CYP 1 A 1 Genetic Polymorphisms and Risk for Esophageal Cancer : a Case-control Study in Central China

Esophageal cancer (EC) is one of the most common malignant diseases worldwide and the sixth leading cause of cancer death, with the majority of cases occurring in developing countries (Parkin et al., 2005). Research showed that risks for EC in different countries or different places were various (Zhuo et al., 1999; Lu et al., 2000; Zhang et al., 2000; Li et al., 2001). China, with about 250,000 cases diagnosed yearly, lies in the “esophageal cancer belt” (Parkin et al., 1988), and contributes to about half of the world’s cases (Yang et al., 2003). The ratio in incidence between highand low-risk areas could be as great as 500:1 (Xing et al., 2003). The high incidence in special areas suggests that the importance of environmental factors in esophageal cancer is developing (Mao et al., 2011). However, only a small part of individuals can develop esophageal cancer under the similar environmental conditions in the highrisk areas, indicating that host susceptibility factors such as the polymorphisms of phase I metabolism enzyme gene CYP1A1, may be risk factors in increasing risk for esophageal cancer.


Introduction
Esophageal cancer (EC) is one of the most common malignant diseases worldwide and the sixth leading cause of cancer death, with the majority of cases occurring in developing countries (Parkin et al., 2005).Research showed that risks for EC in different countries or different places were various (Zhuo et al., 1999;Lu et al., 2000;Zhang et al., 2000;Li et al., 2001).China, with about 250,000 cases diagnosed yearly, lies in the "esophageal cancer belt" (Parkin et al., 1988), and contributes to about half of the world's cases (Yang et al., 2003).The ratio in incidence between high-and low-risk areas could be as great as 500:1 (Xing et al., 2003).The high incidence in special areas suggests that the importance of environmental factors in esophageal cancer is developing (Mao et al., 2011).However, only a small part of individuals can develop esophageal cancer under the similar environmental conditions in the highrisk areas, indicating that host susceptibility factors such as the polymorphisms of phase I metabolism enzyme gene CYP1A1, may be risk factors in increasing risk for esophageal cancer.

CYP1A1 Genetic Polymorphisms and Risk for Esophageal Cancer: a Case-control Study in Central China
Yu-Xia Yun 1,3& , Yan-Ping Wang 1,4& , Peng Wang 1,2,3 , Li-Hong Cui 6 , Kai-Juan Wang 1,2,3 , Jian-Ying Zhang 1,2,3 , Li-Ping Dai 1,2,3 * Esophageal cancer is a complex disease likely resulting from multiple interacting genetic polymorphisms and gene-environment interactions.Most of environmental carcinogens are pre-carcinogens which have carcinogenicity after activated by phase I enzymes.The CYP1A1 gene is closely associated with the metabolism of polycyclic aromatic hydrocarbons (PAHs) carcinogens, which code in the aryl hydrocarbon hydroxyla (AHH) enzyme (Kopf et al., 2010).The enzyme CYP1A1 is involved in the activation of major classes of tobacco procarcinogens, such as polyaromatic hydrocarbons and aromatic amines, and is present in many epithelial tissues (Bartsch et al., 2000).Evidence suggests (Hiyama et al., 2007) that genetic polymorphisms of CYP1A1 may influence the balance between metabolic activation and detoxification of toxicants and thus are related to individual susceptibility to esophageal cancer.
Recently, many investigators have reported association between CYP1A1 polymorphisms and cancers (Dai et al., 2009;Atinkaya et al., 2012;Lopez-Cima et al., 2012;Sergentanis et al., 2012;Ding et al., 2013), especially for two main functional polymorphic sites of CYP1A1 gene (MspI and Ile/Val) and cancer susceptibility.Previous studies (Kawajiri et al., 1990;Nakachi et al., 1991;Hayashi et al., 1992;Xu et al., 1996) have suggested that variant allele of CYP1A1 MspI polymorphisms is associated with malignancies, particularly lung cancer.No association was identified between CYP1A1 MspI and Ile/ Val polymorphisms with esophageal cancer risk in a series of studies done on populations of Caucasians and Japanese (Lucas et al., 1996;Hori et al., 1997;Morita et al., 1997;van Lieshout et al., 1999).However, Nimura et al. (1997) reported that heavy smokers with Val/Val genotype of CYP1A1 Ile/Val had a three-fold risk of developing esophageal cancer as compared to those with Ile/Ile genotype in a case-control study in Chinese population.Roth et al. (2000) carried out a study in Linxian, a region of high esophageal cancer risk in China, and did not find any significant effect between CYP1A1 Ile/Val polymorphism and esophageal cancer.A recent study by Wang et al. (2002a) found that individuals with the CYP1A1 Val/ Val genotype had a higher risk of developing esophageal cancer than those with Ile/Ile (OR:2.48,95%CI:1.12-5.54) in 127 esophageal cancer cases and 101 controls.
Thus, the present study was undertaken to assess the association of genetic polymorphisms of CYP1A1 with esophageal cancer susceptibility in Henan province, the highest incidence area of EC in China.

Study population
We recruited 157 patients with pathologically proven esophageal squamous cell carcinoma before undergoing any treatment at the First Affiliated Hospital of Zhengzhou University, during March 2008 to September 2008.157 healthy population controls with 1:1 matching by age (±5 years) and sex were selected from a census of digestion diseases carried out in Xinxiang County and Xin'an County of Henan Province.All cases were newly diagnosed and primary esophageal squamous cell carcinoma patients.Controls were required to be free of any digestion diseases, having no cancer history and related clinical signs.All of the subjects were unrelated individuals.

Data collection
Uniform trained investigators using a special questionnaire interviewed cases and controls by face to face.The special questionnaire contained information of age, sex, tobacco smoking, alcohol drinking, family history of cancer, etc.The venous blood obtained from the subjects was collected in an EDTA tube and stored at -80℃ for extraction of DNA genome.Tobacco smoking was defined as smoking at least one cigarette per day and persisting for more than one year.Alcohol intake was defined as drinking at least once a week with more than 100 gram every time and persisting for more than six months.We used the medians (18 pack-year) of lifetime consumption of tobacco of control cigarette smokers in distinguishing the moderate and medium heavy smokers.
The study subjects were given the written informed consent before participating in the study.

Genotyping analysis
Genomic DNA was extracted from the blood specimen using the phenol chloroform method for genotyping.All genotyping analyses were PCR-based, with a total volume of 20 μl for each reaction containing 10 μl 2×Tap PCR MasterMix, 0.8 μM each primer, 100 ng DNA, 7.4 μl deionized water.Digestive products were electrophoresed on 3% agarose gel, and photographed.And all assays were repeated at least one time by the same individual to verifying the genotyping results.Genotypes were validated by sequencing through biological technology company.
The PCR products were digested by restriction endonuclease at 37℃ overnight.The digested products    2).

Statistical analysis
χ 2 test was used to detect whether there were significant (α = 0.05) differences in frequencies between cases and controls.
Odds ratios (OR) and 95% confidence intervals (95%CI) from logistic regression models were used for detecting the associations between these two polymorphisms and EC risk.Each analysis was also adjusted for confounding factors (age, gender, smoking, drinking and family history of cancer).
Haplotypes for each individual were inferred using the SNPHAP2.0 software.
All analyses were conducted using SPSS12.0software.All tests were two sided and P values <0.05 was considered statistically significant.

Subject characteristics
Table 1 shows the distribution characteristics of 157 EC patients and 157 controls.The distributions of medium and heavy smoking (P=0.04) and family history of cancer (P=0.00) had significant differences between cases and controls groups.

The relationship between CYP1A1 polymorphisms and esophageal cancer
The frequencies of wild genotype, heterozygous genotype, and homozygous genotype of MspI and Ile/Val polymorphisms among controls were both not departure from Hardy-Weinberg equilibrium (P=0.42 and P=0.14).
For CYP1A1 MspI polymorphism, the distributions of genotypes were not significantly different between the cases and controls groups (χ 2 0.05,2 =5.784, P>0.05).The individuals with T/C genotype had an increased risk for EC, compared with wild genotype T/T (OR: 1.68, 95%CI: 1.04-2.72).But after adjusting for age, gender, smoking, drinking and family history of cancer, there was no significant association between CYP1A1 MspI polymorphism and esophageal cancer.
For CYP1A1 Ile/Val polymorphism, both Ile/Val genotype and Ile/Val+Val/Val combined variant genotype showed increased risk of esophageal cancer (OR: 1.87, 95%CI: 1.17-3.01and OR:1.76, 95%CI:1.12-2.76),compared with wild genotype Ile/Ile.And after adjusting for age, gender, smoking, drinking and family history of cancer, there was also significant association between Ile/ Val genotype, Ile/Val+Val/Val combined variant genotype and esophageal cancer (OR:2.05,95%CI:1. .The distribution of minor allele G had significant difference between the cases and controls groups (OR: 1.43, 95%CI; 1.00-2.04).But no significant difference was observed after adjusting for age, gender, smoking, drinking and family history of cancer.

Haplotype analysis
There were totally four possible haplotypes and the most common haplotype TA containing both major alleles was taken as the reference.As shown in Table 3, the distribution of CG haplotype genotype between the cases and controls reached statically significant difference (P<0.05).But significant difference was null after adjusting for age, gender, smoking, drinking and family history of cancer.

Combined genotypes analysis of MspI and Ile/Val
There were totally nine combination genotypes of MspI and Ile/Val, which were shown in Table 4.The combination genotype TT/AA with wild genotypes of two polymorphisms was taken as referent genotype, and its frequency was 25.5% in cases and 31.2% in controls.The individuals carrying the combined genotype TC/AG including two heterozygosis genotypes showed two-fold increased risk for esophageal cancer (OR:2.12,95%CI:1.16-3.85).And after adjusting for age, gender, smoking, drinking and family history of cancer, there was also significant association between combined genotype TC/AG and esophageal cancer (OR:2.01,95%CI:1.02-4.00).There were no associations between the other seven combined genotypes and esophageal cancer susceptibility.

Discussion
The important phase I enzyme CYP1A1 plays an essential role in the metabolic activation of major classes of procarcinogens such as benzo[a]pyrene, a prototypic polycyclic aromatic hydrocarbon, thus affecting the metabolism of the environmental carcinogens and altering the susceptibility of esophageal cancer.Generally, variation of CYP1A1 gene can alter efficiency of its enzymes which could enhance toxicity of the extraneous stimulating factors that directly influence tissues, thus increasing susceptibility to carcinoma.Two major relevant polymorphic sites of the CYP1A1 gene (Msp1 and Ile/Val) have been suggested to be associated with several types of cancer.The former is located in the 3'-flanking region of the gene (T6235C position) in which the presence of C has been linked with genetic susceptibility to lung cancer.The latter, A4889G, located in the heme-binding region of Ile/Val at codon 462, alters the protein structure by replacing an isoleucine for a valine, making the carriers more susceptible to some cigarette smoking-associated diseases (Wang et al., 2002b).
A case-control study with molecular epidemiology methods was used in the present study to analyze the relationship between CYP1A1 polymorphisms and esophageal cancer risk.Our data showed that the heterozygosis genotype T/C of MspI can increase the risk of esophageal cancer.But associations were null after adjusting for age, gender, smoking, drinking and family history of cancer.This result is consistent with the study of Wu et al. (2002).Guo et al. (2005) found that individuals with the MspI T/C or C/C genotype had a higher risk of developing esophageal cancer than those with the T/T genotype (OR:1.93,95%CI:1.01-3.84).However, the study results of Wang et al. (2003) suggested that the genotype of MspI T/C (OR:0.41,95%CI:0.17-0.99)or (T/C+C/C) (OR:0.41,95%CI:0.17-0.99)might be protective factor for developing esophageal cancer.The heterozygosis genotype T/C of CYP1A1 MspI genetic polymorphism was found to be associated with elevated esophageal cancer risk in the study of van Lieshout et al. (1999).A study of Casson et al. (2003) on Canada population reported no association between the genotype of MspI (T/C+C/C) and risk of esophageal cancer.These different results may be related to sample size, ethnicity and other factors.Therefore, further and large population studies should be carried out to analysis the relationship of CYP1A1 MspI genetic polymorphism and esophageal cancer.
For CYP1A1 Ile/Val polymorphism, most studies of the contribution of CYP1A1 Ile/Val polymorphism to risk of esophageal cancer have provided inconsistent results.The results of our study showed that the Ile/Val (or Ile/Val+Val /Val) genotype can increase the risk of esophageal cancer (OR: 1.87, 95%CI;1.17-3.01)(or OR;1.76, 95%CI;1.12-95%CI;.In contrast, Wang et al. (2003) found cases with CYP1A1 Ile/Val polymorphism had no significant difference of developing esophageal squamous cell carcinoma compared to controls in Northern China.A study of Wang et al. (2002a) (Hayashi et al., 1991).
Tumorigenesis of esophageal cancer is a complex, multistep course that may be multifactorial.The analysis of haplotype and combined genotypes supplied a greater amount of information than a single SNP.According to analysis of haplotype, the CG haplotype was a risk factor of esophageal cancer (OR: 1.54, 95%CI: 1.03-2.31).But significant association was null after adjusting for age, gender, smoking, drinking and family history of cancer.According to analysis of combined genotype of these two polymorphisms, the TC/AG combined genotype, which contains two heterozygosis genotypes for both polymorphisms, can increase the risk to EC (OR: 2.12, 95%CI: 1.16-3.85),even after adjusting for age, gender, smoking, drinking and family history of cancer, there was also significant association between combined genotype TC/AG and esophageal cancer (OR: 2.01, 95%CI: 1.02-4.00).This approach can provide a theoretical basis for the etiology of EC.
Two polymorphisms of CYP1A1 MspI and CYP1A1 Ile/Val have been demonstrated in the CYP1A1 gene: One is a T to C substitution in the 3' flanking region altering protein folding, the other one is an Ile to Val substitution may occur in the heme-binding region of Ile/Val.Both substitutions were considered to result in the enhancement of enzyme activity (Landi et al., 1994), but polymorphisms in the noncoding region of CYP1A1 were unlikely to have direct functional consequences on CYP1A1 activity (Bailey et al., 1998), even the variant of CYP1A1 Ile/ Val was not sure to induce an increased enzyme activity (Zhang et al., 1996).These controversial reports suggested that the effect of CYP1A1 polymorphisms on developing cancer remains to be test and verify.
In conclusion, the current study suggests that the polymorphic metabolic enzymes genes, CYP1A1, may be associated with the risk of esophageal cancer.Although the number of our study was sufficient to reach an adequate statistical power, our results need to be confirmed further by a larger series of study.Future epidemiologic studies should also consider interactions between genetic polymorphisms and exposure to environmental carcinogens to make the tests results more objective and credible.

Table 4 . Combination Analysis of MspI and Ile/Val genotypes
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.11.6507CYP1A1Genetic Polymorphisms and Risk of Esophageal Cancer: a Case-controlStudy in Central China 2.76), which approximate the findings of van LieShout et al. (1999).And after adjusting for age, gender, smoking, drinking and family history of cancer, there was also significant association between Ile/Val genotype, Ile/ Val+Val/Val combined variant genotype and esophageal cancer (OR:2.05,95%CI:1 *Adjusted for age, gender, smoking, drinking and family history of cancer Dai et al. (2009)ribution of the genotype Val/Val had significant difference between cases and controls (P=0.049),suggestingthat the genotype Val/Val can increased susceptibility to EC.A metaanalysis ofDai et al. (2009)showed that genotype Ile/ Val and combined genotype Ile/Val+Val/Val of CYP1A1 Ile/Val polymorphism, compared with wild genotype Ile/ Ile, had association with ESCC risk (OR:1.34,95%CI:1.11-1.61and OR:1.43,95%CI:1.07-1.91).Current study had verified that the gene product of CYP1A1 Val/Val had higher catalytic and carcinogenic activity than that of CYP1A1 Ile/Ile, activating the original carcinogen, increasing individuals' susceptibility to cancers