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: fina@udc.es (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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