Zhang JH, Jin X, Li Y, Wang R, Guo W, Wang N, Wen DG, Chen ZF, Kuang G, Wei LZ, Wang SJ. Epoxide hydrolase Tyr113His polymorphism is not associated with susceptibility to esophageal squamous cell carcinoma in population of North China. World J Gastroenterol 2003; 9(12): 2654-2657 [PMID: 14669306 DOI: 10.3748/wjg.v9.i12.2654]
Corresponding Author of This Article
Professor Shi-Jie Wang, The Fourth Affiliated Hospital, Hebei Medical University, Jiankanglu 12, Shijiazhuang 050011, China. wang.sj@hbmu.edu
Article-Type of This Article
Esophageal Cancer
Open-Access Policy of This Article
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Jian-Hui Zhang, Xia Jin, Yan Li, Wei Guo, Na Wang, Deng-Gui Wen, Zhi-Feng Chen, Gang Kuang, Li-Zhen Wei, Shi-Jie Wang, Hebei Cancer Institute, Hebei Medical University, Shijiazhuang 050011, Hebei Province, China
Rui Wang, Shi-Jie Wang, The Fourth Affiliated Hospital, Hebei Medical University, Shijiazhuang 050011, Hebei Province, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the Scientific Grant of Educational Department of Hebei Province, China, No. 2001150
Correspondence to: Professor Shi-Jie Wang, The Fourth Affiliated Hospital, Hebei Medical University, Jiankanglu 12, Shijiazhuang 050011, China. wang.sj@hbmu.edu
Telephone: +86-311-6085231 Fax: +86-311-6077634
Received: June 16, 2003 Revised: June 25, 2003 Accepted: August 25, 2003 Published online: December 15, 2003
Abstract
AIM: To investigate the possible association of microsomal epoxide hydrolase (mEH) Tyr113His polymorphism with susceptibility to esophageal squamous cell carcinoma (ESCC) in a population of North China.
METHODS: The mEH Tyr113His genotypes were determined by polymerase-chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis in 257 patients with esophageal squamous cell carcinoma (ESCC) and 252 healthy subjects as a control group.
RESULTS: The frequencies for Tyr and His alleles were 44.2%, 55.8% in ESCC patients, and 44.0% and 56.0% in healthy subjects, respectively. No statistic difference in allele distribution was observed between ESCC patients and controls (χ2 = 0.008, P = 0.929). The overall genotype distribution difference was not observed between cancer cases and controls (χ2 = 2.116, P = 0.347). Compared with Tyr/Tyr genotype, neither His/His genotype nor in combination with Tyr/His genotype significantly modified the risk of the development of ESCC, the adjusted odds ratio was 1.076 (95%CI = 0.850-1.361) and 0.756 (95%CI = 0.493-1.157), respectively. When stratified for sex, age, smoking status and family history of upper gastrointestinal cancer, His/His genotype alone or in combination with Tyr/His genotype also did not show any significant influence on the risk of developing ESCC.
CONCLUSION: MEH Tyr113His polymorphism may not be used as a stratification marker in screening individuals at a high risk of ESCC.
Key Words: $[Keywords]
Citation: Zhang JH, Jin X, Li Y, Wang R, Guo W, Wang N, Wen DG, Chen ZF, Kuang G, Wei LZ, Wang SJ. Epoxide hydrolase Tyr113His polymorphism is not associated with susceptibility to esophageal squamous cell carcinoma in population of North China. World J Gastroenterol 2003; 9(12): 2654-2657
China is one of the prevalent areas of esophageal squamous cell cancer (ESCC). Exposure to environmental carcinogens is considered as the main risk factors of ESCC[1,2]. Among them, chemical carcinogens such as polycyclic aromatic hydrocarbons (PAHs) in consumed tobacco or ingested food may contribute to the high incidence of ESCC in China[3,4]. Metabolization of PAHs involves a complex enzymatic mechanism. Microsomal epoxide hydrolase (mEH) is an enzyme that hydrolyzes epoxides such as PHA, yielding corresponding trans-dihydrodiols. Usually, this hydrolysis acts as a detoxifying step, although in some instances, trans-dihydrodiols generated from PAHs are highly toxic and mutagenic. Therefore, mEH plays a dual role in the detoxification and activation of procarcinogens, and its role in carcinogenesis may depend on exposures to different environmental substrates[5].
There are two polymorphic sites that affect the enzyme activity in human mEH gene. One variant is characterized by substitution of histidine for tyrosine at the amino acid position 113, the other is characterized by substitution of arginine for histidine at the position 139. The proteins encoded by polymorphic alleles demonstrated different enzyme activities in vitro[6]. MEH polymorphism has been associated with chemical carcinogen-induced cancers occurring in lung[7,8], ovary[9], colorectum[10] and liver[11]. The correlation of mEH polymorphism with susceptibility to ESCC has not been reported so far. Therefore, the current study investigated the Tyr113His polymorphism in mEH exon 3 in ESCC patients and healthy individuals from North China.
MATERIALS AND METHODS
Subjects
This study included 257 patients with histologically confirmed esophageal squamous cell carcinomas and 252 healthy individuals without overt cancer. The cancer patients were hospitalized for surgery in the Fourth Affiliated hospital, Hebei Medical University between 2001 and 2003. The healthy subjects were from the same hospital for physical examination in the same period. All of the patients and control subjects were from Shijiazhuang city or its surrounding regions. Information of sex, age, smoking habits and family history was obtained from cancer patients and healthy controls by interview following sampling. The smokers were defined as former or current smoking 5 cigarettes per day for at least two years. The individuals with at least one first-degree relative or at least two second-degree relatives having esophageal/cardiac/ gastric cancer were defined as having family history of upper gastrointestinal cancers (UGIC). The smoking status and family history were available from some of the cases and controls. Informed consent was obtained from all the recruited subjects. The study was approved by the Ethics Committee of Hebei Cancer Institute.
DNA extraction
Five ml of venous blood from each subject was drawn in Vacutainer tubes containing EDTA and stored at 4 °C. Genomic DNA was extracted within one week after sampling by using proteinase K digestion followed by a salting out procedure.
MEH genotyping by PCR and restriction fragment analysis
The exon 3 T to C variant in mEH gene, changing tyrosine 113 to histidine, creates an EcoR restriction site (GATATC), which can be exploited for genotyping by PCR and subsequent RFLP analysis[12]. PCR was performed in a 25 µl volume containing 100 ng of DNA template, 2.5 µl of 10 × buffer, 1 U of Taq-DNA-polymerase (BioDev-Tech., Beijing, China), 200 µmol of dNTPs and 200 nmol of sense primer (5’-GATCGATAAGTTCCGTTTCACC-3’) and antisense primer (5’-ATCCTTAGTCTTGAAGTGAGGAT-3’). Initial denaturation at 94 °C for 5 min was followed by 35 cycles at 94 °C for 30 sec, at 56 °C for 30 sec and at 72 °C for 1 min. Subsequently, the PCR products were digested with 10 units of EcoR V (TakaRa Biotechnology Co., Ltd, Dalian, China) overnight at 37 °C and separated on a 3% agarose gel. RFLP bands were visualized through ethidium bromide staining under UV light. Tyr113 wild-type homozygote was characterized by two bands at the position of 140 bp and 22 bp, while His113 homozygotes were identified by a single band (162 bp) and the heterozygotes by three bands (162 bp, 140 bp and 22 bp). For a negative control, each PCR reaction used distilled water instead of DNA in the reaction system. For 10% of the samples, the reaction was repeated once.
Statistical analysis
Statistical analysis was performed using the SPSS10.0 software package (SPSS Company, Chicago, Illinois, USA). Comparison of mEH genotype distribution in the study groups was performed by means of two-sided contingency tables using Chi-square test. A probability level of 5% was considered significant. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model and adjusted by age and sex accordingly.
RESULTS
The mean age of all ESCC cases was 58.5 ± 9.39 years (range 32-85) and that of controls was 49.4 ± 8.56 years (range 29-79 years). The gender distribution in ESCC patients (66.5% men) was comparable to that in healthy controls (58.3% men). Moreover, the proportion of smokers in ESCC patients (52.0%) was also not significantly different from that in healthy controls (49.5%) ( χ2 = 0.283, P = 0.595). In addition, the frequency of the positive family history of UGIC in ESCC patients (44.1%) was significantly higher than that in healthy controls (14.0%) ( χ2 = 49.87, P < 0.0001). Thus, the positive family history of UGIC significantly increased the relative risk to develop ESCC in this population, with an age and sex adjusted odds ratio of 4.06 (95%CI = 2.46-6.69). The demographic distribution of ESCC patients and healthy controls is shown in Table 1.
Table 1 Demographic characteristics and mEH Tyr113His polymorphism in ESCC patients and healthy individuals.
Groups
Control n (%)
ESCC n (%)
Sex
Male
147 (58.3)
171 (66.5)
Female
105 (41.7)
86 (33.5)
Age (mean ± SD)
49.4 ± 8.56
58.5 ± 9.39
Smoking statusa
Ex-or current smoker
110 (49.5)
130 (52.0)
Non-smoker
112 (50.5)
120 (48.0)
Family history of UGICb
Positive
31 (14.0)
104 (44.1)
Negative
191 (86.0)
132 (55.9)
Genotype
Tyr/Tyr
76 (30.2)
49 (37.1)
Tyr/His
71 (28.2)
32 (24.2)
His/His
105 (41.6)
51 (38.7)
Allele type
T
223 (44.2)
226 (44.0)
C
281 (55.8)
288 (56.0)
MEH Tyr113His genotyping was successfully performed in all study subjects. The mEH genotype distribution was not correlated with gender, age and smoking status both in ESCC patients and in healthy controls (data not shown). The Tyr and His allele frequencies were 44.0%, 56.0% in ESCC patients and 44.2%, 55.8% in healthy controls, respectively. There was no statistic difference in allele distribution between ESCC patients and controls ( χ2 = 0.008, P = 0.929). The frequencies of Tyr/Tyr, Tyr/His and His/His genotype were 30.2%, 28.2% and 41.6% in healthy controls, respectively. The overall mEH genotype distribution in ESCC patients was not significantly different from that in healthy controls ( χ2 =2.116, P = 0.347) (Table 1).
By using Tyr/Tyr as the reference genotype, neither His/ His genotype alone nor in combination with Tyr/His genotype significantly modified the risk of ESCC, the adjusted odds ratio was 1.076 (95%CI = 0.850-1.361) and 0.756 (95%CI = 0.493-1.157), respectively. When stratified for sex, age, smoking status and family history of upper gastrointestinal cancer, the frequency of His/His and Tyr/His genotype in ESCC patients was not significantly different from healthy controls. Consistently, His/His alone, or in combination with Tyr/His genotype, did not show any significant influence on the risk of ESCC (Table 2).
Table 2 Influence of mEH Tyr113His polymorphism on ESCC development.
Tyr/Tyr
Tyr/His + His/His
His/His
aOR(95%CI)c
aOR (95%CI)d
Overall
Normal
76 (30.2)
176 (69.8)
105 (41.7)
ESCC
84 (32.7)
173 (67.3)
115 (44.7)
0.756 (0.493~1.157)
1.076 (0.850~1.361)
Male
Normal
44 (30.6)
100 (69.4)
60 (41.7)
ESCC
61 (35.7)
110 (64.3)
73 (42.7)
0.724 (0.427~1.225)
1.087 (0.811~1.458)
Female
Normal
32 (29.6)
76 (70.4)
45 (41.7)
ESCC
23 (26.8)
63 (73.2)
42 (48.8)
0.825 (0.398~1.710)
1.047 (0.704~1.558)
Age ≤ 50
Normal
46 (32.6)
95 (67.4)
60 (42.6)
ESCC
19 (33.9)
37 (66.1)
22 (39.3)
0.867 (0.450~1.671)
1.064 (0.738~1.534)
Age ＞ 50
Normal
30 (27.0)
81 (73.0)
45 (40.6)
ESCC
65 (32.3)
136 (67.7)
93 (46.3)
0.790 (0.472~1.323)
1.018 (0.768~1.348)
Nonsmokera
Normal
31 (27.7)
81 (72.3)
49 (43.7)
ESCC
34 (28.3)
86 (71.7)
60 (50.0)
0.659 (0.338~1.286)
1.135 (0.790~1.631)
Smoker
Normal
35 (31.8)
75 (68.2)
41 (37.3)
ESCC
46 (35.4)
84 (64.6)
52 (40.0)
0.901 (0.494~1.644)
1.022 (0.728~1.433)
Negative family historyb
Normal
59 (30.9)
132 (69.1)
80 (41.9)
ESCC
49 (37.1)
83 (62.9)
51 (38.7)
0.660 (0.385~1.134)
1.237 (0.912~1.678)
Positive family history
Normal
7 (22.6)
24 (77.4)
10 (32.2)
ESCC
29 (27.9)
75 (72.1)
52 (50.0)
0.638 (0.241~1.689)
0.946 (0.546~1.639)
DISCUSSION
Chemical carcinogens in consumed alcohol and tobacco, polluted water, ingested food, are in general considered as the main risk factors of ESCC in China. However, not all individuals exposed to the above exogenous risk factors will develop ESCC, indicating that the host susceptibility factors may play an important role in cancer development. In recent years, many polymorphic carcinogen metabolic enzymes, such as aldehyde dehydrogenase-2 (ALDH2)[13], cytochrome P450(CYP)[14,15], glutathione S-transferase(GST)[15,16], methylenetetrahydrofolate reductase (MTHFR)[17], NAD(P)H, quinone oxidoreductase 1 (NQO1)[18,19] have been found to be able to modify the susceptibility to chemically induced cancers including esophageal and gastric cancer. Therefore, these polymorphic genes, alone or in combination with each other or with other newly developed genetic markers, may be used as predicative parameters for screening individuals at a high risk of ESCC.
MEH is involved in the metabolism of environmental carcinogens. Polymorphisms in mEH gene might affect the enzyme expression and lead to different phenotypes, probably by the alteration of protein stability[6]. Tyr113His substitution in exon 3 could reduce the enzyme expression by about 40%, producing a slow phenotype with a low epoxide hydrolase activity. In contrast, Arg139 His in exon 4 could increase the expression by about 25%, producing a fast phenotype with an increased enzyme activity[6]. The relationship between mEH gene polymorphisms and susceptibility to cancers studied had inconsistent conclusions due to different cancer types and populations. The Tyr allele in exon 3 was reported to increase the risk of several cancer types including ovarian cancer[9], oropharyngeal carcer[20] and acute leukaemia[21], whereas the His allele was associated with increased susceptibility to cancers occurring in colon[10], liver[11] and cervix[22]. In addition, gene-environment interaction was strongly suggested by some investigations, thus, cumulative cigarette smoking might play a pivotal role in association of His homozygous genotype with lung cancer development, altering the direction of risk from a risk factor in nonsmokers to a relatively protective factor in heavy smokers[8].
Recently, a slight decrease in mEH Tyr113 frequency was observed in esophageal adenocarcinoma (42%) compared to controls (53%, P = 0.05)[23]. In the present study, the frequencies of Tyr/Tyr, Tyr/His and His/His genotype in healthy controls were in consistent with a recent report from a Chinese group[24]. The genotype distribution difference was not found in ESCC patients and healthy controls, as well as in stratification comparison according to the sex, age ( > 50 or ≤ 50), smoking status (never smoking or current and ever smoking), and family history of UGIC. The result suggests that although mEH Tyr113His polymorphism is correlated with some cancer types, this genetic alteration may not be associated with susceptibility to ESCC in population of North China.
ACKNOWLEDGEMENTS
We greatly acknowledge Mr. Baoshan Zhao and Mr. Fanshu Meng for their assistance in recruiting study subjects.
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