CYP450 2E1,1A1 Genetic Polymorphisms And Risk Of Colorectal Cancer

【Background】Colorectal Cancer(CRC) is the third common malignant tumor in the world. In 2002, there were over 370, 000 new cases of CRC in Europe, about 12ï¼…in all the tumor, and 200, 000 people died of it. In USA, there were about 155, 000 new cases of CRC and more than 51, 000 people died of it. The incidence of CRC is 10 times in developed country compared to developing country. In China, at present the CRC is the No.5th tumor, however it increased rapidly, especially in metropolis. The incidence of CRC increased 31.95ï¼…in city, and 8.51ï¼…in countryside in last century 90th compared to 70th. CRC accounted for about 140, 000 new cases every year(6.8ï¼…of the tumor total). The mortality of CRC was about 3.54 per 100, 000, occupied 5.29ï¼…and ranked 6th in all tumor mortality.The incidence of CRC is a scribed to multiple factors and stages. Worldwide, the incidence rates vary approximately 25-fold, which could be explained by the environmental factors especially diet factors and the genetic susceptibility. The response of individuals to the environment mutagen may be not completely alike. Hereditary susceptibility of tumor takes an important role in the incidence of CRC. Cytochrome P450s(CYPs) mainly have CYP1,CYP2,CYP3 three gene families, are involved in biological transformation enzymy system of exogenous (hormone,fatty acid) and endogenous(drugs,precarcinogen) compounds in the human, and are closely related to the tumor development. CYP2E1, a member of the cytochrome p-450 superfamily, can induced by alcohol, is involved in the metabolic activation of many precarcinogen compounds such as N-nitrosamines, aniline, vinyl chloride, and urethane. CYP2E1 catalytic activity display individual differences because of protein coding gene is polymorphic. There are many reports about the functional CYP2E1Rsaâ… gene polymorphism might therefore impact on susceptibility for cancer such as esophageal, gastric, lung, and liver cancer. Most of research consider that the polymorphism are related to the susceptibility of tumors, which correlated to the habits of smoking and alcohol drinking, but still have contradictory results in different race,different group.CYP1A1, from the cytochrome p-450 superfamily, is involved in the metabolic activation of many environment precarcinogens/mutagens such as polycyclic aromatic hydrocarbon(PAH), benzo-pyrene(BAP),dimethyl-benzanthracene(DMBA), recently many investigations report it heredity polymorphism also relate to the susceptibility of the lung,liver tumor. Because of heredity polymorphism variation in different race and country(region), The relationship between the genotypes of CYP 1A1 and CRC in Western is discrepancy, There was no such study report based on population in China so far.【Objective and Significance】Because there was no effective method in curing colorectal cancer, it is important to carry out the primary prevention focused to the environmental factors from further study on the risk factors of colorectal cancer, and the secondary prevention aiming directly at the pre-cancer lesions. This study is to reveal the relationship between the genetic polymorphism of CYP2E1 and CYP1A1,smoking,alcohol drinking and the susceptibility of CRC and to offer the basic epidemiological data for further study on the mechanism of CRC occurrence. Finally, this study was set up to explore the relationships of smoking and alcohol drinking environmental exposure factors of colorectal cancer and CYP2E1 and CYP1A1 genotypes by the comparison between case and control groups, which would initially reveal the episode mechanism of CRC. In addition, the distribution of CYP2E1 Rsaâ… and CYP1A1 Mspâ… genotypes in normal population would be known in this study, since control group was a present sample of the normal population in Jiangsu.【Methods】We recruited colorectal cancer cases using data of Cancer Registries in Huian and Jintan Cities of Jiangsu Province of China, and also recruited cases who visited Jiangsu Province Cancer Hospital from these cities from Aug. 2000 to Sept. 2002. All were histopathologically diagnosed as having a primary colorectal cancer. Physicians at the hospital asked eligible cases to participate in our study, and doctors or nurses interviewed the subjects and collected blood samples from a peripheral vein after obtaining informed consent. Population-based controls were selected from healthy residents in eight villages or towns of Huian and Jintan Cities. Doctors of the public health center randomly selected one or two controls for each case, after matching for ethnicity, sex and age within 2 years using the records of residents at the local governmental office, and then asked eligible residents for their participation. Interviews and blood collection were performed as for the cancer cases. The items of our questionnaire covered smoking and drinking habits. Smokers were divided into never-and ever-smokers (current and former). Drinkers also were divided into two groups(≥2 times/month and＜2 times/month) according to drinking frequency.Whole blood was collected into EDTA-coated tubes and centrifuged for 15 min, and the buffy coat layer was isolated. Genomic DNA was extracted from 200μl of huffy coat using a Qiagen QIAamp DNA Blood Mini Kit(QIAGEN Inc., Valencia, CA). The laboratory assays were organized two parts. Part One: To explore the relationship between the genetic polymorphism of CYP2E1 Rsaâ… , smoking, alcohol drinking and the susceptibility of colorectal cancer. PCR was used to amplify the transcription regulation region of CYP2E1 that includes the Rsaâ… enzyme recognition site. The up-stream primer: 5′-TTCATTCTGTCTTCTAACTGG-3' and down-stream primer: 5′-CCAGTCGAGTCTACATTGTCA-3′were used to amplify the genomic CYP2E1 gene of participants; And the restriction enzyme of Rsaâ… were applied to identify the CYP2E1 polymorphisms. The authenticity of this assay was confirmed by DNA sequencing, and using 2ï¼…agarose gel electrophoresis(AGE) to detect the genetype of CYP2E1 Rsaâ… polymorphism. Part Two: To reveal the relationship between the genetic polymorphism of CYP1A1, smoking, alcohol drinking and the susceptibility of CRC. The up-stream primer: 5′-CAGTGAAGAGGTGTAGCCGCT-3′and down-stream primer: 5′TAGGAGTCTTGTCTCATGCCT-3′were used to amplify the genomic CYP1A1 gene of participants; And the restriction enzyme of Mspâ… were applied to identify the CYP1A1 polymorphisms. Using 2ï¼…AGE to detect the genetype of CYP1A1 Mspâ… polymorphism. The statistical analysis used unconditional logistic regression to adjust odds ratios(ORs) for the matching variables(age, sex), as well as for the variables found to be associated with risk. Theχ² test was used to compare the observed genotype distributions with those expected by the Hardy-Weinberg equilibrium. All analyses were performed with SAS and Excel 2000. All statistical tests are two-sided.【Results】1. Smoking,alcohol drinking habit and colorectal cancerMultiplicity display that smoking was not associated with risk of CRC. OR=1.01(95ï¼…CI: 0.69～1.48, P=0.9579). Individuals which drunk alcohol were at a significantly increased risk of CRC(OR=1.94, 95ï¼…CI: 1.33～2.84).2 CYP2E1,CYP1A1 gene polymorphism and CRC2.1 Relationship between CYP2E1 Rsaâ… gene polymorphism and CRC: The frequencies of the CYP2E1 Rsaâ… c1/c1,c1/c2 and c2/c2 genotypes were 59.1ï¼…,33.9ï¼…and 7.0ï¼…in colorectal cancer cases, and 61.4ï¼…,35.6ï¼…and 3.0ï¼…in controls, the genetype frequency distribution of two groups have significant variance (χ²_MH=6.58, P=0.037); The frequencies of the CYP2E1 Rsaâ… c1/c1,c1/c2 and c2/c2 genotypes were 60.6ï¼…,33.7ï¼…and 5.8ï¼…in colon cancer cases, compared with the control, the genetype frequency distribution without significant variance (χ²_MH=1.91, P=0.385); and the frequencies of the CYP2E1 Rsaâ… c1/c1, c1/c2 and c2/c2 genotypes were 58.4ï¼…, 34.0ï¼…and 7.7ï¼…in rectal cancer cases, compared with the control, it have notable difference(χ²=7.07, df=2, P=0.029). As compared with individuals who had CYP2E1 c1/c1 genotype, individuals who had CYP2E1 c2/c2 genotype was at an increased risk of developing CRC(OR=1.64, 95ï¼…CI: 1.12～2.41).2.2 Relationship between CYP1A1 Mspâ… gene polymorphism and CRC: The frequencies of the CYP1A1 A (m1m1),B(m1m2) and C(m2m2) genotypes were 45.19ï¼…, 43.91ï¼…and 10.9ï¼…in colorectal cancer cases, and 41.38ï¼…, 45.75ï¼…and 12.87ï¼…in controls, Two groups have no significance(χ²=1.338, P=0.512). As compared with individuals who had CYP1A1 A genotype, individuals who had CYP1A1 C genotype had no significance of risk of developing CRC(OR=0.85, 95ï¼…CI: 0.66～1.08); Further more in colon caner and rectal cancer, it also had no significance (OR=0.87(95ï¼…CI: 0.60～1.26), OR=0.84(95ï¼…CI: 0.63～1.10, respectively).3 Interaction between smoking,alcohol drinking and CYP2E1 gene3.1 Interaction between smoking, alcohol drinking and CYP2E1 gene Rsaâ… polymorphism: Among the smokers, as compared with individuals who had c1 genetype, individuals who had c2/c2 genotype had no significance of developing CRC(sex-, age-adjusted OR==1.41(95ï¼…CI: 0.50～3.96)); Among the alcohol drinkers, compared with those with CYP2E1 Rsaâ… c1/c1 genotypes, individuals with CYP2E1 Rsaâ… c2/c2 genotypes were at an increased risk of colorectal cancer(OR=5.42(95ï¼…CI: 1.65～17.40)), and OR=4.74(95ï¼…CI: 1.10～20.40)in colon cancer, OR=5.75(95ï¼…CI: 1.65～20.05) in rectal cancer, respectively. There is a significantly cooperated effect between CYP2E1 Rsaâ… polymorphism and alcohol drinking habit in developing colorectal cancer.【Conclusions】There are some conclusions gained from this study as follows: Smoking had no significant association with risk of CRC, whereas alcohol drinking was associated with increased risk of CRC. Polymorphisms of the CYP2E1 Rsaâ… influence susceptibility of individuals to colorectal cancer and have significantly synergic effect combined with alcohol drinking in the developing rectal cancer. No significant association was found between CYP1A1 Mspâ… gene polymorphism and CRC. There search of genetic polymorphisms improved the deeper of environment-individual susceptibility interaction in molecular machine and explored the risk factors of CRC form smoking and alcohol drinking and genetic polymorphisms of CYP2E1 and CYP1A1.