Susceptibility to Esophageal Cancer and Genetic Polymorphisms in Glutathione S-Transferases

Genetic polymorphisms in enzymes involved in carcinogen metabolism have been shown to influence susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is primarily responsible for the bioactivation of many low molecular weight carcinogens, including certain nitrosamines, whereas glutathione S-transferases (GSTs) are involved in detoxifying many other carcinogenic electrophiles. Esophageal cancer, which is prevalent in China, is hypothesized to be related to environmental nitrosamine exposure. Thus, we conducted a pilot casecontrol study to examine the association between CYP2E1, GSTM1, GSTT1, and GSTP1 genetic polymorphisms and esophageal cancer susceptibility. DNA samples were isolated from surgically removed esophageal tissues or scraped esophageal epithelium from cases with cancer (n = 45), cases with severe epithelial hyperplasia (n 45), and normal controls (n 46) from a high-risk area, Linxian County, China. RFLPs in the CYP2EJ and the GSTPJ genes were determined by PCR amplification followed by digestion with RsaI or DraI and A1w261, respectively. Deletion of the GSTM1 and GSTTJ genes was examined by a multiplex PCR. The CYP2E1 polymorphism detected by RsaI was significantly different between controls (56%) and cases with cancer (20%) or severe epithebial hyperplasia (17%; P < 0.001). Persons without the RsaI variant alleles had more than a 4-6-fold risk of developing severe epithebiab hyperplasia (adjusted odds ratio, 6.0; 95% confidence interval, 2.3-16.0) and cancer (adjusted odds ratio, 4.8; 95% confidence interval, 1.8-12.4). Polymorphisms in the GSTs were not associated with increased esophageal cancer risk. These results indicate that CYP2E1 may be a genetic susceptibility factor involved in the early events leading to the development of esophageal cancer. Received 5/29/98; revised 8/25/98; accepted 9/1/98. 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. I To whom requests for reprints should be addressed. at Division of Molecular Epidemiology, National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, AR 72079-9502. Introduction Cancer of the esophagus is one of the most common fatal diseases in certain regions of China such as Linxian County, where the mortality rate is as high as 150/100,000 (1). The risk for esophageal cancer in these areas has been associated with exposure to environmental carcinogens, particularly nitrosamines (2-4). However, even in high-risk regions, only certain individuals develop esophageal cancer. Most people live a normal life span, suggesting that host susceptibility factors may play an important role in cancer development. Most chemical carcinogens require metabolic activation for DNA-damaging capabilities, a step widely believed to be essential in carcinogenesis (5, 6). On the other hand, carcinogens may also be detoxified before damaging DNA by in vivo metabolic detoxifying systems (7, 8). Thus, it has been suggested that individuab differences in carcinogen metabolism, which may be heritable or due to sustained environmental exposure to agents that affect the expression of enzymes involved in carcinogen activation or detoxification, may determine the susceptibility to chemically induced cancers. The major enzymes involved in the metabolic activation of chemical carcinogens are CYPs2 (4), a multigene superfamily of enzymes. Of the 25 or more CYP enzymes known to be expressed in human tissues, CYP2E1 is believed to be involved in the activation of most carcinogenic nitrosamines (9-12). Furthermore, this enzyme has the ability to metabolically activate many low molecular weight carcinogens (9) and to produce reactive free radicals from ethanol (13, 14), which also might be of importance in cancer etiology. CYP2E1 represents a major CYP isoform in the human liver and is also expressed in extrahepatic tissues (15-17). Although the enzyme can be induced by certain chemicals such as ethanol, large interindividual variations have been observed before and after induction (18-20), suggesting that the variation may be due to genetic polymorphisms. RFLPs of the human CYP2EJ gene have been identified, and a polymorphism in the upstream region of the gene detected by RsaI has been shown to be associated with the transcriptional regulation of gene expression (21). Another genetic polymorphism that is detectable with DraI is located in intron 6 (22), and this allele has been found to contain a number of functional mutations affecting protein expression and catalytic activity (23). Associations between these genetic polymorphisms in CYP2EJ and the susceptibility to some types of cancer have been reported in case-control studies (22, 24-29), although some negative results have been reported (30-40). To date, one study in Japan has evaluated the role of the RsaI polymorphism in relation to 2 The abbreviations used are: CYP. cytochrome P450: GST, glutathione Stransferase; OR, odds ratio: CI, confidence interval: dNTP, deoxynucleotide triphosphate. on August 15, 2017. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from = 1014 CYP2E1 and Esophageal Cancer Susceptibility 3 W. Tan, Z. Li, and P. Lin, unpublished observations. esophageal cancer and found no association (36). However, an increased risk of both oral (41) and nasopharyngeal (42) cancer has been associated with the prevalence of this variant allele in Taiwan. The GSTs are a family of multifunctional enzymes that play a central robe in the detoxification of toxic and carcinogenic ebectrophiles (43). Individuals who are homozygous for the null GSTMJ or GSTTJ alleles lack the respective enzyme function (44, 45), and these genotypes have been associated with an increased risk of cancer at many sites (44, 46-49). GSTP1, which is a major GST isozyme expressed in the human esophagus (17, 50), has also been shown to be genetically polymorphic (5 1,52). A mutation of A to G within exon 5 results in an I to V change at position 105 in the amino acid sequence of the protein, which alters the specific activity and affinity for the electrophilic substrates of the enzyme (52, 53). Therefore, the GSTP1 polymorphism may also have potential effects on cancer susceptibility (51). Whereas numerous studies have been undertaken to examine the association between genetic polymorphisms in CYPs and/or GSTs and cancer susceptibility (vide supra), there is limited information on their association with esophageal cancer (36, 54). In this study, we analyzed genetic polymorphisms in GSTMJ, GS77’l, GSTPJ, and CYP2EJ in subjects with esophageal cancer and severe epithelial hyperplasia, which is a precancerous lesion, and frequency-matched controls from Linxian County, China, an area where dietary nitrosamine exposures are known to be high (3). Materials and Methods Study Subjects. A pilot case-control study was designed to evaluate the possible robe of genetic polymorphisms in carcinogen-metabobizing enzymes in the susceptibility to esophageal cancer. All subjects were residents of Linxian County, Henan Province, China. Smoking status was not available; however, recent studies3 with a similar population indicate that smoking prevalence is comparable between cases and controls. The diagnoses of esophageal cancer and epithelial hyperplasia were confirmed either histologically or cytologically. Cases were grouped as follows: (a) those with advanced dysplasia or severe hyperplasia (n = 45); (b) those with squamous cell carcinoma or adenocarcinoma (n = 45); and (c) those with squamous cell carcinoma, adenocarcinoma, or advanced dysplasia (n = 62). DNA samples were isolated from surgically removed esophageal tissues using standard methods (55) or from epithelial cells scraped from the esophagus. The yields of DNA varied with the amount of tissue available, and the analysis of each sample for all genotypes was not possible. GSTMJ and GSTTJ Genotyping. GSTMJ and GS17’l genotyping for gene deletions was carried out by a multiplex PCR using primer pairs 5’-GAACTCCCTGAAAAGCTAAAGC-3’ and 5’-GTTGGGCTCAAATATACGGTGG-3’ for GSTMJ, which produced a 219-bp product, and 5’-TTCCTTACTGI and 5’ -TCACCGGATCATGGCCAGCA-3’ for GSJ7’J, which produced a 459-bp product. Amplification of the albumin gene (5’-GCCCTCTGCTAACAAGTCCTAC-3’ and 5’-GCCCTAAAAAGAAAATCCCCAATC-3’) was used as an internal control and produced a 350-bp product (56). PCR was performed in a 50l mixture consisting of sample DNA (0. 1 pg), dNTP (0.2 mrvt; Boehringer Mannheim, Indianapolis, IN), MgCb2 (2.5 mM), M 1234567