Cytochrome P 450 1 A 1 , 2 E 1 and GSTM 1 Gene Polymorphisms and Susceptibility to Colorectal Cancer in the Saudi Population

Colorectal cancer (CRC) is one of the most frequent causes of cancer death in industrialized countries with a yearly incidence of 50 new cases for every 100,000 people in the population (Boyle and Ferlay, 2005). CRC is the third most common cancer in the world and its prevalence has been steadily increasing over the last century, while mortality rates have declined as a result of improved treatment and efficient screening and surveillance (Heavey et al., 2004; Parkin et al., 2005). CRC is traditionally classified into sporadic and familial or hereditary forms and represents a complex disease which development is mediated by genetic and environmental factors (Potter, 1999; Hemminki and Czene, 2002). Several studies have reported the association between polymorphisms for gene encoding enzymes involved in biotransformation of xenobiotics and susceptibility to cancers (Induski and Lutz, 2000; Chang et al., 2003; Terry et al., 2003). The human body is exposed daily to a number of xenobiotics


Introduction
Colorectal cancer (CRC) is one of the most frequent causes of cancer death in industrialized countries with a yearly incidence of 50 new cases for every 100,000 people in the population (Boyle and Ferlay, 2005).CRC is the third most common cancer in the world and its prevalence has been steadily increasing over the last century, while mortality rates have declined as a result of improved et al., 2004;Parkin et al., 2005).CRC is traditionally and represents a complex disease which development is mediated by genetic and environmental factors (Potter, reported the association between polymorphisms for gene encoding enzymes involved in biotransformation of xenobiotics and susceptibility to cancers (Induski and Lutz, 2000;Chang et al., 2003;Terry et al., 2003).The human body is exposed daily to a number of xenobiotics including drugs, dietary compounds and environmental carcinogens which are metabolized by a variety of enzymes through phase I and phase II reactions (Bozina et al., 2009).These enzymes participate in the conversion of xenobiotics to more water soluble metabolites which are readily excreted from the body.During metabolism of some of these xenobiotics a variety of unstable and reactive intermediates can be formed which could attack et al., 2009).The key enzyme system involved in such activation process are phase I xenobiotic metabolizing enzymes (XMEs) such as cytochrome P450s (CYPs) xenobiotics, including environmental carcinogens, chemotherapeutic agents and reactive oxygen species (Taspinar et al., 2008).Mutations in the genes that code for these enzymes can affect the metabolism of chemical carcinogens and which alters susceptibility to different important for the conversion of carcinogenic polycyclic long arm of chromosome 15q22-qter (Corchero et after exposure to inducer than the non exposed group (Kellerman et al., 1973).In genotyping studies, two been well studied in Caucasian and Oriental populations, increased sensitivity to inducer in some studies and the exon 7 Ile-Val substitution that appears to result in higher higher rates of carcinogen activation than individuals with the wild-type allele.The CYP2E1 enzyme, a member of the cytochrome P450 superfamily, is a natural ethanolinducible enzyme that is involved in the metabolic oxidation of low molecular weight carcinogens such as N-nitrosoamines, benzene and vinyl chloride (Liu et al., 2009).CYP2E1 gene is located on the 10q24.3-qter.It is 18,754 bp long consisting of nine exons and eight introns, which encodes a 493 amino acid protein.CYP2E1 gene contains six restriction fragment length polymorphisms, region has been shown to affect its transcriptional level.The variant type of this polymorphic site can enhance the transcription and increase the level of CYP2E1 activity 6413432).The effect of this mutation on enzyme activity plasma ratios in healthy subjects possessing at least one the mutated genotype in comparison with the wild-type conjugation of a variety of different compounds with the between individuals, making them liable to the toxic effects of environmental carcinogens.Elevated levels cancer tumours compared with normal tissues (Ketterer et enzyme activity.Individuals possessing this genotype theoretically at a high risk of cancer development.Case control studies have shown that some of the mentioned increase in the risk of cancer including CRC in some populations (Chang and Yang, 2000).cancer incidence has not been investigated previously.The aim of the present study was to test for potential

Materials and Methods
This study was conducted after review and approval of the Institutional Review Board of the Ethics Committee cancer patients (65 males and 27 females, age range, 26-80 years; mean age, 58.4 years) and 79 healthy controls matched for age and sex.The control samples were collected from subjects referred to the hospital for general medical checkups.Colon cancer tissue samples were also collected from 12 unrelated colorectal cancer patients 8 males and 4 females and histologically normal tissues in the distant margin to the tumour were collected at the time of surgery from the patient who undergoing resection of colorectal tumours.The diagnosis of cancer was based on standard clinical, endoscopic, radiological, and histological criteria.Clinical and demographic characteristics were recorded, including age at diagnosis, gender, family history, smoking habits, disease behaviour, disease location, and need for surgery.Tissue samples to were homogenized in RLT lyses buffer (Qiagen Co., instructions.Elution was performed with 50 µl nucleasefree water.Concentration, purity, and quality of the 10X and 40X.Five images of representative areas were acquired for each specimen. equilibrium (http://ihg2.helmholtz-muenchen.de/cgi-bin/ hw/hwa1.pl).Case-control and other genetic comparisons were performed using the chi-square test and allelic odds

Results
It should be noted that the molecular tools used for the genotyping do not determine whether other polymorphisms in the same gene are also present in the tested subject.The wild type (wt) denotation thus refers to the wild type allele at the investigated polymorphic site only, regardless of other unstudied polymorphisms for that particular gene.Furthermore, in this study, there were no for the three investigated genes when male groups were compared with females groups (data not shown).
tested subjects and statistical analysis of the obtained data are detailed in Table 1.The frequency of the 2 =7.59 different from the control healthy individuals (OR=4.17; 2 =9.63 and p=0.0019).and B. The distribution of CYP2E1 genotypes among the tested subjects and the statistical analysis of the obtained data are detailed in Table 1.The frequency of the CRC patients but not in the control group.The was not statistically different from that of healthy controls 0.81-3.05).Moreover, the results remained statistically considered together as one group (OR=1.29 at CI 0.68- polymorphic sites in the three investigated genes was conducted (data not shown).None of the tested subjects carried the variant allele for the three gene polymorphisms genotypes of the three genes showed no significant variation from the results of the analysis of each gene separately.
and twelve normal tissues in the distant margin to the highly expressed in colon cancer tissues as compared with normal control adjacent tissues and their expression levels were found to be 4, 4.2 and 4.8 times as compared to the lines (Figure 3B).On the other hand, detectable levels of under investigation (Figure 3B).
T h e r e l i a b i l i t y o f t h e e x p r e s s i o n q P C R immunohistochemistry on colon cancer tumour samples be highly expressed in colon cancer tissues as indicated by strong staining (Figure 4C) when compared with control tissues (polyp) (weak staining) (Figure 4B).Overtumour progression indicating a potential role in tumour development.

Discussion
factorial disease involving inherited and environmental factors, the present study focused on the genetic basis nucleotide polymorphism are the most abundant forms among variations in human genomes, and it has been with pathophysiological properties of individuals such as responses to medications and mortalities of hereditary of markers for individual medications (Carlson et al., 2001).Moreover, susceptibility to cancer is determined by the activation of enzymes involved in carcinogens genes encoding the enzymes, possibly by altering their expression levels and functions, may increase or decrease at nucleotide 3801 in the 3' non-coding region containing a single T to C substitution that results in a polymorphic polymorphism, rs4646903).The MspI restriction site polymorphism results in three genotypes: a predominant TT), the heterozygote (type B, TC) and a homozygous rare m 2 allele with the MspI site (type C, CC) (Zhou et associated experimentally with increased catalytic activity to exhibit high rates of carcinogen activation (Landi et al., 1994).The present study showed that the variant agreement with studies in other populations, for example shown in Italy (Boccia et al., 2007), the United Kingdom It has been reported that the detection of an association in Caucasian populations because of the low frequency been associated experimentally with increased catalytic expected to exhibit high rates of carcinogen activation (Lindi et al., 1994).The present study showed that the CRC compared to control normal tissues as measured by with normal tissues.This finding was confirmed by immunohistochemistry study using anti-cytochrome P450 than normal adjacent control tissues.Many studies have investigated the relationship between CYP2E1 gene variation and the risk of CRC.The indicated that the polymorphism caused by the presence or absence of a DraI site in CYP2E1 intron 6 has no This is in agreement with a previous study about CRC in a Taiwanese population (Dil LIio et al., 1987) and Lebanese population (Darazy et al., 2011).The effect of this mutation on CYP2E1 enzyme activity is still not well established; however, an increased risk for CRC was detected among Italian population ever drinkers carrying frequency of this allele and the low number of samples individuals.The present study demonstrated that the times higher in CRC patients (Figure 3C) compared generally considered as a protective enzyme because it in larger tumour could comply with an adaptive cellular response to disease progression and this response was expression varies widely inter-individually and this may linked and may affect an individual's susceptibility for cancer (Katoh et al., 1996;Rawal et al., 1999 al., 1999;Piao et al., 2009).Meta analysis of 49 published case-control and cohort studies showed that there was with CRC in Caucasians but not in other ethnic groups in term of association might be due to a difference in the frequency of this polymorphism among these populations as well as to other factors related to the diet and the environment.In addition, the presence of other genetic might also affect the strength of the association.For of chemical carcinogens, also exhibits a gene deletion associated with a 1.91-fold increase in the risk of CRC, but genotypes was associated with an even higher OR of 4.98 In conclusion, the work undertaken in this study raises a number of interesting observations.First, the factors in the analyzed population.Third, all genes under investigation were found to be highly expressed in CRC patients which is likely to be an important determinant in predicting the outcome of cancer chemotherapy.Finally, large-scale studies with more CRC patients are required to draw a clearer picture about the genetic factors of CRC according to instruction provided by the manufacturer Integrity Numbers, RIN were ranged from 6.4-8.6 in CRC by reverse transcription and used as a template for expression levels.LoVo (human colon supraclavicular lymph nodeResearch Laboratories, King Faisal hospital Riyadh, 185TM).These cell lines were cultured in Dulbecco's medium (DMEM) supplemented with 100 IU/mL of changed three times a week and when the culture reached rs4646903) was achieved by polymerase chain reactionrestriction fragment length polymorphism (PCRpolymorphic MspI restriction site, corresponding to the gel electrophoresis and the size of the products were Version 2.0.0).Finally, 20 µl of each PCR product restriction enzyme.The digestion products were subjected (Pharmacia Biotech.)electrophoresis system at 350 V and rs6413432) was also achieved by PCR-RFLP (Darazy the polymorphic DraI restriction site in intron 6 of the gel.genotype) was performed by PCR as previously described an internal positive control.The PCR mixture containedThe same PCR program was applied as for the other two Co.).The chain termination sequencing reaction was conducted utilizing the DYEnamic ET terminator kit as Quantitative PCR (qPCR) was carried out as previously was performed in a 7500 fast real time PCR Thermal expression levels varying by less than 0.5 CTs between sample conditions and was therefore used as a reference the 2 (Livak) relative expression method.xylenefor 5 min each.They were hydrated in decreasing hydrophobic barrier created around the section using an retrieval was performed by immersing the slides in 0detection system was employed with reagent (biotinylated anti-rabbit IgM antibody) for 1 slides were incubated with the peroxidase-conjugated streptavidin label for 20 min.The sections were again were washed in two changes of water for 8 min each and then dehydrated, cleared in xylene, and mounted with supplies, UK).

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Figure 1.PCR Products of CYP1A1 in Colon Cancer Patients

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; Martinez elevated risk of CRC in several populations such as the an elevated risk of gastric cancer in Turkish and Iranian Other studies investigating the association between the