Cancer Epidemiology Biomarkers & Prevention Vol. 15, 1043-1045, May 2006
© 2006 American Association for Cancer Research
No Association between Polymorphisms in CYP2E1, GSTM1, NAT1, NAT2 and the Risk of Gastric Adenocarcinoma in the European Prospective Investigation into Cancer and Nutrition
Antonio Agudo1,
Núria Sala2,
Guillem Pera1,
Gabriel Capellá3,
Antonio Berenguer1,
Nadia García2,3,
Domenico Palli4,
Heiner Boeing5,
Giuseppe Del Giudice6,
Calogero Saieva4,
Fatima Carneiro7,
Franco Berrino8,
Carlotta Sacerdote9,
Rosario Tumino10,
Salvatore Panico11,
Göran Berglund12,
Henrik Simán12,
Roger Stenling13,
Göran Hallmans14,
Carmen Martínez15,
Pilar Amiano16,
Aurelio Barricarte17,
Carmen Navarro18,
Jose R. Quirós19,
Naomi Allen20,
Tim Key20,
Sheila Bingham21,
Kay-Tee Khaw21,
Jakob Linseisen22,
Gabriele Nagel22,
Kim Overvad23,
Anne Tjonneland24,
Anja Olsen24,
H. Bas Bueno-de-Mesquita25,
Hendriek C. Boshuizen26,
Petra H. Peeters27,
Mattijs E. Numans27,
Françoise Clavel-Chapelon28,
Marie-Christine Boutron-Ruault28,
Antonia Trichopoulou29,
Eiliv Lund30,
Hendrik Bläker31,
Mazda Jenab32,
Pietro Ferrari32,
Teresa Norat32,
Elio Riboli33 and
Carlos A. González1
1 Unit of Epidemiology, Catalan Institute of Oncology; 2 Medical and Molecular Genetics Center, Oncology Research Institute; 3 Laboratory of Translational Research, Catalan Institute of Oncology, Institut d’Investigació Biomèdica de Bellvitge, Hospitalet de Llobregat, Spain; 4 Epidemiology Unit, Centre for Cancer Research and Prevention, Florence, Italy; 5 German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany; 6 Immunobiological Research Institute of Siena, Chiron SPA, Siena, Italy; 7 Institute of Molecular Pathology and Immunology, University of Porto and Medical Faculty, Porto, Portugal; 8 Department of Epidemiology, National Cancer Institute, Milan, Italy; 9 Unit of Cancer Epidemiology, University of Turin and CPO-Piemonte, Turin, Italy; 10 Cancer Registry, Azienda Ospedaliera Civile - M.P. Arezzo, Ragusa, Italy; 11 Department of Clinical and Experimental Medicine, Federico II University, Medical School, Naples, Italy; 12 Malmö Diet and Cancer Study, Lund University, Malmö, Sweden; 13 Department of Medical Biosciences, Pathology, University of Umea, Sweden; 14 Department of Public Health and Clinical Medicine, Nutritional Research, University of Umea, Sweden; 15 Andalusian School of Public Health, Granada, Spain; 16 Fundación Vasca de Innovación e Investigación Sanitarias, Sondika, Bizkaia, Spain; 17 Public Health Institute, Navarra, Spain; 18 Consejería de Sanidad y Consumo, Murcia, Spain; 19 Dirección General de Salud Pública, Consejería de Salud y Servicios Sanitarios, Asturias, Spain; 20 Cancer Research UK Epidemiology Unit, University of Oxford, United Kingdom; 21 Medical Research Council Dunn Human Nutrition Unit, Cambridge, United Kingdom; 22 Division of Clinical Epidemiology, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 23 Department of Clinical Epidemiology, Aalborg Hospital and Aarhus University Hospital, and Department of Epidemiology and Social Medicine, University of Aarhus, Denmark; 24 Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark; 25 Centre for Nutrition and Health and 26 Centre for Information Technology and Methodology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands; 27 Julius Center for Health Sciences and Primary Care, University Medical Center, Utrecht, The Netherlands; 28 Institut National de la Santé et de la Recherche Médicale, Institut Gustave Roussy, Villejuif, France; 29 Department of Hygiene and Epidemiology, Medical School, University of Athens, Greece; 30 Institute of Community Medicine, University of Tromso, Norway; 31 Division of General Pathology, University of Heidelberg, Heidelberg, Germany; 32 Unit of Nutrition and Cancer, IARC, Lyon, France; and 33 Cancer Epidemiology and Prevention, Department of Epidemiology and Public Health, Faculty of Medicine, Imperial College London, London, United Kingdom
Requests for reprints: Antonio Agudo, Unit of Epidemiology. Catalan Institute of Oncology, Av. Gran Via s/n km 2.7, 08907 L'Hospitalet de Llobregat, Spain. Phone: 34-932607401; Fax: 34-932607787. E-mail: a.agudo{at}ico.scs.es
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Introduction
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Metabolizing enzymes of many procarcinogens present in tobacco smoke often display genetic polymorphisms; thereby, genetic variation of such enzymes can modulate susceptibility to gastric cancer risk. The ethanol-inducible enzyme cytochrome P450 2E1 (CYP2E1) metabolizes dietary and tobacco-specific nitrosamines; N-acetyltransferases (NAT1 and NAT2) play a role in the metabolism of aromatic amines and heterocyclic amines; finally, subjects with deletion of the phase II enzyme glutathione S-transferase (GST) have a decreased capability of detoxifying several carcinogens (1). Despite the biological plausibility and epidemiologic studies investigating the relationship between cancer in humans and these enzymes, there are still conflicting results regarding their etiologic relevance in gastric carcinogenesis (2). We have reported separately the association between gastric cancer risk and genetic variation in CYP1A1 and CYP1A2, microsomal epoxide hydrolase (EPHX1), and GSTT1 involved in the metabolism of polycyclic aromatic hydrocarbons. Here, we present the results concerning the relationship between polymorphisms in CYP2E1, NAT1, NAT2, and GSTM1 and the risk of gastric adenocarcinoma, as well as potential effect modification of such association by tobacco smoking.
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Materials and methods
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Subjects
The study subjects were selected according to a nested case-control design from the EPIC cohort, which includes about half million individuals recruited between 1992 and 1998 in 23 centers in 10 European countries (3). Cases were subjects newly diagnosed during the follow-up of gastric adenocarcinoma, confirmed and validated by an independent panel of pathologists. For each case, up to four controls were randomly selected among cohort members alive and free of cancer at the time of diagnosis of the case, with blood samples available, matched by gender, age, center, and date of blood collection. The final study group consisted of 243 cases and 946 matched controls.
Genotyping
Genomic DNA was extracted from a 0.5-mL aliquot of buffy coat as reported.34 Single nucleotide polymorphisms (SNP) genotyping was done in a LightCycler instrument (Roche Diagnostics, Mannheim, Germany) by melting curve analysis of a fluorescently labeled sensor probe specific for each PCR amplified variant. The four NAT2 polymorphisms were analyzed by use of the LC NAT2 Mutation Detection kit (Roche Molecular Biochemicals, Mannheim, Germany). Primers and hybridization probes were obtained from published literature (4, 5). Genes have been named according to the HUGO Gene Nomenclature committee (http://www.genomic.unimelb.edu.au), and polymorphisms have been identified according to the SNP500Cancer database (http://snp500cancer.nci.nih.gov/home.cfm) and to the ID numbering of the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP).
Statistical Analysis
Hardy-Weinberg equilibrium for each polymorphism was tested separately for cases and controls. Association between each SNP and gastric cancer risk was assessed by the odds ratio (OR) with corresponding 95% confidence interval estimated by logistic regression (6). Effect modification by smoking status was assessed by the likelihood ratio test.
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Results
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In our study, 56% cases (and matched controls) were males, with average age at recruitment of 59 years. Among controls, all the genotype distributions were in Hardy-Weinberg equilibrium. The genotype distribution and ORs for all the analyzed SNPs are shown in Table 1
. No significant associations were seen when each genotype was compared with the homozygous group of the most frequent allele, or by combining heterozygotes and homozygotes for the variant allele (dominant model); in addition, no significant associations appeared carrying out the analysis according to a recessive model (data not shown). There was an increased risk in gastric cancer among ever smokers associated with the T allele of CYP2E1 –1054C>T and the A allele of NAT2 *7A/B, but none of them was statistically significant (Table 2
). There were no significant interactions between smoking status and any of the analyzed SNP.
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Table 1. Frequency distribution of genotypes and ORs for polymorphisms in metabolic genes in gastric adenocarcinoma cases and controls
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Discussion
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CYP2E1 is involved in the metabolism of low molecular weight compounds, such as nitrosamines. Our results show that the frequency of the –1054C>T variant (allele *5B, RsaI sensitive) is lower in Caucasians than in Asian populations. Although some studies have reported lack of association with gastric cancer risk (2, 7), others observed a protective effect for the uncommon T variant (7-9), and another study reported a significant increased risk (OR, 2.9) for the homozygous variant (10). A recent meta-analysis (11) estimated an overall 24% increase in gastric cancer risk for GSTM1 deletion, only marginally significant overall, and slightly lower in Caucasian populations. Acetylation may have substrate-specific effects: whereas it is seen as an activating mechanism for heterocyclic amines, it has detoxifying effect for aromatic amines (12). All the SNPs analyzed are associated with fast NAT1 enzymatic activity and slow acetylation by NAT2. Regarding NAT2 and gastric cancer risk, there are controversial results: two Japanese studies (8, 13) reported significant increased risks of about 60% for slow enzymatic activity, whereas a significant OR of 0.37 was observed in a study in Spain (14) and nonsignificant OR of 0.46 in the United Kingdom (15). The latter also reported a significant increased risk (OR, 2.5) for the rapid phenotype *10 in NAT1.
Taking into account the prevalence of the analyzed variants in our population (Table 1), we had always a power > 80% to detect an OR of >1.5 or <0.67, except for CYP2E1, with power < 50% because of the small frequency of variant allele T. The power was also compromised (slightly below 50%) to detect an OR of 1.24 as estimated in a meta-analysis for GSTM1, in spite of high proportion of deletions. Both for NAT1 and NAT2, the power to detect ORs in the range of those reported in previous studies (0.4-2.5) was very high, always >90%.
In conclusion, by analyzing a large European population essentially of Caucasian origin, our study did not find any association between gastric cancer risk and several polymorphisms in genes involved in the metabolism of tobacco carcinogens. Most studies dealing with these genes have been done on Asian populations showing controversial results.
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Acknowledgments
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We thank the members of the pathologist panel for their valuable work: Dr. Johan Offerhaus (Amsterdam, The Netherlands), Dr. Vicki Save (Cambridge, United Kingdom), Dr. Julio Torrado (San Sebastian, Spain), Dr. Gabriella Nesi (Firenze, Italy), Dr. U. Mahlke (Potsdam, Germany), Dr. Claus Fenger (Denmark), and Dr. Dimitrious Roukos (Ioannina, Greece); Catia Moutinho (Porto, Portugal) for her technical work in the preparation of pathologic material; and Francisco Rico and Fátima Marín for their technical assistance in the genotyping analysis.
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Footnotes
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Grant support: The EURGAST project received financial support from the FP5 of the European Commission grant QLG1-CT-2001-01049. The EPIC study was funded by Europe Against Cancer Programme of the European Commission, SANCO; Ligue Contre le Cancer, France; Société 3M, France; Mutuelle Générale de l'Education Nationale; Institut National de la Santé et de la Recherche Médicale; German Cancer Aid; German Cancer Research Center; German Federal Ministry of Education and Research; Danish Cancer Society; Health Research Fund of the Spanish Ministry of Health grants RCESP-C03/09 and RTICCC 03/10; AGAUR, Generalitat de Catalunya grant 2002-PIR-00333; the participating regional governments and institutions of Spain; Cancer Research UK; Medical Research Council, UK; the Stroke Association, United Kingdom; British Heart Foundation; Department of Health, United Kingdom; Food Standards Agency, United Kingdom; the Wellcome Trust, United Kingdom; Greek Ministry of Health; Greek Ministry of Education; Italian Association for Research on Cancer; Italian National Research Council; Dutch Ministry of Public Health, Welfare and Sports; Dutch Ministry of Health; Dutch Prevention Funds; LK Research Funds; Dutch ZON, Zorg Onderzoek Nederland; World Cancer Research Fund; Swedish Cancer Society; Swedish Scientific Council; Regional Government of Skane, Sweden; and Norwegian Cancer Society.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
34 A. Agudo et al. Polymorphisms in metabolic genes related to tobacco smoke and the risk of gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST). Cancer Epidemiol Biomarkers Prev, submitted for publication. 
Received 1/30/06;
accepted 3/ 2/06.
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A. Agudo, N. Sala, G. Pera, G. Capella, A. Berenguer, N. Garcia, D. Palli, H. Boeing, G. Del Giudice, C. Saieva, et al.
Polymorphisms in Metabolic Genes Related to Tobacco Smoke and the Risk of Gastric Cancer in the European Prospective Investigation into Cancer and Nutrition
Cancer Epidemiol. Biomarkers Prev.,
December 1, 2006;
15(12):
2427 - 2434.
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Introduction
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