Toxicology Letters 151 (2004) 273–282

Association of cytochrome P450 2E1 polymorphismsand head and neck squamous cell cancer

Thomas Neuhausa, Yon-Dschun Koa,∗, Katrin Lorenzena, Stefan Fronhoffsa,Volker Harthb, Peter Brödec, Hans Vettera, Hermann M. Boltc,

Beate Pescha,b, Thomas Brüningb,c

a Medizinische Universitäts–Poliklinik Bonn, Universität Bonn, Wilhelmstr. 35-37, D−53111 Bonn, Germanyb Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin (BGFA), Ruhr-Universität Bochum,

Bürkle-de-la-Camp-Platz 1, D-44789 Bochum, Germanyc Institut für Arbeitsphysiologie an der Universität Dortmund (IfADo), Ardeystr. 67, D−44139 Dortmund, Germany

Received 20 August 2003; received in revised form 2 September 2003; accepted 2 September 2003

Dedicated to the late Christian Hodel

Available online 20 April 2004

Abstract

The development of head and neck squamous cell cancer (HNSCC) is known to be strongly associated with tobacco use.One of the main enzymes for bioactivation of tobacco-related substances is the cytochrome 450 (CYP)2E1, of which differentgenetic variants are described. Analyzing a correlation between certain neoplasia and alteration of the CYP2E1 gene, moststudies focus on the polymorphisms−1053C>T and 7632T>A, but recently another polymorphism, named−71G>T, withenhanced transcriptional activity, has been identified. In the current case-control study we investigate the putative association ofthe mentioned CYP2E1 polymorphisms on the risk of HNSCC. Comparing 312 German individuals with HNSCC to 299 controlswe found a significantly enhanced risk for the development of that neoplasia in smoking carriers of−71G>T heterozygosity, whilein −1053C>T and 7632T>A polymorphisms a corresponding correlation was absent. Since a coincidence of an aberratedp53gene and CYP2E1 mutations has been described, we choose a subgroup of 140 patients with HNSCC for analyzing an associationof mutations in these two genes. However, no such association could be found in either of the mentioned polymorphisms. Furtherstudies have to focus on the−71G>T polymorphism and its possible linkage to cancers, in which smoking is a known risk-factor,as well as its functional relevance concerning the bioactivation of tobacco-related substances.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: CYP2E1; Polymorphism; Head and neck cancer

∗ Corresponding author. Tel.:+49-228-287-2263;fax: +49-228-287-2266.

E-mail address: y.ko@jk-bonn.de (Y.-D. Ko).

1. Introduction

CYP2E1 is an ethanol-inducible cytochrome P-450isoenzyme that is involved in the metabolic activa-tion of many low-weight-substances, e.g. acetone,

0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.toxlet.2003.09.017

274 T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282

paracetamol or gaseous anesthetics (Guengerich et al.,1991; Kharasch and Thummel, 1993; Ingelman-Sundberg et al., 1994; Lechevrel and Wild, 1997;Novak and Woodcroft, 2000; Tanaka et al., 2000; Boltet al., 2003). Due to its ability to bioactivate com-pounds which are potentially carcinogenic, like vinylchloride (Whysner et al., 1996) or tobacco-associatednitrosamines (Yang et al., 1990), CYP2E1 is thoughtto be linked to the development of human cancers(Bolt et al., 2003).

Several studies dealt with associations betweenpolymorphisms of the CYP2E1 gene (for currentnomenclature of CYP2E1 alleles seeBolt et al., 2003)and incidences of different neoplasias. Most papershave focused on the−1053C>T (RsaI) polymorphismlocated in the 5′-flanking region of the CYP2E1 gene(Hayashi et al., 1991). ThoughHayashi et al. (1991)described a 10-fold enhancement of the CYP2E1transcription rate in homozygous carriers of this se-quence variant, others failed to demonstrate such anincrease (Lucas et al., 1995; Powell et al., 1998)and Marchand et al. (1999)even found a decreasedactivity in carriers of the variant allele, using theclearance of chlorzoxazone as a phenotypic probefor CYP2E1 activity. The frequency of this particularpolymorphism shows significant interethnic variation(Stephens et al., 1994). The −1053C>T variant isfound in 2–8% of Caucasians, while about 25–36%of East Asians carry this allele (Kato et al., 1992,1995; London et al., 1996; Agundez et al., 1997).

Another important polymorphism, 7632T>A, de-tectable with Dra I restriction enzymes, is locatedin intron 6. The distribution of the variant genotypewithout the Dra I site, again, depends on the ethnic-ity. While the reference genotype could be found inover 80–90% of Caucasians, only 50–60% of the EastAsians carry this genotype. The frequency of the het-erozygous genotype was 7–20%, for the homozygousvariant genotype 1–2% in Caucasians (Hirvonen et al.,1993; Persson et al., 1993; Carriere et al., 1996), inEast Asians the corresponding numbers were 30–43%and 3–14% (Stephens et al., 1994; Uematsu et al.,1994).

As mentioned initially, different studies havedescribed the influence of polymorphisms of theCYP2E1 gene on the development of various types ofcancer. However, the results are contradictory. Sev-eral studies have failed to show a linkage of either

RsaI- and/or Dra I-polymorphism with lung can-cer (Hirvonen et al., 1992; Kato et al., 1992, 1994;Sugimura et al., 1995; Watanabe et al., 1995;London et al., 1996), hepatocellular carcinoma (Katoet al., 1995; Wong et al., 2000), urothelial cancer(Anwar et al., 1996; Farker et al., 1998), nasopha-ryngeal or laryngeal carcinoma (Jahnke et al., 1996;Lucas et al., 1996; Matthias et al., 1998), squamouscell carcinoma of the esophagus (Lucas et al., 1996;Hori et al., 1997; Morita et al., 1997), gastric can-cers (Kato et al., 1995, 1996, 1997), prostate cancer(Murata et al., 2001) or cervix carcinoma (Kim et al.,2000). In contrast, other studies described significantassociations between CYP2E1 polymorphism and theincidences of different carcinomas, at least if factorslike smoking or drinking alcohol were additionallytaken into account, e.g. in lung cancer (Uematsu et al.,1994; Oyama et al., 1997), nasopharyngeal carcinoma(Hildesheim et al., 1997), hepatocellular carcinoma(Ladero et al., 1996), breast cancer (Shields et al.,1996), oral cancer (Hung et al., 1997) and colorectalcancer (Kiss et al., 2000). Interestingly, some authorseven described a protective effect of variant alleles ofthe CYP2E1 gene for risks of developing carcinoma(Persson et al., 1993; Yu et al., 1995; Wu et al., 1997;Marchand et al., 1998; Tan et al., 2000).

Recently, novel polymorphisms of the CYP2E1gene had been detected, of which the−71G>T poly-morphism in exon 1 was correlated with enhancedtranscriptional activity in HepG2 cells (Fairbrotheret al., 1998). Data concerning the allele frequency inCaucasians are very rare and comprise a population ofless than 200 persons in total, where heterozygosityfor the −71G>T polymorphism was found in about10% (Fairbrother et al., 1998; Thier et al., 2002).So far, a study analyzing the association betweenthis particular polymorphism and the incidence ofneoplasia is missing.

As tobacco smoking and high-percentage alcoholdrinking are considered main risk factors for headand neck squamous cell cancer (HNSCC) (Wynderand Stellman, 1977; Tuyns, 1988; Boyle et al., 1990)and, as mentioned initially, both factors are reason-ably linked with CYP2E1, an association betweenmutations of the CYP2E1 gene and the incidenceof HNSCC is not unlikely. In this study, we investi-gated the link between three polymorphisms of theCYP2E1 gene,−1053C>T (RsaI), 7632T>A (Dra

T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282 275

I) and −71G>T, on the development of HNSCC insmokers and non-smokers.

Mutations of drug-metabolizing enzymes may belinked with changes in chemically induced somatic al-terations of genes, which play a pivotal role in cellgrowth and differentiation, likep53 does. Since mu-tations in thep53 gene are often found in HNSCC(Weber et al., 2002) and an association between a mu-tant allele of CYP1B1 and a mutation ofp53 gene inHNSCC has been described (Ko et al., 2001), we alsoanalyzed the combined occurrence of mutations of thegenes of CYP2E1 and ofp53 within the tumor tissue.

2. Material and methods

2.1. Study group

Details of the study population have been reportedpreviously (Ko et al., 2001). In brief, 312 Germansubjects (mean age: 60 years, 251 males) with histo-logically confirmed HNSCC were enrolled. The con-trol group consisted of 299 unrelated healthy controls(mean age: 47 years, 176 males), all without a historyof cancer. A structured questionnaire was applied torecord smoking habits and other risk factors. Informedconsent of the study subjects was obtained, and theEthics Committee of the University of Bonn had givenits approval. Smokers were defined as individuals hav-ing ever smoked five cigarettes or more (or cigars orpipes) per day, for at least 4 years. Non-smokers wereindividuals who had never smoked or had smoked lessthan one pack per year. Data on 7632T>A polymor-phism were available for a subgroup, consisting of 272people with HNSCC (mean age: 60 year, 223 males)and 292 controls (mean age: 46 year, 174 males).

Table 1PCR primers and hybridization probes for CYP2E1 polymorphisms

PCR Primers Hybridization Probes

−71G>T 5′-TTGTCTAACCAGTGCCAAAG-3′ 5′-LCR-CTCCTTCTCAGAACACATTATAAAAA-3′5′-GGAGGACAATCCTGTGGAAA-3′ 5′-GCAAGAGGGCATTGGTTGGTGGGTCA-FL-3′

−1053C>T 5′-AGATGGCATAACTCAAAATCC-3′ 5′-LCR-CAACCTATGAATTAAGAACTTCTATATATTGCCAG-3′5′-CAGACCCTCTTCCACCTTCTAT-3′ 5′- TTCATTGTTAATATAAAAGTACAAAATT-FL-3 ′

The PCR primers and hybridization probes were newly constructed. The fluorescein-labeled oligo-probe hybridizes around the G→ T sitefor the −71G>T and around the C→ T site for the –1053C>T polymorphism. LCR, Light-CyclerTM—Red; FL, Fluorescein.

2.2. Genotyping

Peripheral blood samples were collected in EDTAtubes, and genomic DNA was isolated from wholeblood using the QIAmp DNA Blood Maxi Kit (Qia-gen, Hilden, Germany). For genotyping the CYP2E1polymorphisms−1053C>T and−71G>T real-timePCR analysis was used (Light-CyclerTM, Roche,Mannheim, Germany), hybridization probes were ap-plied in combination with a Light-CyclerTM DNAMaster Hybridization Probes Kit (Roche, Mannheim,Germany). Both, the PCR primers and the fluorescent-labeled detection probes were synthesized by TIBMOLBIOL, Berlin, Germany. The structures of PCRprimers and fluorescent-labeled detection probes forthe variant CYP2E1 alleles are given inTable 1.For PCR, conditions were 4 mM MgCl2, 1 pmol ofeach hybridization probe, 20 pmol of the two PCRprimers each, 2�l of Light-CyclerTM DNA MasterHybridization Mix (Roche, Mannheim, Germany)and 100 pg to 10 ng DNA in a final volume of 20�l.After 2 min of denaturation (at 95◦C), 45 PCR cycleswere performed with 5 s denaturation at 95◦C, 20 sannealing at 55◦C and 25 s extension at 72◦C. Dif-ferentiation of the CYP2E1 alleles was performed bydetermination of melting curves after PCR. Meltingcurves were obtained following a denaturation periodof 5 s at 95◦C at a start temperature of 45◦C and afinal temperature of 80◦C, with a temperature gra-dient of 0.4◦C/s. PCR and melting procedure weredetected online with the Light-CyclerTM instrument.As an internal control, in 10 randomly chosen assayssequencing on an automated DNA sequencer wasperformed (by Qiagen), which was compared to theresults by the Light-CyclerTM technique. For eachsample, melting curves were produced, showing a

276T.

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/Toxicology

Letters

151(2004)

273–282

Fig. 1. Online differentiation of CYP2E1 polymorphisms−1053C>T and−71G>T by melting curves analysis. (a) shows, separated between−1053C>T and−71G>T,representative online melting curves, generated from temperature-dependent decrease in fluorescence intensity. In (b) differential analysis of these melting curves are shown,offering the opportunity to clearly discriminate between reference genotype, variant genotype and heterozygous individuals.

T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282 277

temperature-dependent decrease in fluorescence in-tensity (Fig. 1a). The melting curve analysis showsdifferent melting maxima (−dF/dT) for the hybridiza-tion probes, depending on the genotype. Concerning−71G>T, there was a single melting maximum of64.2◦C in the case of the wild-type and two meltingmaxima of 60.6 and 64.2◦C in heterozygous DNA.Homozygous variant genotypes were missed in thestudy group. For−1053C>T, a single melting maxi-mum of 56.2◦C was found in the case of the variantgenotype and of 60.2◦C in the case of referencegenotype. In heterozygotes, the two melting maximawere located at 56.2 and 60.2◦C, as shown inFig. 1b.Thus, the differentiation of genotypes was possible bymelting curve analysis. For internal control, 10 ran-domly selected samples were sequenced and showed100% concordance with the Light-CyclerTM data.

2.3. SNaPshotTM

Since Light-CyclerTM hybridization probes did notfit 7632T>A polymorphism, this allele was analyzedby SNaPshotTM technique (Norton et al., 2002).However, 28 samples did not yield a conclusive re-sult and thus these samples were not used for thestatistical analysis. For primer extension 4�l of thePCR-fragments were prepared with 1 U Exonuclease(Roche Diagnostics) and 1 U shrimp alkaline phos-phatase (New England Biolabs) for 60 min at 37◦C ona thermocycler followed by 15 min at 72◦C. Primerextension was performed by combining 1�l primer(5′-cac cca gct gat taa aaa tt-3′), 5�l SNaPshot readyreaction premix (Applied Biosystems), 0.15 pmol/�lprimer and 3�l water. This reaction mix was incu-bated at 96◦C for 10 min and then was subject to 25cycles of 96◦C for 10 s, 50◦C for 5 s and 60◦C for30 s. To prevent unincorporated fluorescent dNTPswhich would obscure the primer extension productsduring electrophoresis, the reactions were treatedwith 1 U shrimp alkaline phosphatase for 60 min at37◦C, followed by 15 min at 72◦C. Aliquots of 1�lSNaPshot product and 10�l Hi-Di foramide wereloaded onto a 310 DNA sequencer (Applied Biosys-tems). Electrophoresis was done on a capillary arrayat 95◦C by using POP4 polymer and GeneScan RunModule E5 (Applied Biosystems). Electrophoresisdata were processed by GeneScan Analysis, version3.7 (Applied Biosystems).

2.4. p53 sequence analysis

A subset of 140 HNSCC cases was available foranalysis ofp53 somatic mutations and comparisonwith CYP2E1 genotypes. Details of tissue samplepreparation andp53 sequence analysis have beenreported previously (Ko et al., 2001).

2.5. Statistical analysis

Unconditional logistic regression models wereapplied to estimate odds ratios (OR) and 95% confi-dence intervals (CI) for the genetic risk factor in thetotal study population and the subgroups of smokers,adjusted for gender and age. Homogeneity betweendistributions was analyzed with the Wald’sχ2-test.Computations were carried out using the statisticalsoftware SAS, version 8.2.

3. Results and discussion

The genotype distributions of the CYP2E1 polymor-phisms examined in controls and in cases with HN-SCC are shown inTable 2. In controls, heterozygosityfor the 7632T>A polymorphism was found in 16.5%,for the−1053C>T polymorphism in 4.4% and for the−71G>T mutation in 7.4%, which correspond to re-ported data in Caucasians (Garte et al., 2001, Thieret al., 2002). No variant genotype was found concern-ing −71G>T allele. In less than 1% of controls, vari-ant genotypes were found for both−1053C>T and7632T>A polymorphisms, somewhat below the ex-pected frequencies.

In HNSCC patients the heterozygous−1053C>Tand 7632T>A genotypes were found in 14.1% and2.6%, respectively. The genotype distribution of bothpolymorphisms showed no significant differences be-tween cases and controls, neither for smokers nor fornon-smokers. In contrast, the−71G>T polymorphismrevealed a significantly higher frequency of the het-erozygous genotype in the HNSCC group (P = 0.04).This significance was just caused by the smoking in-dividuals (P = 0.01), whilst in non-smokers the es-timated risk for HNSCC was not elevated (P = 0.8,data not shown).

As mentioned above, the functional relevance of the−71G>T polymorphism is still unknown.Fairbrother

278 T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282

Tabl

e2

Dis

trib

utio

nof

CY

P2E

1ge

noty

pes

amon

gco

ntro

lin

divi

dual

san

dH

NS

CC

-cas

esan

dO

Rs

(95%

CI)

for

HN

SC

Cin

all

subj

ects

and

insm

oker

s

All

subj

ects

Sm

oker

s

HN

SC

C-c

ases

N(%

)C

ontr

olsn

(%)

OR

(95%

CI)a

P-v

alue

HN

SC

C-c

asesN

(%)

Con

trol

sn(%

)O

R(9

5%C

I)aP

-val

ue

CY

P2E

176

32T

>A

Ref

eren

cege

noty

pe22

5(8

5.9)

196

(83.

1)13

6(8

4.0)

120

(87.

6)H

eter

ozyg

ous

37(1

4.1)

39(1

6.5)

1.02

(0.5

6–1.

84)

0.95

26(1

6.0)

17(1

2.4)

0.69

(0.3

2–1.

49)

0.34

Varia

ntge

noty

pe0

(0.0

)1

(0.4

)0

(0.0

)0

(0.0

)

CY

P2E

1−1

053C

>T

Ref

eren

cege

noty

pe30

4(9

7.4)

282

(94.

9)19

1(9

7.9)

166

(94.

3)2.

55(0

.68–

9.60

)0.

17H

eter

ozyg

ous

8(2

.6)

13(4

.4)

1.58

(0.5

6–4.

49)

0.39

4(2

.1)

9(5

.1)

Varia

ntge

noty

pe0

(0.0

)2

(0.7

)0

(0.0

)1

(0.6

)

CY

P2E

1−7

1G>

TR

efer

ence

geno

type

276

(88.

5)27

7(9

2.6)

167

(85.

6)16

6(9

4.3)

Het

eroz

ygou

s36

(11.

5)22

(7.4

)0.

49(0

.25–

0.98

)0.

0428

(14.

4)10

(5.7

)0.

33(0

.14–

0.79

)0.

01Va

riant

geno

type

0(0

.0)

0(0

.0)

0(0

.0)

0(0

.0)

aC

ompa

rison

refe

renc

ege

noty

pevs

.he

tero

zygo

usor

varia

ntge

noty

pead

just

edfo

rag

ean

dge

nder

.

et al. (1998) described that constructs containingthe polymorphisms−71G>T and−333T>A showedincreased transcriptional activity compared to thevariant genotype or−333T>A alone, but they failedto detect an influence of−71G>T mutation on the ki-netics of chlorzoxazone elimination. AlsoThier et al.(2002)found no association between−71G>T alleleand the levels ofN-(cyanoethyl)valine in industrialworkers handling acrylonitrile. Our data suggest thatthe −71G>T polymorphism could be involved in thebioactivation of a carcinogenic, tobacco-related sub-stance, e.g. diethylnitrosamine (Thier et al., 2001).Thus, further studies should focus on a putative as-sociation of this CYP2E1 polymorphism and othermalignancies in which smoking is an important riskfactor, like squamous cell cancer of the lung.

As previously described, combined polymorphismsof different genes may act synergistically on the can-cer risk (Ko et al., 2001). For this we performed ananalysis of the effect of a combination of the CYP2E1polymorphisms on the development of HNSCC. How-ever, an increased risk for HNSCC could not be foundin either combination (data not shown).

To examine associations between mutation ofp53gene and CYP2E1 genotypes, tumor DNA from 140HNSCC patients was analyzed forp53 aberrations. Aspreviously described (Ko et al., 2001), tumor-specificaberrations were found in 47.1%, and the frequencyof somatic mutations ofp53 gene in smokers was 1.9times higher than in non-smokers.

As indicated inTable 3, no association appearedbetween−1053C>T or−71G>T polymorphisms andaberration of thep53 gene, independent of the smok-ing habits. Conversely, thep53 mutation was linked tothe reference genotype of 7632T>A polymorphism.Although this association is significant (P = 0.02)in the total study group, it is mainly caused by thesmokers, but, because of the small number of cases,the association in this subgroup is not statisticallysignificant (P = 0.09). As on the one hand, thereare actually two studies describing an increased riskof the development of lung cancer in carriers of7632T>A reference genotype especially in smok-ers (Marchand et al., 1998; Wu et al., 1998), whichwould be in line with the data shown here, and onthe other hand there are lots of others, as mentionedabove, that missed such an association, the rele-vance of this association betweenp53 mutation and

T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282 279

Tabl

e3

Ass

ocia

tion

betw

een

p53-

mut

atio

nsan

dC

YP

2E1

geno

type

sin

indi

vidu

als

with

HN

SC

C(a

llsu

bjec

tsan

dsm

oker

s)

HN

SC

C-c

ases

HN

SC

C,

smok

ers

Nor

mal

p53

n(%

)M

utan

tp5

3n(%

)O

R(9

5%C

I)aP

-val

ueN

orm

alp5

3n(%

)M

utan

tp5

3n(%

)O

R(9

5%C

I)aP

-val

ue

CY

P2E

176

32T

>A

Ref

eren

cege

noty

pe48

(78.

7)48

(92.

3)4.

69(1

.33–

16.5

8)0.

0217

(70.

8)31

(88.

6)3.

30(0

.82–

13.2

5)0.

09H

eter

ozyg

ous

13(2

1.3)

4(7

.7)

7(2

9.2)

4(1

1.4)

CY

P2E

1−1

053C

>T

Ref

eren

cege

noty

pe71

(95.

9)64

(97.

0)1.

15(0

.17–

7.81

)0.

8929

(96.

7)45

(97.

8)1.

44(0

.09–

24.0

3)0.

80H

eter

ozyg

ous

3(4

.1)

2(3

.0)

1(3

.3)

1(2

.2)

CY

P2E

1−7

1G>

TR

efer

ence

geno

type

64(8

6.5)

58(8

7.9)

1.64

(0.5

7–4.

71)

0.36

23(7

6.7)

39(8

4.8)

1.68

(0.5

2–5.

50)

0.39

Het

eroz

ygou

s10

(13.

5)8

(12.

1)7

(23.

3)7

(15.

2)

aA

djus

ted

for

age

and

gend

er.

reference genotype of 7632T>A polymorphism re-mains open.

There are very few studies that were engaged inanalyzing a correlation between polymorphism ofCYP2E1 and mutation of thep53 gene. While twogroups missed such an association (Przygodzki et al.,1998; Kim et al., 2000), two others described a corre-lation between the homozygous variant genotype for−1053C>T andp53 aberrations in workers with vinylchloride monomer exposure (Wong et al., 2002) andin patients with squamous cell carcinoma of the lung(Oyama et al., 1997). Here no patient with homozy-gous variant genotype for−1053C>T was identifiedand for this we were not able to support the findingsof one of the works presented.

In essence, we show that smoking carriers of het-erozygosity of the CYP2E1−71G>T allele displaya significantly elevated risk for the development ofHNSCC, which should induce studies focusing on thefunctional and epidemiological relevance of this poly-morphism.

Acknowledgements

The excellent technical assistance of ElisabethGrünewald and Silke Schöneborn is greatly appreci-ated.

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