Genetic Polymorphisms in Antioxidant Enzymes Modulate Hepatic Iron Accumulation and Hepatocellular Carcinoma Development in Patients with Alcohol-Induced Cirrhosis Angela Sutton, 1,4 Pierre Nahon, 1,6 Dominique Pessayre, 7 Pierre Rufat, 8 Aure ´lie Poire ´, 1 Marianne Ziol, 2,5 Dominique Vidaud, 9 Nathalie Barget, 6 Nathalie Ganne-Carrie ´, 3,6 Nathalie Charnaux, 1,4 Jean-Claude Trinchet, 3,6 Liliane Gattegno, 1,4 and Michel Beaugrand 1,6 1 UPRES 3410, 2 UPRES 3406, and 3 UPRES 3409, UFR Sante´, Me´decine et Biologie Humaine,Universite´ ParisXIII, Bobigny, France; 4 Service de Biochimie and 5 Service d’Anatomopathologie, Ho ˆpital Jean Verdier; 6 Service d’He ´pato-Gastroente´rologie,Ho ˆpital Jean Verdier, Bondy, France; 7 Institut National de la Sante et de la Recherche Medicale U481, Faculte´ de Me ´decine Xavier Bichat; 8 De ´partement MSI, Groupe HospitalierPitie´-Salpeˆtrie`re,Paris,France;and 9 Service de Biochimie, Ho ˆpital Beaujon, Clichy, France Abstract Manganese superoxide dismutase (MnSOD) converts the superoxide anion into H 2 O 2 , which, unless it is detoxified by glutathione peroxidase 1 (GPx1), can increase hepatic iron and can react with iron to form genotoxic compounds. We investigated the role of Ala/Val-MnSOD and Pro/Leu-GPx1 polymorphisms on hepatic iron accumulation and hepatocel- lular carcinoma development in patients with alcoholic cirrhosis. Genotypes were determined in 162 alcoholic patients with cirrhosis but without hepatocellular carcinoma initially, who were prospectively followed up for hepatocellu- lar carcinoma development. We found that patients with two Val-MnSOD alleles (slow H 2 O 2 production) and two Pro-GPx1 alleles (presumably quick H 2 O 2 detoxification) had a lower risk of hepatocellular carcinoma development than other patients (C 2 trend test, P = 0.001; log-rank, P = 0.0009). Indeed, hepatocellular carcinoma percentage was 0% in subjects with this ‘‘2Val-MnSOD/2Pro-GPx1’’ genotype versus 16%, 27%, and 32% in ‘‘2Val-MnSOD/1or2Leu-GPx1,’’ ‘‘1or2Ala- MnSOD/2Pro-GPx1,’’ and ‘‘1or2Ala-MnSOD/1or2Leu-GPx1’’ patients, respectively. The percentage of patients with stainable hepatic iron increased progressively with these genotypic associations: 22%, 28%, 50%, and 53%, respectively (C 2 trend test, P = 0.005). Stainable iron was a risk factor for hepatocellular carcinoma (log-rank, P = 0.0002; relative risk, 3.40). In conclusion, polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in French alcoholic patients with cirrhosis. (Cancer Res 2006; 66(5): 2844-52) Introduction Hepatocellular carcinoma is the fifth cause of cancer worldwide and the third cause of cancer-related deaths (1). Its incidence is rising in Western countries due to the recent hepatitis C epidemics (1). However, alcohol abuse still remains the prevailing cause of hepatocellular carcinoma in some countries, including France (1). Alcohol intoxication increases reactive oxygen species (ROS) formation in several cell compartments, including mitochondria (2, 3), causing oxidative stress and mitochondrial damage (4, 5). ROS and ROS-induced cytokines (6) lead to the apoptosis of some hepatocytes (7–9) followed by inflammation, fibrogenesis (10), and carcinogenesis (11). Mitochondrial ROS are detoxified by the successive action of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx1; ref. 12). MnSOD dismutates the superoxide anion into H 2 O 2 , which GPx1 detoxifies into water (12). GPx1 is the main glutathione peroxidase in the mammalian liver (13, 14). It is encoded by nuclear DNA, with an internal, incomplete mitochondrial targeting sequence, whose optional use targets GPx1 to either mitochondria or cytosol (13, 14). The active site of GPx1 contains a selenocysteine encoded by an UGA codon (15). Albeit usually a stop codon, UGA encodes for selenocysteine in GPx1, thanks to the formation of a quaternary structure involving the ribosome, a stem-loop structure in the 3V -untranslated region of the GPx1 mRNA, an RNA-binding protein, and selenocysteinyl-tRNA Sec (15). Insufficient selenium concentrations impair GPx1 mRNA stability, mRNA translation, and GPx1 activity (16). A genetic polymorphism encodes for either proline (Pro) or leucine (Leu) at codon 198 of human GPx1 (reference SNP cluster identifier number: 1050450; refs. 17, 18). Although basal GPx1 activities were similar in breast carcinoma cells transfected with either Leu-GPx1 or Pro-GPx1 variants, selenium at concentrations anticipated in the human serum increased the activity of the Pro-GPx1 variant more than the Leu-GPx1 variant (19). The less active Leu-GPx1 variant was associated with lung (17), prostate (18), and breast (19) cancer. Furthermore, loss of heterozygosity affecting GPx1 can occur in the tumors of patients with breast cancer (19) or cancer of head and neck (20). MnSOD is encoded by nuclear DNA and is inducible by ROS, cytokines, and ethanol (21–23). MnSOD is synthesized with a mitochondrial targeting sequence, which drives its mitochondrial import (24). In the matrix, the targeting sequence is cleaved, and the mature protein assembles into the active tetramer (24). A genetic polymorphism incorporates either alanine (Ala) or valine (Val) in the targeting sequence of MnSOD (reference SNP cluster identifier number: 1799725; ref. 24). The Ala-MnSOD variant, whose presequence has an a-helix structure, is easily imported and achieves high mitochondrial activity, whereas the Val-MnSOD variant, whose presequence may have a partial h-sheet structure, is partly stuck within the narrow inner membrane import pore (24), and is partly degraded in the proteasome (25). Furthermore, its mRNA is rapidly degraded, so that the Val-MnSOD variant only achieves low activity (25). Note: A. Sutton and P. Nahon contributed equally to this study. Requests for reprints: Angela Sutton, UPRES EA 3410, UFR SMBH, Universite´ Paris XIII, 74 Rue Marcel Cachin, 93017 Bobigny Cedex, France. Phone: 33-1-48-38-76- 97; Fax: 33-1-48-02-65-03; E-mail: angela.sutton@jvr.ap-hop-paris.fr. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2566 Cancer Res 2006; 66: (5). March 1, 2006 2844 www.aacrjournals.org Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/66/5/2844/2560818/2844.pdf by guest on 05 October 2022