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

Toxicology Letters 151 (2004) 273–282

Association of cytochrome P450 2E1 polymorphisms and head and neck squamous cell cancer Thomas Neuhaus a , Yon-Dschun Ko a,∗ , Katrin Lorenzen a , Stefan Fronhoffs a , Volker Harth b , Peter Bröde c , Hans Vetter a , Hermann M. Bolt c , Beate Pesch a,b , Thomas Brüning b,c a

c

Medizinische Universitäts–Poliklinik Bonn, Universität Bonn, Wilhelmstr. 35-37, D−53111 Bonn, Germany b Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin (BGFA), Ruhr-Universität Bochum, Bürkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany 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 different genetic variants are described. Analyzing a correlation between certain neoplasia and alteration of the CYP2E1 gene, most studies focus on the polymorphisms −1053C>T and 7632T>A, but recently another polymorphism, named −71G>T, with enhanced transcriptional activity, has been identified. In the current case-control study we investigate the putative association of the mentioned CYP2E1 polymorphisms on the risk of HNSCC. Comparing 312 German individuals with HNSCC to 299 controls we found a significantly enhanced risk for the development of that neoplasia in smoking carriers of −71G>T heterozygosity, while in −1053C>T and 7632T>A polymorphisms a corresponding correlation was absent. Since a coincidence of an aberrated p53 gene and CYP2E1 mutations has been described, we choose a subgroup of 140 patients with HNSCC for analyzing an association of mutations in these two genes. However, no such association could be found in either of the mentioned polymorphisms. Further studies 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

1. Introduction ∗

Corresponding author. Tel.: +49-228-287-2263; fax: +49-228-287-2266. E-mail address: [email protected] (Y.-D. Ko).

CYP2E1 is an ethanol-inducible cytochrome P-450 isoenzyme that is involved in the metabolic activation 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

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paracetamol or gaseous anesthetics (Guengerich et al., 1991; Kharasch and Thummel, 1993; IngelmanSundberg et al., 1994; Lechevrel and Wild, 1997; Novak and Woodcroft, 2000; Tanaka et al., 2000; Bolt et al., 2003). Due to its ability to bioactivate compounds which are potentially carcinogenic, like vinyl chloride (Whysner et al., 1996) or tobacco-associated nitrosamines (Yang et al., 1990), CYP2E1 is thought to be linked to the development of human cancers (Bolt et al., 2003). Several studies dealt with associations between polymorphisms of the CYP2E1 gene (for current nomenclature of CYP2E1 alleles see Bolt et al., 2003) and incidences of different neoplasias. Most papers have focused on the −1053C>T (RsaI) polymorphism located in the 5 -flanking region of the CYP2E1 gene (Hayashi et al., 1991). Though Hayashi et al. (1991) described a 10-fold enhancement of the CYP2E1 transcription rate in homozygous carriers of this sequence variant, others failed to demonstrate such an increase (Lucas et al., 1995; Powell et al., 1998) and Marchand et al. (1999) even found a decreased activity in carriers of the variant allele, using the clearance of chlorzoxazone as a phenotypic probe for CYP2E1 activity. The frequency of this particular polymorphism shows significant interethnic variation (Stephens et al., 1994). The −1053C>T variant is found 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, detectable with Dra I restriction enzymes, is located in intron 6. The distribution of the variant genotype without the Dra I site, again, depends on the ethnicity. While the reference genotype could be found in over 80–90% of Caucasians, only 50–60% of the East Asians carry this genotype. The frequency of the heterozygous genotype was 7–20%, for the homozygous variant genotype 1–2% in Caucasians (Hirvonen et al., 1993; Persson et al., 1993; Carriere et al., 1996), in East Asians the corresponding numbers were 30–43% and 3–14% (Stephens et al., 1994; Uematsu et al., 1994). As mentioned initially, different studies have described the influence of polymorphisms of the CYP2E1 gene on the development of various types of cancer. However, the results are contradictory. Several studies have failed to show a linkage of either

RsaI- and/or Dra I-polymorphism with lung cancer (Hirvonen et al., 1992; Kato et al., 1992, 1994; Sugimura et al., 1995; Watanabe et al., 1995; London et al., 1996), hepatocellular carcinoma (Kato et al., 1995; Wong et al., 2000), urothelial cancer (Anwar et al., 1996; Farker et al., 1998), nasopharyngeal or laryngeal carcinoma (Jahnke et al., 1996; Lucas et al., 1996; Matthias et al., 1998), squamous cell carcinoma of the esophagus (Lucas et al., 1996; Hori et al., 1997; Morita et al., 1997), gastric cancers (Kato et al., 1995, 1996, 1997), prostate cancer (Murata et al., 2001) or cervix carcinoma (Kim et al., 2000). In contrast, other studies described significant associations between CYP2E1 polymorphism and the incidences of different carcinomas, at least if factors like smoking or drinking alcohol were additionally taken 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 colorectal cancer (Kiss et al., 2000). Interestingly, some authors even described a protective effect of variant alleles of the 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 CYP2E1 gene had been detected, of which the −71G>T polymorphism in exon 1 was correlated with enhanced transcriptional activity in HepG2 cells (Fairbrother et al., 1998). Data concerning the allele frequency in Caucasians are very rare and comprise a population of less than 200 persons in total, where heterozygosity for the −71G>T polymorphism was found in about 10% (Fairbrother et al., 1998; Thier et al., 2002). So far, a study analyzing the association between this particular polymorphism and the incidence of neoplasia is missing. As tobacco smoking and high-percentage alcohol drinking are considered main risk factors for head and neck squamous cell cancer (HNSCC) (Wynder and Stellman, 1977; Tuyns, 1988; Boyle et al., 1990) and, as mentioned initially, both factors are reasonably linked with CYP2E1, an association between mutations of the CYP2E1 gene and the incidence of HNSCC is not unlikely. In this study, we investigated the link between three polymorphisms of the CYP2E1 gene, −1053C>T (RsaI), 7632T>A (Dra

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

I) and −71G>T, on the development of HNSCC in smokers and non-smokers. Mutations of drug-metabolizing enzymes may be linked with changes in chemically induced somatic alterations of genes, which play a pivotal role in cell growth and differentiation, like p53 does. Since mutations in the p53 gene are often found in HNSCC (Weber et al., 2002) and an association between a mutant allele of CYP1B1 and a mutation of p53 gene in HNSCC has been described (Ko et al., 2001), we also analyzed the combined occurrence of mutations of the genes of CYP2E1 and of p53 within the tumor tissue.

2. Material and methods 2.1. Study group Details of the study population have been reported previously (Ko et al., 2001). In brief, 312 German subjects (mean age: 60 years, 251 males) with histologically confirmed HNSCC were enrolled. The control group consisted of 299 unrelated healthy controls (mean age: 47 years, 176 males), all without a history of cancer. A structured questionnaire was applied to record smoking habits and other risk factors. Informed consent of the study subjects was obtained, and the Ethics Committee of the University of Bonn had given its approval. Smokers were defined as individuals having ever smoked five cigarettes or more (or cigars or pipes) per day, for at least 4 years. Non-smokers were individuals who had never smoked or had smoked less than one pack per year. Data on 7632T>A polymorphism were available for a subgroup, consisting of 272 people with HNSCC (mean age: 60 year, 223 males) and 292 controls (mean age: 46 year, 174 males).

275

2.2. Genotyping Peripheral blood samples were collected in EDTA tubes, and genomic DNA was isolated from whole blood using the QIAmp DNA Blood Maxi Kit (Qiagen, Hilden, Germany). For genotyping the CYP2E1 polymorphisms −1053C>T and −71G>T real-time PCR analysis was used (Light-CyclerTM , Roche, Mannheim, Germany), hybridization probes were applied in combination with a Light-CyclerTM DNA Master Hybridization Probes Kit (Roche, Mannheim, Germany). Both, the PCR primers and the fluorescentlabeled detection probes were synthesized by TIB MOLBIOL, Berlin, Germany. The structures of PCR primers and fluorescent-labeled detection probes for the variant CYP2E1 alleles are given in Table 1. For PCR, conditions were 4 mM MgCl2 , 1 pmol of each hybridization probe, 20 pmol of the two PCR primers each, 2 ␮l of Light-CyclerTM DNA Master Hybridization 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 cycles were performed with 5 s denaturation at 95 ◦ C, 20 s annealing at 55 ◦ C and 25 s extension at 72 ◦ C. Differentiation of the CYP2E1 alleles was performed by determination of melting curves after PCR. Melting curves were obtained following a denaturation period of 5 s at 95 ◦ C at a start temperature of 45 ◦ C and a final temperature of 80 ◦ C, with a temperature gradient of 0.4 ◦ C/s. PCR and melting procedure were detected online with the Light-CyclerTM instrument. As an internal control, in 10 randomly chosen assays sequencing on an automated DNA sequencer was performed (by Qiagen), which was compared to the results by the Light-CyclerTM technique. For each sample, melting curves were produced, showing a

Table 1 PCR primers and hybridization probes for CYP2E1 polymorphisms PCR Primers

Hybridization Probes

−71G>T

5 -TTGTCTAACCAGTGCCAAAG-3

5 -GGAGGACAATCCTGTGGAAA-3

5 -LCR-CTCCTTCTCAGAACACATTATAAAAA-3 5 -GCAAGAGGGCATTGGTTGGTGGGTCA-FL-3

−1053C>T

5 -AGATGGCATAACTCAAAATCC-3 5 -CAGACCCTCTTCCACCTTCTAT-3

5 -LCR-CAACCTATGAATTAAGAACTTCTATATATTGCCAG-3 5 - TTCATTGTTAATATAAAAGTACAAAATT-FL-3

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

276 T. Neuhaus et al. / 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

temperature-dependent decrease in fluorescence intensity (Fig. 1a). The melting curve analysis shows different melting maxima (−dF/dT) for the hybridization probes, depending on the genotype. Concerning −71G>T, there was a single melting maximum of 64.2 ◦ C in the case of the wild-type and two melting maxima of 60.6 and 64.2 ◦ C in heterozygous DNA. Homozygous variant genotypes were missed in the study group. For −1053C>T, a single melting maximum of 56.2 ◦ C was found in the case of the variant genotype and of 60.2 ◦ C in the case of reference genotype. In heterozygotes, the two melting maxima were located at 56.2 and 60.2 ◦ C, as shown in Fig. 1b. Thus, the differentiation of genotypes was possible by melting curve analysis. For internal control, 10 randomly selected samples were sequenced and showed 100% concordance with the Light-CyclerTM data.

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2.4. p53 sequence analysis A subset of 140 HNSCC cases was available for analysis of p53 somatic mutations and comparison with CYP2E1 genotypes. Details of tissue sample preparation and p53 sequence analysis have been reported previously (Ko et al., 2001). 2.5. Statistical analysis Unconditional logistic regression models were applied to estimate odds ratios (OR) and 95% confidence intervals (CI) for the genetic risk factor in the total study population and the subgroups of smokers, adjusted for gender and age. Homogeneity between distributions was analyzed with the Wald’s χ2 -test. Computations were carried out using the statistical software SAS, version 8.2.

2.3. SNaPshotTM Since Light-CyclerTM hybridization probes did not fit 7632T>A polymorphism, this allele was analyzed by SNaPshotTM technique (Norton et al., 2002). However, 28 samples did not yield a conclusive result and thus these samples were not used for the statistical analysis. For primer extension 4 ␮l of the PCR-fragments were prepared with 1 U Exonuclease (Roche Diagnostics) and 1 U shrimp alkaline phosphatase (New England Biolabs) for 60 min at 37 ◦ C on a thermocycler followed by 15 min at 72 ◦ C. Primer extension was performed by combining 1 ␮l primer (5 -cac cca gct gat taa aaa tt-3 ), 5 ␮l SNaPshot ready reaction premix (Applied Biosystems), 0.15 pmol/␮l primer and 3 ␮l water. This reaction mix was incubated at 96 ◦ C for 10 min and then was subject to 25 cycles of 96 ◦ C for 10 s, 50 ◦ C for 5 s and 60 ◦ C for 30 s. To prevent unincorporated fluorescent dNTPs which would obscure the primer extension products during electrophoresis, the reactions were treated with 1 U shrimp alkaline phosphatase for 60 min at 37 ◦ C, followed by 15 min at 72 ◦ C. Aliquots of 1 ␮l SNaPshot product and 10 ␮l Hi-Di foramide were loaded onto a 310 DNA sequencer (Applied Biosystems). Electrophoresis was done on a capillary array at 95 ◦ C by using POP4 polymer and GeneScan Run Module E5 (Applied Biosystems). Electrophoresis data were processed by GeneScan Analysis, version 3.7 (Applied Biosystems).

3. Results and discussion The genotype distributions of the CYP2E1 polymorphisms examined in controls and in cases with HNSCC are shown in Table 2. In controls, heterozygosity for 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 reported data in Caucasians (Garte et al., 2001, Thier et al., 2002). No variant genotype was found concerning −71G>T allele. In less than 1% of controls, variant genotypes were found for both −1053C>T and 7632T>A polymorphisms, somewhat below the expected frequencies. In HNSCC patients the heterozygous −1053C>T and 7632T>A genotypes were found in 14.1% and 2.6%, respectively. The genotype distribution of both polymorphisms showed no significant differences between cases and controls, neither for smokers nor for non-smokers. In contrast, the −71G>T polymorphism revealed a significantly higher frequency of the heterozygous genotype in the HNSCC group (P = 0.04). This significance was just caused by the smoking individuals (P = 0.01), whilst in non-smokers the estimated 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

0.01

0.17

Comparison reference genotype vs. heterozygous or variant genotype adjusted for age and gender. a

276 (88.5) 36 (11.5) 0 (0.0) CYP2E1 −71G>T Reference genotype Heterozygous Variant genotype

277 (92.6) 22 (7.4) 0 (0.0)

0.49 (0.25–0.98)

0.04

167 (85.6) 28 (14.4) 0 (0.0)

166 (94.3) 10 (5.7) 0 (0.0)

0.33 (0.14–0.79)

2.55 (0.68–9.60) 166 (94.3) 9 (5.1) 1 (0.6) 191 (97.9) 4 (2.1) 0 (0.0) 0.39 1.58 (0.56–4.49) 304 (97.4) 8 (2.6) 0 (0.0) CYP2E1 −1053C>T Reference genotype Heterozygous Variant genotype

282 (94.9) 13 (4.4) 2 (0.7)

0.69 (0.32–1.49) 120 (87.6) 17 (12.4) 0 (0.0) 136 (84.0) 26 (16.0) 0 (0.0) 0.95 1.02 (0.56–1.84) 196 (83.1) 39 (16.5) 1 (0.4) 225 (85.9) 37 (14.1) 0 (0.0)

OR (95% Controls n (%) HNSCC-cases N (%)

CYP2E1 7632T>A Reference genotype Heterozygous Variant genotype

OR (95% CI)a Controls n (%) HNSCC-cases N (%) P-value

Smokers

CI)a All subjects

Table 2 Distribution of CYP2E1 genotypes among control individuals and HNSCC-cases and ORs (95% CI) for HNSCC in all subjects and in smokers

0.34

T. Neuhaus et al. / Toxicology Letters 151 (2004) 273–282 P-value

278

et al. (1998) described that constructs containing the polymorphisms −71G>T and −333T>A showed increased transcriptional activity compared to the variant genotype or −333T>A alone, but they failed to detect an influence of −71G>T mutation on the kinetics of chlorzoxazone elimination. Also Thier et al. (2002) found no association between −71G>T allele and the levels of N-(cyanoethyl)valine in industrial workers handling acrylonitrile. Our data suggest that the −71G>T polymorphism could be involved in the bioactivation of a carcinogenic, tobacco-related substance, e.g. diethylnitrosamine (Thier et al., 2001). Thus, further studies should focus on a putative association of this CYP2E1 polymorphism and other malignancies in which smoking is an important risk factor, like squamous cell cancer of the lung. As previously described, combined polymorphisms of different genes may act synergistically on the cancer risk (Ko et al., 2001). For this we performed an analysis of the effect of a combination of the CYP2E1 polymorphisms on the development of HNSCC. However, an increased risk for HNSCC could not be found in either combination (data not shown). To examine associations between mutation of p53 gene and CYP2E1 genotypes, tumor DNA from 140 HNSCC patients was analyzed for p53 aberrations. As previously described (Ko et al., 2001), tumor-specific aberrations were found in 47.1%, and the frequency of somatic mutations of p53 gene in smokers was 1.9 times higher than in non-smokers. As indicated in Table 3, no association appeared between −1053C>T or −71G>T polymorphisms and aberration of the p53 gene, independent of the smoking habits. Conversely, the p53 mutation was linked to the reference genotype of 7632T>A polymorphism. Although this association is significant (P = 0.02) in the total study group, it is mainly caused by the smokers, but, because of the small number of cases, the association in this subgroup is not statistically significant (P = 0.09). As on the one hand, there are actually two studies describing an increased risk of the development of lung cancer in carriers of 7632T>A reference genotype especially in smokers (Marchand et al., 1998; Wu et al., 1998), which would be in line with the data shown here, and on the other hand there are lots of others, as mentioned above, that missed such an association, the relevance of this association between p53 mutation and

1.68 (0.52–5.50)

0.39

reference genotype of 7632T>A polymorphism remains open. There are very few studies that were engaged in analyzing a correlation between polymorphism of CYP2E1 and mutation of the p53 gene. While two groups missed such an association (Przygodzki et al., 1998; Kim et al., 2000), two others described a correlation between the homozygous variant genotype for −1053C>T and p53 aberrations in workers with vinyl chloride monomer exposure (Wong et al., 2002) and in patients with squamous cell carcinoma of the lung (Oyama et al., 1997). Here no patient with homozygous variant genotype for −1053C>T was identified and for this we were not able to support the findings of one of the works presented. In essence, we show that smoking carriers of heterozygosity of the CYP2E1 −71G>T allele display a significantly elevated risk for the development of HNSCC, which should induce studies focusing on the functional and epidemiological relevance of this polymorphism.

23 (76.7) 7 (23.3) 0.36

39 (84.8) 7 (15.2)

0.80 1.44 (0.09–24.03) 29 (96.7) 1 (3.3) 0.89

45 (97.8) 1 (2.2)

0.09 3.30 (0.82–13.25) 17 (70.8) 7 (29.2) 0.02

31 (88.6) 4 (11.4)

Normal p53 n (%) P-value

HNSCC, smokers

279

1.64 (0.57–4.71)

1.15 (0.17–7.81)

4.69 (1.33–16.58)

The excellent technical assistance of Elisabeth Grünewald and Silke Schöneborn is greatly appreciated.

Adjusted for age and gender. a

64 (86.5) 10 (13.5) CYP2E1 −71G>T Reference genotype Heterozygous

58 (87.9) 8 (12.1)

71 (95.9) 3 (4.1) CYP2E1 −1053C>T Reference genotype Heterozygous

64 (97.0) 2 (3.0)

48 (78.7) 13 (21.3)

48 (92.3) 4 (7.7)

References

CYP2E1 7632T>A Reference genotype Heterozygous

Normal p53 n (%)

Mutant p53 n (%)

OR (95%

CI)a

Acknowledgements

HNSCC-cases

Table 3 Association between p53-mutations and CYP2E1 genotypes in individuals with HNSCC (all subjects and smokers)

Mutant p53 n (%)

OR (95% CI)a

P-value

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