N-Acetyltransferase-2, glutathione S-transferase M1 and T1 genetic polymorphisms, cigarette smoking and hepatocellular carcinoma: A case-control study Umberto Gelatti 1 * , Loredana Covolo 1 , Renato Talamini 2 , Alessandro Tagger 3 , Fabio Barbone 4 , Claudia Martelli 5 , Francesca Cremaschini 1 , Silvia Franceschi 6 , Maria Lisa Ribero 7 , Seymour Garte 8 , Giuseppe Nardi 1 , Valter Donadon 9 and Francesco Donato 1 1 Institute of Hygiene, University of Brescia, Brescia, Italy 2 Oncologic Referral Center of Aviano, Pordenone, Italy 3 Institute of Virology, University of Milan, Milan, Italy 4 Institute of Hygiene, Department of Experimental and Clinical Pathology and Medicine, University of Udine, Udine, Italy 5 Hospital S. Orsola Fatebenefratelli, Brescia, Italy 6 International Agency for Research on Cancer, Lyon, France 7 Institute of Hygiene, University of Milan, Milan, Italy 8 Genetics Research Institute, Milan, Italy 9 Unit of Internal Medicine, Hospital of Pordenone, Pordenone, Italy Our aim was to evaluate the role of N-acetyltransferase (NAT2) and glutathione S-transferase M1 and T1 (GSTM1 and GSTT1) polymorphisms in hepatocellular carcinoma (HCC) according to cigarette smoking, taking into account hepatitis B (HBV) and C (HCV) viral infection as well as alcohol consumption. A hospital- based case-control study was conducted in 2 areas of north Italy. Cases (n = 200) were patients hospitalized for HCC, and controls (n = 400) were patients admitted for reasons other than liver disease, neoplasms and tobacco- and alcohol-related diseases. Genotypes were determined using PCR and the PCR/restriction fragment length polymorphism–based method. The putative risk genotypes NAT2 slow acetylator, GSTM1 null and GSTT1 null were not associated with HCC (OR = 1.3, 95% CI 0.8–2.0; OR = 1.0, 95% CI 0.6–1.5; OR = 0.8, 95% CI 0.4–1.4, respectively). Although not statistically significant, an increase in HCC risk was observed among light smokers (1–20 pack-years) carrying GSTT1 null (OR = 1.7, 95% CI 0.6–4.7) and NAT2 slow acetylator (OR = 1.3, 95% CI 0.6–3.0) genotypes. In conclusion, there was no evidence for a gene–environment interaction in HCC risk for GSTM1, GSTT1 and NAT2 genotypes. ' 2005 Wiley-Liss, Inc. Key words: liver cancer; case-control study; epidemiology; gene– environment interaction HCC is one of the most common neoplasms in the world, and it is estimated that half a million cases occur annually. 1 HBV and HCV infections 2 and alcohol drinking 3 have been recognized as the major risk factors for HCC in developed countries. Tobacco smok- ing has also been implicated as a risk factor for liver cancer, though some studies did not find such an association with HCC. 4 The dis- crepancies among the results are possibly due to confounding by the main risk factors for HCC, particularly alcohol consumption and hepatitis viral infections. Indeed, a significant positive relation- ship between cigarette smoking and HCC was found only among subjects without HBV and/or HCV infection in a study in Greece. 5 Individual cancer susceptibility following exposure to carcino- gens may be based on differences in the capacity of metabolic enzymes to activate or deactivate xenobiotic compounds. Gluta- thione S-transferases 6 and N-acetyltransferase are the main enzymes responsible for the detoxification of carcinogenic com- pounds such as polycyclic aromatic hydrocarbon and aromatic amines, which are largely present in cigarette smoke. 7 GSTM1 and GSTT1, which are important members of the GST multigene family, are polymorphic in humans. Homozygous deletion of part of these genes (null genotype) results in enzyme deficiency and, thus, an impaired ability to eliminate carcinogenic compounds metabolically. 8 NAT2 is a polymorphic enzyme involved in the activation and deactivation of aromatic and heterocyclic amines. Polymorphisms of the NAT2 gene result in slow and fast acetyla- tors of potentially toxic substances. 7 A significant relation was found between GSTM1 null genotype and various types of cancer among smokers, including cancers of the lung, 9 bladder, 10 rectum 11 and esophagus. 12 Only 2 studies have investigated the role of GSTM1 and GSTT1 in HCC develop- ment in relation to cigarette smoking and found no associa- tion. 13,14 Both studies, however, were small in size and therefore not powerful enough to detect a weak association. An interaction between NAT2 slow acetylator genotype and smoking was found for breast cancer, 15 colorectal adenoma 16 and bladder cancer. 17 Regarding HCC, some studies have observed a significant association with NAT2 genetic polymorphisms, for both slow 18,19 and rapid 20 acetylator genotypes. Our aim was to investigate whether GSTM1, GSTT1 and NAT2 genotypes influence susceptibility to HCC in relation to smoking habits, taking into account HBV and HCV infection as well as alcohol consumption. Material and methods The study had a hospital-based case-control design. From March 1999 to July 2002, we enrolled 200 cases admitted for HCC and 400 controls hospitalized for reasons other than liver disease, neoplasms and tobacco- or alcohol-related disease. The absence of liver disease was checked by clinical record examina- tion. The size of the study was established to estimate an OR for HCC of 2 for each genotype investigated with a prevalence of 15–75% in the general population, with an a error of 0.05 and a power of 0.90. Enrollment of both cases and controls took place in 2 areas in north Italy. Half of the cases and controls were recruited in the province of Brescia (Lombardy region) and the other half in the province of Pordenone (Friuli-Venezia-Giulia region). Cases and controls were enrolled at 3 major hospitals in Brescia, at the Abbreviations: CI, confidence interval; HBsAg, hepatitis B surface antigen; HBV/HCV, hepatitis B virus/C virus; HCC, hepatocellular carci- noma; GSTM1, glutathione S-transferase M1; GSTT1, glutathione S-trans- ferase T1; NAT2, N-acetyltransferase 2; OR, odds ratio; RFLP, restriction fragment length polymorphism. Grant sponsor: Ministero dell’Istruzione, dell’Universita ` e della Ricerca; Grant sponsor: Associazione Italiana per la Ricerca sul Cancro. *Correspondence to: Cattedra di Igiene, Universita ` di Brescia, Viale Europa 11, 25123 Brescia, Italy. Fax: þ39-030-3701404. E-mail: gelatti@med.unibs.it Received 2 July 2004; Accepted after revision 24 November 2004 DOI 10.1002/ijc.20895 Published online 1 February 2005 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 115, 301–306 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer