic activation (Norppa and Sorsa,
1993; Scott and Preston, 1994). Styrene-7,8-oxide
(SO) is the first metabolite of styrene, produced by
cytochrome P450 monooxygenases (CYPs). SO is
thought to be responsible for the genotoxic effects
of styrene exposure and many in vitro studies have
demonstrated its ability to induce SCE, MN,chromosomal aberrations and DNA damage
(Scott and Preston, 1994; Laffon et al., 2001a,
2002) and to alter the expression of certain
apoptosis-related genes (Laffon et al., 2001b). SO
is then hydrolyzed by epoxide hydrolase (EH) or,
to a minor extent, conjugated with glutathione by
glutathione S-transferases (GSTs) (Sumner and
Fennell, 1994). The International Agency forResearch on Cancer (IARC, 1994) has classified
styrene as possible human carcinogen (group 2B)
and SO as probable human carcinogen (group 2A).
Biotransformation of chemicals, involving acti-
vation and detoxification pathways, plays a pri-
mary role in chemical carcinogenesis. The human
genome contains at least 50 different P450 genes,
subdivided into ten different families and theyfunction as the terminal oxidase in an electron
transport chain (Autrup, 2000). These enzymes are
involved in the metabolism of fatty acids, steroids
and xenobiotics. EH metabolizes a range of alkene
and arene oxides to dihydrodiols and, as such, play
an important role in the metabolism of toxic,
highly reactive intermediates formed by CYP-
mediated reaction to less toxic metabolites. GSTsare key phase II enzymes and play critical roles in
protection against products of oxidative stress and
electrophiles. They conjugate hydrophobic and
electrophilic compounds with reduced glutathione.
Many of these genes, encoding for xenobiotic-
metabolizing enzymes, have been found to be
polymorphic and some of these polymorphisms
affect the function of the encoded proteins. Thesepolymorphisms may be important in determining
an individual’s ability to biotransform environ-
mental carcinogens or mutagens and therefore, to
modulate the toxicological outcome.
The objective of this study was to investigate if
the polymorphisms of metabolic traits modulate
the genotoxic potential of styrene. CYP1A1 (m1,
m2, m4), CYP2E1 (Rsa I, Dra I), EH (exons 3 and4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1
polymorphisms were determined and single-strand
breaks, measured by means of alkaline comet
assay, were evaluated as a marker of genetic
damage to explore the relationship of the cited
polymorphisms and styrene-induced DNA da-
mage.
2. Materials and methods
2.1. Samples and chemicals
Thirty human volunteers were used for this
study, 15 females and 15 males, mean age 27.13
(range 18�/48). All subjects filled out a detailedquestionnaire on consumer habits to ensure they
were not alcohol or coffee addicts and to check
that they had not been on any medical treatment
or exposed to any known mutagen in the 3-month
period prior to sample collection. Two blood
fractions were obtained from each donor, one in
a lithium heparin container, processed for MN
cultures and lymphocyte isolation and another inan EDTA container, for DNA extraction and
genotyping.
Styrene (CAS No. 100-42-5) and acetone were
purchased from Sigma Chemicals Co. (St. Louis,
MO). Styrene was dissolved in acetone and 10 ml of
each solution were added to the culture media to
obtain final concentrations of 5 and 10 mM
styrene, using 1% acetone as negative solventcontrol.
2.2. Comet assay
2.2.1. Leukocyte isolation and treatment
Mononuclear leukocytes were isolated by means
of a Ficoll density gradient. Heparinized whole
blood diluted (1:1) with phosphate buffer solution
(PBS) pH 7.4 was centrifuged at 2100 rpm for 30min over half a volume of lymphocyte isolation
medium (Rafer, Zaragoza, Spain). The buffy coat
was removed and washed twice in PBS at 1500
rpm for 10 min.
Leukocytes were then resuspended to obtain
5�/105 cells/ml in RPMI 1640 medium containing
B. Laffon et al. / Toxicology 186 (2003) 131�/141132
the different styrene treatments or 1% acetone and
maintained at 37 8C for 30 min. Cells were then
collected by centrifugation at 9000 rpm for 3 min
and washed in PBS. Cell viability was estimated by
Trypan blue exclusion, �/90% in the controls and
�/80% in all the exposed leukocytes. These
viability results are in the range recommended by
Henderson et al. (1998) (�/75%) to avoid false-
positive responses due to cytotoxicity.
2.2.2. Cell embedding and electrophoresis
The comet assay was performed under alkaline
conditions, basically as described by Singh et al.
(1988) with minor modifications. Briefly, 80 ml of
0.5% low-melting-point agarose (LMA) (Gibco
BRL, Paisley, UK), in PBS at pH 7.4 containing
:/2.5�/105 cells, was dropped onto a slide pre-
coated with a 150 ml layer of 1% normal-melting-
point agarose (Gibco BRL) and dehydrated at
65 8C for 15 min. Slides were placed on ice for 10
min and a third layer of 80 ml LMA was applied
and allowed to solidify again on ice. Coverslips
were then removed and slides were immersed in
freshly prepared lysing solution (2.5 M NaCl, 100
mM Na2EDTA, 250 mM NaOH, 10 mM Tris�/
HCl, pH 10, with 1% Triton X-100 added just
before use) for 1 h at 4 8C. After lysis, slides were
placed on a horizontal electrophoresis tank into an
ice bath. The tank was filled with freshly made
alkaline electrophoresis buffer (1 mM Na2EDTA,
300 mM NaOH, pH �/13) to cover the slides and
they were left for 20 min in the dark, to prevent
additional DNA damage, to allow DNA unwind-
ing and expression of alkali-labile sites. Electro-
phoresis was carried out for 20 min at 0.83 V/cm,
also in the dark. The slides were then washed three
times for 5 min each with neutralizing solution (0.4
M Tris�/HCl, pH 7.5) and stained with 60 ml of 5
mg/ml 4,6-diamidino-2-phenylindole (DAPI) in
antifade solution. The preparations were kept in
a humidified sealed box to prevent drying of the
gel and analyzed within 24 h. Two slides were
prepared for each treatment and donor. Slides
from three donors were processed in each electro-
phoresis.
2.2.3. Imaging and analysis
The slides were coded and examined by a ‘blind’
scorer using a magnification of 400�/. One
hundred randomly selected cells (50 per replicate
slide) were examined from each sample. Image
capture and analysis were performed using the
QWIN Comet software (Leica Imaging Systems,
Cambridge, UK) and tail length, measured from
the estimated center of the cell (Olive et al., 1990),was measured for each cell as DNA damage
parameter.
2.3. Genotype analysis
All genotype analyses were performed at least in
duplicate. PuregeneTM DNA isolation kit (Gentra
Systems, Minneapolis) was used to isolate DNAfrom the donor samples.
2.3.1. CYP1A1
CYP1A1 mutations were characterized by poly-
merase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) techniques. For de-
termination of m1 (T3801C, 3?-flanking region), a
247 bp fragment was amplified using 0.75 U Taq
polymerase, 0.5 mM of each primer (5?-GTG CAC
TGG TAC CAT TTT GTT -3? and 5?-TCT TGT
CTC ATG CCT GTA ATC C-3?), 0.2 mM
deoxynucleoside triphosphates (dNTPs), 1.5 mM
MgCl2 and 30 ng genomic DNA in a total volume
of 30 ml. PCR conditions were 35 cycles of 30 s at
94 8C, 30 s at 63 8C and 45 s at 72 8C, preceded
by an initial denaturation of 90 s at 94 8C andfollowed by a final extension of 5 min at 72 8C.
Ten microliters of the PCR product were digested
with 10 U MspI, generating fragments of 206 and
41 bp in the case of mutation and remaining
unchanged with 247 bp length in the case of wild
type. Fragments were evaluated on a 2.5% agarose
electrophoresis gel and stained with 0.5 mg/l
ethidium bromide. CYP1A1 mutations m2(A2455G, exon 7) and m4 (C2453A, exon 7),
were examined as described by Cascorbi et al.
(1996).
2.3.2. CYP2E1
CYP2E1 Rsa I site polymorphism (C-1053T, 5?-flanking region) and CYP2E1 Dra I site poly-
B. Laffon et al. / Toxicology 186 (2003) 131�/141 133
morphism (T7632A, intron 6) were analyzedfollowing Tan et al. (2000) and Lin et al. (1998),
respectively.
2.3.3. EH
Two PCR-RFLP assays, following Smith andHarrison (1997), were used to detect the T to C
mutation in exon 3 (Tyr113His) and the A to G
transition in exon 4 (His139Arg). Individuals were
then classified according to the expected EH
activity, on the basis of their exon 3 and exon 4
genotypes (Sarmanova et al., 2000):
�/ Low activity: His/His-His/His; His/His-His/
Arg; Tyr/His-His/His; His/His-Arg/Arg.
�/ Medium activity: Tyr/Tyr-His/His; Tyr/His-His/Arg; Tyr/His-Arg/Arg.
�/ High activity: Tyr/Tyr-Arg/Arg; Tyr/Tyr-His/
Arg.
2.3.4. GSTP1
PCR-RFLP techniques, basically as described
by Saarikoski et al. (1998), were followed to detect
polymorphisms in exons 5 (Ile105Val) and 6
(Ala114Val) of the GSTP1 gene. Amino acid
change Ile105Val is present in GSTP1*B and
GSTP1*C alleles, while amino acid change
Ala114Val is present in GSTP1*C alleles.
2.3.5. GSTM1 and GSTT1
The presence of GSTM1 and GSTT1 genes was
detected by means of a multiplex PCR method,
using the b-globin gene as internal control to verify
the proper functioning of the amplification reac-tion. An initial 5 min at 94 8C denaturation step
was undertaken, followed by 32 cycles of 20 s at
94 8C, 30 s at 55 8C and 30 s at 72 8C and a final
extension of 5 min at 72 8C. The PCR mixture
was composed of 40 ng genomic DNA template,
0.67 mM of each GSTM1 primer (5?-CGC CAT
CTT GTG CTA CAT TGC CCG-3? and 5?-TTC
TGG ATT GTA GCA GAT CA-3?), 0.5 mM ofeach GSTT1 primer (5?-GCC CTG GCT AGT
TGC TGA AG-3? and 5?-GCA TCT GAT TTG
GGG ACC ACA-3?), 0.25 mM of each b-globin
primer (5?-CAA CTT CAT CCA CGT TCA CC-3?and 5?-GAA GAG CCA AGG ACA GGT AC-
3?’), 0.2 mM dNTPs, 3.3 mM MgCl2 and 0.75 U
Taq polymerase in a final volume of 30 ml. Theamplification reaction gave fragments of 230 bp
for GSTM1, 112 bp for GSTT1 and 268 bp for b-
globin that were resolved in a 2.5 agarose electro-
phoresis gel and stained with 0.5 mg/l ethidium
bromide. Genotypes were classified as positive (at
least one undeleted allele) or null (both alleles
deleted).
2.4. Statistical evaluation
Kolmogorov�/Smirnov goodness of fit test was
used to compare the distribution of the data
obtained with the normal distribution. Since
none departed significantly from normality, para-
metric tests were used for the statistical analysis.
The contribution of genotypes and other variables(sex, smoking, age) to the inter-individual varia-
bility of tail length was evaluated by means of
analysis of variance (ANOVA) and then Student’s
t-test, when the overall F -test was significant. The
association between styrene doses and comet tail
length was analyzed by Pearson’s correlation. The
level of significance was set at 0.05. All statistical
evaluations were performed with the SPSS forWindows statistical package, version 11.0 (IL).
3. Results
The possible association between polymorph-
isms in CYP1A1, CYP2E1, EH, GSTP1, GSTM1
and GSTT1 genes and the extent of styrene-
induced DNA damage, evaluated as comet taillength, in human leukocytes was studied in this
work. Among the donors used, only the wild type
homozygotes and heterozygotes were assessed,
given variant homozygote individuals could not
be obtained, except for one EH exon 4 (Arg/Arg).
Frequencies of CYP1A1 m1, m2 and m4 muta-
tions obtained in this work (0.17, 0.07 and 0.08,
respectively) were higher than those described byCascorbi et al. (1996) (0.08, 0.03 and 0.03,
respectively), m2 always being linked to m1;
however, for CYP2E1 c2 and C alleles, the
frequencies (0.020 and 0.100, respectively) in the
present work were similar to those reported by
Sarmanova et al. (2000) (0.023 and 0.077, respec-
B. Laffon et al. / Toxicology 186 (2003) 131�/141134
tively). For EH, rate of mutation in exon 3 (0.18)
was lower than that reported in the study of
Sarmanova et al. (2000) (0.38), yet the same rate
of mutation was observed in exon 4 (0.20).
Frequencies of GSTP1 *A, *B and *C alleles
were 0.63, 0.30 and 0.07, respectively, similar to
those described by Welfare et al. (1999) (0.67, 0.23
and 0.10, respectively). For GSTM1 and GSTT1,
frequencies of null genotypes (0.43 and 0.20,
respectively) were in the range described by
Seidegard and Pero (1985) and Pemble et al.
(1994) (0.4�/0.6 and 0.2�/0.4, respectively). All the
reports cited were carried out in Caucasian popu-
lations.
Table 1 displays comet tail lengths classified for
smoking habits, sex and age of the donors. Both
styrene concentrations induced significant in-
creases in DNA damage compared to unexposed
controls in all donors groups and significant dose�/
response relationship was obtained at 0.01 (Pear-
son’s correlation coefficient r�/0.156). Eleven of
the 30 donors were smokers, seven smoked more
than ten cigarettes per day (heavy smokers) and
four smoked ten or less cigarettes per day (light
smokers). Five smokers had been smoking for 5
years or less (short-time smokers) and six for over
5 years (long-time smokers). DNA damage in-
creased significantly with tobacco consumption in
control and exposed cells in heavy smokers and
with length of time smoked. With regard to sex,
longer comet tail lengths were obtained in leuko-
cytes from females, in controls and at both styrene
concentrations. Age did not affect the results in
any of the treatments.
Table 2 describes some characteristics of the
selected genetic polymorphisms and the predicted
effect on the corresponding enzymatic activity and
Table 3 shows the influence of these polymorph-
isms on comet tail length induced by styrene.
Heterozygote individuals for m1 and m2 poly-
morphisms of the CYP1A1 gene had higher values
of tail length than wild type homozygotes, these
values increasing with increasing styrene concen-
tration. The high values of the mean S.E. for m2
heterozygotes are a consequence of the low
number of individuals included in this group
(only four). In contrast, m4 heterozygotes showed
less DNA damage than wild type homozygotes in
the styrene-treated leukocytes. For CYP2E1 Rsa I
polymorphism, only one heterozygote donor was
obtained, so no statistically significant effect was
detected in the analysis of this polymorphism.
Table 1
Styrene-induced DNA damage in human leukocytes
Donors N TL (mm, mean9/S.E.)
Control 5 mM 10 mM
All donors 30 54.99/0.9 70.89/1.6a 98.19/3.0a
Smoking
Non-smokers 19 49.79/0.3 57.59/0.4a 72.29/0.6a
Light smokers 4 51.19/0.7 61.19/1.0a,b 62.29/1.0a
Heavy smokers 7 71.59/3.7b 112.59/6.5a,b 188.99/12.2a,b
Short-time smokers 5 53.39/0.6b 60.29/0.8a,b 67.39/1.0a
Long-time smokers 6 73.09/4.3b 121.89/7.5a,b 205.89/14.1a,b
Sex
Males 15 51.29/0.3 57.69/0.4a 71.29/0.7a
Females 15 58.79/1.7c 84.09/3.1a,c 125.09/5.9a,c
Age
5/30 years 23 55.49/1.2 72.59/2.1a 103.89/3.9a
�/30 years 7 53.39/0.5 65.29/0.7a 79.49/1.0a
a Significant difference (P 5/0.05) with regard to the corresponding controls.b Significant difference (P 5/0.05) with regard to non-smokers.c Significant difference (P 5/0.05) with regard to males.
B. Laffon et al. / Toxicology 186 (2003) 131�/141 135
With regard to the Dra I polymorphism of the
same gene, longer tails were observed for hetero-
zygote individuals in controls and styrene-treated
cells.
Regarding EH, increases in DNA damage were
detected for the group of expected medium enzyme
activity with respect to low and high activitydonors, the differences being higher with increas-
ing doses. For GSTP1, increases in tail length were
found for both *A/*B and *A/*C heterozygote
variants with regard to the wild type homozygote
*A/*A, *A/*B genotype cells being significant. As
for GSTM1 and GSTT1, significantly lower comet
tail length values were obtained for the null
genotypes in controls and both styrene treatments.
4. Discussion
Many environmental agents need to be activated
metabolically before exerting their genotoxic ef-fects and thus, it seems reasonable to propose that
genetic polymorphisms in metabolizing enzymes
that may affect catalytic activity could lead to
individual differences in the extent of DNA
damage induced. In this study, we investigated
the possible influence of functional genetic poly-
morphisms in a broad spectrum of relevant meta-
bolic genes, including CYP1A1, CYP2E1, EH,
GSTP1, GSTM1 and GSTT1, on DNA damage
induced in styrene-exposed human leukocytes.The results obtained for styrene-mediated DNA
damage are consistent with the findings of Norppa
et al. (1983) and Bernardini et al. (2002), who
reported that styrene induced a significant increase
in SCE in cultured human lymphocytes without
adding a metabolizing enzyme system. Neverthe-
less, styrene doses used by these researchers (0.5,
1.5 and 2 mM) were lower since they tested whole
blood lymphocyte cultures and the erythrocytes
present in the cultures may activate styrene to SO
(Norppa et al., 1983). In the study presented here,
isolated leukocytes were used, these known to be
less able to activate styrene. Norppa and Tursi
(1984) described significant SCE increments in
CHO cells treated with styrene, without an exogen
activating system, at doses similar to those used in
the present study (5, 8 and 10 mM). Moreover,
duration of styrene exposure in SCE test was much
longer than that in the comet assay (usually 48 h
versus 30 min).
Tobacco smoke contains numerous substances,
most being mutagenic and carcinogenic (Hoff-
mann and Hecht, 1990). Many reports describe
the effect of tobacco smoke on comet assay results
(Betti et al., 1994; Palus et al., 1999; Zhu et al.,
1999), which are in accordance with the results
obtained in this study. Moller et al. (2000) suggest
that the existing discrepancies in some comet assay
works on the effect of smoking may be due to their
low statistical power, since DNA damage in
smokers is slight. Regarding sex, usually no effect
of this parameter is detected in genotoxicity tests
(Landi et al., 1999; Zhu et al., 1999), or, as in the
present study, greater damage is associated with
females (Fenech, 1993; Fenech et al., 1999);
increases in the damage are rarely attributed to
males (Betti et al., 1994). Age had no effect on tail
length, as in most comet assay studies (Betti et al.,
1994; Zhu et al., 1999), yet it must be considered
that there was a small number of individuals in the
older-age group (only seven) and that the age of
these older subjects was not advanced (31�/48
years old).
Table 2
Characteristics of the selected xenobiotic metabolizing enzymes
Gene Variant Amino acid change Influence on enzyme
CYP1A1 m1 3?-UTRa Increased inducibility
m2 Ile462Val Increased activity
m4 Thr461Asp Decreased activity
CYP2E1 c2 5?-UTRa Contradictory effect
C Intron 6 Unknown
EH 113H Tyr113His Low activity
139R His139Arg High activity
GSTP1 *B Ile105Val Substrate-dependent
alteration
*C Ile105Val
�/Ala114Val
GSTM1 Nullb No protein Lack of activity
GSTT1 Nullb No protein Lack of activity
a UTR, untranslated region.b Null, homozygote for the deletion.
B. Laffon et al. / Toxicology 186 (2003) 131�/141136
Although CYP1A1 is not one of the main CYP
isozymes involved in styrene metabolism, it is one
of the most abundant CYP isozymes in lungs and
thus polymorphisms affecting this gene may be
important in styrene-induced genotoxicity since
human exposure to this chemical takes place by
inhalation. It has been reported that T�/C variant
in the 3?-noncoding region of the CYP1A1 gene
(m1) is associated with increased inducibility and
that the amino acid change Ile462Val (located in
the conserved heme-binding region) in m2 variant
drives to double enzymatic activity (Smith et al.,
1998). In concordance with these facts, higher
values of comet tail length were observed in
heterozygote individuals for CYP1A1 m1 and m2
polymorphisms than in wild type homozygotes,
since the increase in enzymatic activity produces
higher quantities of the genotoxic styrene metabo-
lite SO. In contrast, the m4 variant has been
associated with decreased enzyme activity (Brock-
stedt et al., 2002) and thus more DNA damage was
found in CYP1A1 m4 heterozygote subjects, due
to their decreased enzymatic production of SO.
CYP2E1 represents a major CYP isoform in the
liver (Tan et al., 2000) and is the main isozyme
involved in styrene oxidation (Guengerich et al.,
1991). The variant c2 allele, recognized by RsaI
digestion in the 5?-flanking region of the gene,
appears to be associated with decreased enzyme
activity or noninducibility (Carriere et al., 1996),
but Watanabe et al. (1994) reported higher mRNA
expression for c2 allele than for c1 allele. In this
study, no statistically significant results were
detected in the analysis of CYP2E1 Rsa I poly-
Table 3
Influence of the selected polymorphisms on comet tail length induced by styrene
Gene Polymorphism Genotype N TL (mm, mean9/S.E.)
Control 5 mM 10 mM
CYP1A1 m1 wt/wt 20 51.09/0.3 60.09/0.4a 72.09/0.6a
wt/m1 10 62.89/2.6b 92.49/4.6a,b 150.29/8.7a,b
m2 wt/wt 26 50.89/0.2 58.99/0.3a 72.59/0.5a
wt/m2 4 81.69/6.3b 148.59/11.0a,b 264.19/20.5a,b
m4 wt/wt 25 55.39/1.1 73.09/1.9a 104.69/3.6a
wt/m4 5 53.09/0.7 59.99/0.8a,b 65.49/1.1a,b
CYP2E1 Rsa I c1/c1 29 55.09/0.9 71.39/1.7a 98.59/3.1a
c1/c2 1 52.59/1.2 56.99/1.4 85.69/2.0a
Dra I D/D 24 49.89/0.2 58.49/0.4a 70.69/0.5a
D/C 6 75.69/4.3b 120.59/7.5a,b 207.99/14.1a,b
EH Exon 3 and exon 4 High activity 6 49.99/0.5c 55.09/0.6a,c 77.69/1.2a,c
Medium activity 16 57.39/1.5 79.19/2.6a 115.39/5.0a
Low activity 5 52.99/0.6 61.79/0.8a,c 67.09/1.0a,c
GSTP1 Exon 5 and exon 6 *A/*A 8 50.29/0.4 60.29/0.7a 70.69/0.9a
*A/*B 18 57.19/1.5b 77.69/2.6a,b 114.39/5.0a,b
*A/*C 4 54.69/0.7 61.49/0.8a 80.29/1.2a
GSTM1 Gene deletion Positive 15 58.59/1.5 81.29/2.8a 125.89/5.2a
Null 12 50.39/0.4d 57.29/0.5a,d 61.99/0.6a,d
GSTT1 Gene deletion Positive 21 56.79/1.1 73.89/2.0a 102.59/3.8a
Null 6 47.89/0.4d 58.79/0.7a,d 80.39/1.1a,d
a Significant difference (P 5/0.05) with regard to the corresponding control cells.b Significant difference (P 5/0.05) with regard to the wild type homozygous genotype.c Significant difference (P 5/0.05) with regard to the medium activity genotype.d Significant difference (P 5/0.05) with regard to the positive genotype.
B. Laffon et al. / Toxicology 186 (2003) 131�/141 137
morphism, since only one c1/c2 heterozygotedonor was obtained. With regard to the CYP2E1
Dra I polymorphism, the presence of the variant C
allele has been associated with lung cancer sus-
ceptibility in several populations (Hirvonen et al.,
1992; Persson et al., 1993), but the results of these
studies are inconclusive and no metabolic rationale
has been proposed to support the observed asso-
ciations (Smith et al., 1998). Since lung cancer isrelated in part to environmental mutagen expo-
sures (such as tobacco smoke) and given the higher
levels of styrene-induced DNA damage observed
in heterozygote D/C individuals, perhaps indivi-
duals with the C allele may be more sensitive to the
damage induced by environmental xenobiotics.
Moreover, in a study with styrene-exposed indivi-
duals, Vodicka et al. (2001) found a significantassociation between heterozygosity in CYP2E1
Dra I polymorphism and HPRT gene mutant
frequencies. In any case, the effect of the
CYP2E1 Dra I polymorphism on the enzymatic
function remains unclear.
Mutations in exon 3 (Tyr/His) of EH gene
confer low enzyme activity and mutations in
exon 4 (His/Arg) confer high activity (Hasset etal., 1994). On the basis of this fact, individuals are
classified by expected EH enzyme activity accord-
ing to their exon 3 and 4 alleles (Sarmanova et al.,
2000). Since EH acts by detoxifying SO, it seems
reasonable that EH genotypes with high activity
have less damage and EH genotypes with low
activity have more damage than those genotypes
with medium activity, respectively. However, in-creased tail length values have been observed for
the EH medium activity group when compared to
the other two groups. Wenker et al. (2000) found
no relationship between enzymatic activity of EH
protein over SO and EH genotype; Hasset et al.
(1997) concluded that the variation in enzyme
activity could not be explained on the basis of the
two mentioned polymorphisms only and Raaka etal. (1998) identified polymorphic loci in the
regulatory part of the gene which are likely to be
a contribution factor to EH activity variation.
Therefore, if the assignation of expected enzyme
activity with respect to exon 3 and 4 polymorph-
isms is not entirely correct, this may be the reason
for the incoherent results obtained.
GSTP1 has particular importance in the detox-ification of inhaled toxicants since it is the most
abundant GST isoform in the lungs (Saarikoski et
al., 1998). Polymorphisms in exon 5 (Ile105Val)
and exon 6 (Ala114Val) of this gene produce
enzymes with different thermal stability and sub-
strate affinity (Sarmanova et al., 2000), both being
affected codons in the electrophile binding site of
the enzyme (Ali-Osman et al., 1997). In this work,increase in DNA damage was observed for the
variant heterozygote genotypes *A/*B and *A/*C
with regard to the wild type homozygote, signifi-
cant for the first one. This could be a consequence
of an alteration in the affinity of the enzyme for
SO, that leads to a decrease in its detoxifying
activity. Our results agree with those of Vodicka et
al. (2001), who reported a significant associationbetween mutant frequencies at the HPRT gene and
heterozygosity in the GSTP1 gene (exon 5, poly-
morphism present in both *B and *C alleles) in the
previously mentioned group of styrene-exposed
workers.
Both GSTM1 and GSTT1 genes are affected by
a deletion polymorphism, resulting in the total
lack of enzyme activity in the homozygous geno-type, with frequencies of 40�/60% and 20�/40%,
respectively, in Caucasians (Seidegard and Pero,
1985; Pemble et al., 1994). A previous study, using
whole blood lymphocyte cultures from 24 donors,
reported higher SCE induction by 1.5 mM styrene
in those individuals lacking both GSTM1 and
GSTT1 genes than in subjects having both genes,
with intermediate SCE induction in donors nullfor only one of the genes (Bernardini et al., 2002).
Nevertheless, the results presented here show
greater damage induction in those individuals
having positive genotypes, in opposition with the
theory that, whether GSTM1 and GSTT1 enzymes
act by detoxifying SO, null genotypes might be
associated with greater damage. It must be taken
into account that GST-mediated conjugation con-stitutes only a minor pathway in styrene metabo-
lism, estimated to contribute in B/1% of SO
detoxification (Ghittori et al., 1996) and that in
the mentioned work of Bernardini et al. (2002)
polymorphisms in CYP isozymes, that may influ-
ence damage induction by styrene as our results
show, are not considered. In addition, one of the
B. Laffon et al. / Toxicology 186 (2003) 131�/141138
derivatives of SO-glutathione conjugation hasbeen suggested to have certain genotoxic potential
(Zhang et al., 1993) and thus it may accumulate in
the culture media in the presence of the conjuga-
tion enzymes and lead to more DNA damage than
that in cells lacking these enzymes.
In summary, the present in vitro findings
suggest that CYP1A1 m1, m2 and m4, CYP2E1
Dra I and GSTP1 (exons 5 and 6) polymorphismsmay affect styrene induction of DNA damage in
human leukocytes. Contradictory results were
obtained, in comparison to previously published
reports, regarding the influence of GSTM1 and
GSTT1 polymorphisms on styrene individual
sensitivity. Nevertheless, these results need to be
confirmed in variant homozygote individuals, not
analyzed in this study due to the relatively smallnumber of individuals included, by means of a
larger population study. In addition, increasing
the population size may allow to carry out a
stratified analysis, taking into account the distri-
bution of smokers and non-smokers among geno-
types, since the importance of smoking habits has
been demonstrated and an uneven distribution of
smokers among genotypes may influence theresults obtained.
Acknowledgements
A grant from the Xunta de Galicia (XUGA
10605B98) funded this investigation.
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