Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle, Fujian Province

Abstract

AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2E1 polymorphisms are associated with risks of gastric cancer.

METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including demographic characteristcs, diet intake, and alcohol and tobacco consumption of indivduals in our study were completed by a standardized questionnaire. PCR-RFLP revealed three genotypes:heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1.

RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 indivduals in gastric cancer group (6.6%), whereas there was only one in the control group (1.1%). However, there was no statistically significan difference between the two groups (two-tailed Fisher’s exact test, P = 0.066). Indivduals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR = 1.50) and C2/C2 (OR = 7.34) than indivduals in control group (χ² = 4.597, for trend P = 0.032). The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele (C1/C1 genotype) among indivduals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that indivduals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer.

CONCLUSION: Polymorphism of CYP2E1 gene may have some effct in the development of gastric cancer in Changle county, Fujian Province.

Key Words: gastric neoplasm/genetics; gastric neoplasm/etiology; Cytochrome P-450 2E1; CYP2E1/genetics; genotype; human; FUJIAN

INTRODUCTION

Fujian Province is one of the highest risk areas of gastric cancer in China [1,2]. Gastric cancer is the major cause of cancer mortality in Changle County, Fujian Province[3-5]. Epidemio logical studies have shown that an increased risk of developing gastric cancer is associated with diet and tobacco smoking[6-8]. One particular hypothesis, which has been paid more attention, is that N-nitroso compounds from dietary sources are involved in carcinogenesis of gastric cancer[9-10]. Recent advances in cancer research have revealed that the main etiology of human cancers are genetic changes in cancer related genes caused by carcinogens in the environment[11-14]. It is known that most of exogenous (xenobiotics) and endogenous chemical carcinogens require biotransformation to activated forms to be carcinogenic[15,16]. Most of the human metabolizing enzymes are genetically polymorphic, and those polymorphisms may affect the enzyme activity or inducibility[17-20]. The sensibitity to procarcinogen differs among individuals, which may have substantial importance in carcinogenesis[21-24]. Cytochromes P450 (CYPs) play an important role on metablism of several aspects of cancer. Cytochrome P450 2E1 (CYP2E1) is primarily responsible for the bioactivation of many low molecular weight carcinogens, and is involved in the metabolic oxidation of carcinogenic nitroso compounds, including N-nitrosoamines[25-26]. This study was designed to in vestigate the relationship between polymorphism of CYP 2E1 and gastric cancer.

MATERIALS AND METHODS

Subjects

All indivduals in our study are the residents of Changle county, Fujian Province of China. Ninety-one patients with pathologic diagnosis as primary gastric cancer between January 1996 to March 1998 and 94 age-and sex matched healthy controls were included in this study. Each indivdual was personally interviewed to obtain information on demographic characteristics, habits of cigarette smoking, alcohol drinking, and dietary consumption frequency. Blood specimens from them were also obtained.

DNA isolation and PCR-RFLP analysis

Genomic DNA was isolated from white blood cells by extraction using phenol/chloroform and precipitation using ethanol. PCR was performed using the primers 5’-CCAGTCGAGTCTACATTGTCA-3’ (1370-1349) and 5’-TTCATTCTGTCTTCTAACTGG-3’ (999-978). The amplification reaction was conducted in a 50 (L solution containing PCR buffer (1.5 mmol•L¯¹ MgCl₂, 50 mmol•L¯¹ KCl, 10 mmol·L ^-1 Tris-HCl, pH8.3), 200 μmol•L¯¹ dNTP, 1 μmol•L¯¹ primer, 200 ng template DNA, and 2.5 μTaq DNA polymerase (Promega Corp). Those reactions were performed about 35 cycles at conditions following as denaturation for 1 min at 95 °C, annealing for 1 min at 55 °C, extension for 1 min at 72 °C and a final extension for 10 min at 72 °C. The PCR products were digested with PstI or RsaI (Fermantas-MBI. Vilnius, Lithuania) for 18 h at 37 °C. The restriction sites was identified by 2.2% agarose gel electrophoresis. The genotypes of CYP2E1 were classified as following: a predominant homozygote (C1/C1), a heterozygote (C1/C2) and a rare homozygote (C2/C2). Figure 1, Figure 2.

Figure 1 The PCR products of CYP2E1 gene. M: markers.

Figure 2 PCR-RFLP with Pst1. Lanes 2, 12, 14: C1/C1; Lanes 4, 6, 10: C1/C2; Lane 8: C2/C2; Lanes 1, 3, 5, 7, 9, 11, 13: Sample controls.

Statistical analysis

Fisher’s exact tests were determined by standard methods. The relationships between CYP 2E1 genotypes and putative risk factors were measured using the odds ratios (ORs) and their 95% confidence intervals (95%CIs).

RESULTS

The age (mean ± SD) of indivduals in gastric cancer group and control group were 58.4 ± 11 years and 58.2 ± 11 years, respectively. The distributions of age and sex were similar in cases and controls. The risks of gastric cancer were related to smoking, alcohol drinking, and fish sauce intake (a condiment commonly used by local residents), Table 1.

Table 1 Distribution of selected variables in gastric cancer group and group.

	Gastric cancer (n = 91)	Control (n = 94)	P value
	n (%)	n (%)
Age/y
< 50	21 (23.1)	22 (23.4)
50-59	22 (24.2)	22 (23.4)
60-69	32 (35.2)	34 (36.2)
70+	16 (17.6)	16 (17.0)	0.9983
mean ± SD	58.4 ± 10.9	58.2 ± 11.0
Range	32-78	34-79
Gender
Male	77 (84.62)	82 (87.23)
Female	14 (15.38)	12 (12.77)	0.6094
Education
College	1 (1.1)	1 (1.1)
High school	15 (16.5)	63 (67.0)
Elementary school	57 (62.6)	22 (23.4)
Illiterate	18 (19.8)	8 (8.5)	0.0000
Smoking/y
0	31 (34.1)	60 (63.8)
< 10	2 (2.2)	16 (17.0)
10-	16 (17.6)	7 (7.5)
20-	22 (24.2)	4 (4.3)
30-	20 (22.0)	7 (7.5)	0.0000
Alcohol drinking/y
0	40 (44.0)	66 (70.2)
< 10	3 (3.3)	13 (13.8)
10-	14 (15.4)	8 (8.5)
20-	34 (37.4)	7 (7.5)	0.0000
Fish sauce intake
Low (< 3 times/w)	17 (18.68)	69 (73.40)
High (≥ 3 times/w)	76 (81.32)	25 (26.60)	0.0000

The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer group and control group was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 of gastric cancer group (6.6%), but in 1 of the controls (1.1%). However, the result showed no statistical difference. Fisher’s exact test P = 0.066 (T able 2). The allele frequencies of CYP2E1 fit with Hardy-Weinberg equilibrium (χ² = 0.242, P > 0.05) (Table 2).

Table 2 Cytochrome P450 2E1 genotype and risks of gastric cancer.

CYP2E1	Gastric cancer		Control (n = 94)		OR (95%CI)
	n	(%)	n	(%)
C1/C1	58	(63.7)	71	(75.5)	1.00
C1/C2	27	(29.7)	22	(23.4)	1.50 (0.74-3.07)
C2/C2	6	(6.6)	1	(1.1)	7.34 (0. 84-166.60)

Fisher’s test P = 0.066 vs controls χ² trend = 4.597 P = 0.032 vs controls Gastric cancer indivduals were more likely to carry genotype C1/C2 and C2/C2 than indivduals in control group. Individuals carried at least one C2 allele (genotypes C1/C2 or C2/C2) seemed to have an increased risk of gastric cancer (OR = 1.86, 95%CI 1.07～3.25), Table 3.

Table 3 The allele frequencies of CYP2E1 in gastric cancer group and control group.

	C1		C2
	n	Frequencies	n	Frequencies
Gastric cancer	143	0.7857	39	0.21 43
Controls	164	0.8723	24	0.1276

χ² = 4.91 P < 0.05 vs controls; OR = 1.86 95%CI 1.07～3.25.

The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2) were compared with those of genotypes without C2 allele (C1/C1) among indivduals in gastric cancer and control group according to the pattern of gastric cancer risk factors of smoking, alcohol drinking and fish sauce intake. Indivduals who have been exposed to those risk factors of gastric cancer and carried the C2 allele seemed to have a higher risk of developing gastric cancer (Table 4).

Table 4 Relationships between CYP2E1 together with selected variables and risk of gastric cancer.

CYP2E1	Variables Smoking	Gastric cancer	Controls	OR (95%CI)
C1/C1	No	21	48	1.00
C1/C1	Yes	37	23	3.68 (1.67-8.19)
C1/C2 or C2/C2	No	10	12	1.90 (0.64-5.69)
C1/C2 or C2/C2	Yes	23	1 1	4.78 (1.82-12.78)
Alcohol drinking
C1/C1	No	26	51	1.00
C1/C1	Yes	32	20	3.14 (1.42-6.99)
C1/C2 or C2/C2	No	14	15	1.83 (0.70- 4.77)
C1/C2 or C2/C2	Yes	19	8	4.66 (1.65-13.53)
Fish sauce intakea
C1/C1	Low	11	53	1.00
C1/C1	High	47	18	12.58 (5.40-29.34)
C1/C2 or C2/C2	Low	6	16	1.81 (0.58-5.66)
C1/C2 or C2/C2	High	27	7	18.58 (6.47-53.37)

^aHigh ≥ 3 times/w; Low < 3 times/w

DISCUSSION

Epidemiological studies have shown that up to 90% of all cancers are related to environmental factors. Most of the environmental carcinogens need to be metabolically activated to exert their carcinogenic effects[27-30]. Genetic polymorphisms in enzymes involved in carcinogen metabolism has shown to influence the susceptibility to cancer[31-33]. Cytochrome P4502E1 (CYP2E1) plays an important role in this process. It participates in the metabolic activation of carcinogenic nitrosamines. Several recent studies show that the genetic polymorphisms of metabolizing enzymes are associated with some cancers such as lung cancer[34-36], esophageal cancer[37-40] and colorectal cancer[41]. But the results of those studies of the relation between CYP2E1 and cancer susceptibility are inconsistent[42].

The possibility that N-nitrosated compounds are involved in gastric cancer has been an issue for many years. Nitrosamines occur in tobacco smoke and in some kinds of food, which are also formed endogenously in the stomach. In this study, fish sauce intake, cigarette smoke, and alcohol drinking were positively associated with gastric cancer. Fish sauce is a condiment that is particularly favored by the local residents in Fujian Province[43-45]. Many N-nitro compound precursors have been found in fish sauce [46,47]. Tobacco smoke contains many potential carcinogens, also including nitroso compounds[48]. CYP2E1 are known to vary and are induced by ethanol consumption. Several studies reported that the variant C2 was associated with enhanced enzyme activity. Hayashi et al[49] reported that enhanced activity of C2/C2 DNA was about 1 0 times than that of C1/C1 DNA. This difference in the transcriptional activities might associate with the susceptibility in human carcinogenesis. There is evidence suggesting that there may be a gene-environment interaction in the development of cancer so that cancer risk associated with a given exposure is modified by the genotype of the host[50]. In this study, a much higher risk was observed from those who exposed to the risk factors of gastric cancer and carried the C1/C2 or C2/C2 genotypes. The results suggest that polymorphic genes that code for tobacco carcinogen and alcohol metabolizing enzymes may play a role in susceptibility of gastric cancer. The intervention against cigarette smoking, alcohol drinking, bad eating habits may be important for the prevention of gastric cancer in high-incidence areas.

Because of the limited number of samples in this study, determination of precise dose-response relations with respect to a sufficient number of dose level for each of genotype groups may not be able to be con ducted. Further studies with a larger number of samples are needed to confirm the role of genetic polymorphism of human CYP2E1 in gastric cacinogenesis.