Polymorphisms in glutathione S‐transferases GSTM1, GSTT1 and GSTP1 and cytochromes P450 CYP2E1 and CYP1A1 and susceptibility to cirrhosis or pancreatitis in alcoholics

Regislaine Valeria Burim 2 Renata Canalle 2 Ana de Lourdes Candolo Martinelli 1 Catarina Satie Takahashi 0 2 0 Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto, Sao Paulo University , Ribeirao Preto, Sao Paulo, Brazil 1 Department of Internal Medicine, Faculty of Medicine of Ribeirao Preto, Sao Paulo University , Av. Bandeirantes 3900, 14049-900 Ribeirao Preto, Sao Paulo, Brazil 2 Department of Genetics - Excessive alcohol consumption may cause the development of pathologies in the liver and pancreas and various digestive tract cancers. The enzymes GSTM1, GSTT1, GSTP1, CYP1A1 and CYP2E1 are involved in the bioactivation and detoxification of a variety of xenobiotics present in food, organic solvents, tobacco smoke, drugs, pesticides, environmental pollutants and alcoholic drinks. Polymorphisms in the genes coding for these enzymes have been associated with susceptibility to different diseases, including ethanol-related diseases. To investigate whether these polymorphisms represent risk-modifying factors for ethanol-related diseases, a study was conducted involving 120 Brazilian alcoholics and 221 controls with similar ethnic backgrounds. The distribution of alcoholics groups was as follows: 65 with liver cirrhosis, 14 with chronic pancreatitis and 41 without cirrhosis or pancreatitis. The data revealed that carriers of the rare GSTP1 Val allele were at higher risk of liver cirrhosis and pancreatitis, since we found higher frequencies of the Val/Val genotype in alcoholics with liver cirrhosis (15.4%) and pancreatitis (28.6%) in comparison with alcoholics without disease (7.3%). No differences were found in the prevalences of the GSTM1 and GSTT1 null genotypes between alcoholics and the controls and no association was found between the rare CYP2E1 c2 allele and liver cirrhosis and pancreatitis. However, when the mutant CYP1A1 allele was compared between alcoholics and controls, the m2/m2 genotype was more prevalent in the liver cirrhosis alcoholics (7.7%) than in the controls (1.4%) and this difference was statistically significant (P = 0.03, OR = 5.33). In conclusion, our data indicate an association between occurrence of the Val/Val GSTP1 genotype and chronic pancreatitis and an association between the m2/m2 CYP1A1 genotype and alcoholic liver cirrhosis. This could indicate that persons with these genotypes are genetically more prone to the development of alcoholic pancreatitis and alcoholic cirrhosis, respectively. Introduction Excessive alcohol consumption causes physical, psychological and social problems. Alcohol abuse and dependence are major factors of morbidity and mortality in various countries (Greenfield et al., 2000). Moreover, alcohol abuse may cause the development of pathologies in the liver and pancreas and various digestive tract cancers (Longnecker, 1995). However, the vulnerability to ethanol hepatotoxicity varies significantly; 20% of chronic alcoholics develop liver cirrhosis, whereas 20% survive the chronic toxic effects without developing any pathology of the liver (Lelbach, 1975; Sorensen et al., 1984; Savolainen et al., 1993, 1995). Lack of a doseresponse effect of alcohol on the liver may include multiple explanations concerning environmental and nutritional factors and infectious agents acting separately or in combination (Day and Bassendine, 1992). Twin studies have, however, convincingly shown that individual susceptibility to liver cirrhosis is at least partly genetically determined (Hrubec and Omenn, 1981). Ethanol, similarly to most environmental toxins, requires metabolic activation and subsequent detoxification by a series of enzymes. Glutathione S-transferases (GSTs) are phase II xenobiotic metabolizing enzymes that catalyze the conjugation of electrophilic compounds with reduced glutathione to produce less toxic or readily excretable metabolites (Boyer, 1989). In more than 90% of cases the substrates for this reaction are provided by phase I xenobiotic metabolizing cytochrome P450 isozymes (CYPs). Although many chemicals are detoxified via this route, some compounds, including carcinogenic chemicals, undergo metabolic activation to give rise to ultimate carcinogens (Lewis et al., 1998). Therefore, the levels and duration of bioactivated toxic or carcinogenic compounds in an organ depend on the interplay of both biotransforming enzyme systems. Clearly, the yield of these metabolites would be higher, if cells were rich in phase I bioactivating enzymes but poor in detoxifying phase II enzymes (Standop et al., 2002). Phase I and phase II biotransformation enzymes are characterized by zone-specific expression in the liver (Oinonen and Lindros, 1995) and the pancreas (Standop et al., 2002). Indeed, it is well known that long-term ethanol exposure induces various xenobiotic metabolizing enzymes, including CYPs (Buhler et al., 1991; Takahashi et al., 1993) and GSTs (Vanhaecke et al., 2000). Because both GSTs and CYPs are expressed zone-specifically and are induced by exposure to ethanol, an imbalance between these enzymes in determined areas of the liver or the pancreas might have a toxic effect (Standop et al., 2002). Thus, because GSTs play an important role in protecting the cell against cytotoxic and carcinogenic agents (Hayes and Pulford, 1995), the toxic load caused by CYP-mediated products might lead to a more rapid depletion of GSTs in areas with lower concentrations of these enzymes and, consequently, to a pronounced toxic effect, which might cause diseases such as cirrhrosis and chronic pancreatitis (Standop et al., 2002). Individual variations in the metabolic activation and detoxification of chemical carcinogens and genotoxins, such as ethanol, are likely to be one of the major determinants of inter-individual differences in susceptibility to environmentally induced cancers. The genetic constitution seems to play the most important role in this context. An increasing number of xenobiotic metabolizing enzymes, such as GSTs and CYPs, have been shown to be polymorphic (Hirvonen, 1995). Homozygous gene deletion has been associated with deficiencies in GSTM1 and GSTT1 enzyme activity and thus potential increases in the levels of toxic metabolites resulting from chronic ethanol administration (Seidegard et al., 1986). Therefore, because of its patho-biological association with alcohol-induced liver damage, genetic polymorphism of the GST1 locus may be important in the inherited susceptibility to liver cirrhosis (Savolainen et al., 1996). Several studies have, indeed, suggested that the GSTM1 and GSTT1 null genotypes are risk factors for tumors such as bladder, breast, oral, lung, head and neck cancers (Schnakenberg et al., 2000; Mitrunen et al., 2001; Buch et al., 2002; Matthias et al., 2002; Hung et al., 2003). Polymorphisms of the GSTP1 gene were first reported by Board et al. (1989). They consisted of an AfiG transition of nucleotide 313 in exon 5 (GSTP1*B) and a GfiT transition of nucleot (...truncated)