Cancer Investigation, 32:53–62, 2014 ISSN: 0735-7907 print / 1532-4192 online C 2014 Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357907.2013.867972
ORIGINAL ARTICLE
GSTM1-null Genotype as a Risk Factor for Sporadic Colorectal Cancer in a Romanian Population. Association with the NAT2-rapid-acetylator Phenotype and Exposure to Environmental Factors Lucia Maria Procopciuc1 and Gelu Osian2 Department of Medical Biochemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, 2 3rd Surgical Clinic, Cluj-Napoca, Romania
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of CRC occur as part of hereditary disorders: familial adenomatous polyposis 1% and hereditary nonpolyposis cancer (HNPCC; Lynch syndromes) 3–4%. In these situations, the genetic mutations involved are known, the APC gene in the case of familial adenomatous polyposis, and mismatch repair genes in HNPCC (2). The rest 95% of CRC are sporadic cancers (3). Unlike familial CRC that occurs with a high frequency in the relatives of the affected patient and has hereditary transmission, sporadic CRC involves interactions between external chemical factors and the genetic susceptibility to CRC of the individuals concerned. Thus, sporadic CRC is considered a multifactorial disease that appears due to the exposure of individuals with specific genetic deficiencies to compounds with a carcinogenic effect (4). Environmental risk factors include dietary habits consisting of high fried red meat intake and low fiber, vegetable and fruit consumption, smoking behavior, and alcohol use. There are a lot of carcinogenic compounds in meat processed by pyrolysis and in tobacco smoke, such as polycyclic aromatic hydrocarbons [PAH], heterocyclic amines or nitrosamines (5–7). The role of smoking in CRC was confirmed by many studies (8). Other studies failed to find any association (9, 10). One of the protective mechanisms against cancer includes the efficient metabolization of carcinogens that enter our bodies from diet and tobacco smoke. Thus, the polymorphic genes that encode enzymes involved in the metabolism of these carcinogens are very important susceptibility genes in cancer, including CRC (7, 11, 12). One of these enzymes is glutathione S-transferase (GST, chromosome 1p13.3), a phase II enzyme that catalyzes the metabolic transformation of carcinogens in the reaction with glutathione into inactive products. These products are incapable of interacting with the DNA, with the possibility to be excreted in urine (13–15). There are seven cytosolic GST isoenzymes, the most important being GSTM, GSTP, and GSTT(1, 13). The deletion of the GSTM1 gene in both alleles (homozygotes or GSTM1-null genotype) results in the loss of enzyme activity
We evaluated the association between the presence of the GSTM1-null genotype and the combined presence of the GSTM1-null genotype/NAT2 rapid acetylator phenotype and the risk of developing sporadic colorectal cancer (CRC), as well as their interaction with environmental risk factors. One hundred and fifty patients with sporadic CRC and 162 controls were genotyped using PCR-RFLP analysis. For testing and quantification of the simple effect (main effect) and of the gene–gene and gene–environment interaction (modification effect), univariate and multivariate logistic regression was used. In the multiplicative model, from the genetic factors, GSTM1-null and NAT2∗ 6B had a statistically significant influence on the risk for CRC, while from the environmental factors, smoking and diet had similar effects. The combination of GSTM1-null/NAT2 rapid acetylator phenotype/smoking behavior or GSTM1-null/NAT2 rapid acetylator phenotype/diet rich in fried red meat was not found to influence the sporadic CRC risk in Romanians, but the GSTM1-null genotype, NAT2 rapid acetylator phenotype influenced the sporadic CRC risk differently depending on the gender of the patient. Keywords: Sporadic colorectal cancer, GSTM1-null, NAT2-rapid acetylator phenotype, Smoking, Diet rich in fried red meat, Multiplicative model, Romania
INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in both women and men, with a higher frequency in European countries, representing 13% of all cancers. In Romanians, CRC is the most frequent gastrointestinal cancer, with an incidence of 41.2% in males and 27.9% in females and a mortality rate of 25.2% in males and 14.7% in females [data refers to the Estimated ASRs (European standard) of cancer incidence and mortality by sex, cancer site, and country, 2008]. In 2008, 45.900 new cases of CRC in males and 41.400 in females were estimated(1). Three to 5%
Correspondence to: Procopciuc Lucia Maria, Department of Medical Biochemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy, Padis Street no. 8/14, Cluj-Napoca 400452, Romania. email: [email protected] Received 1 April 2012; revised 26 October 2013; accepted 15 November 2013.
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L. M. Procopciuc and G. Osian
and thus in a possibly higher risk to develop CRC (12, 16). The frequency of the GSTM1-null genotype in the African population is 23–48% and increases to 33–63% and 39–62%, respectively, in Asian and European populations (17–19). Some studies evidenced a strong association between the GSTM1-null genotype and CRC, but other studies failed to find any association (20–24). The study performed by Wang et al (2011) in a Indian population showed a 1.55 risk for CRC in patients with the GSTM1-null genotype(11). In one meta-analysis conducted in 2010 by Economopoulos et al on 11.998 Caucasian individuals, the risk for CRC associated with this genotype was 1.15 (14). The relation between CRC and the GSTM1-null genotype is not very well established, possibly because of the gene–environment interaction. Another phase II enzyme involved in the chemical detoxification is N-acetyl transferase (NAT2, chr 8p22). It acts by the O-acetylation of heterocyclic aromatic amines (HAA) from tobacco smoke and cooked meals (25–27). Two of the genetic variations that influence acetylation activity are NAT2∗ 5A (C481T, P161L) and NAT2∗ 6B (G590A, R197Q). Individuals heterozygous or homozygous for the negative allele are rapid acetylators, while individuals with two positive alleles are slow acetylators (25). Some studies, such as the study carried out by Hein et al (2002), have shown that rapid acetylators have an increased risk for developing CRC (26). Other studies do not confirm these results (28, 29). There are studies confirming the risk of sporadic CRC in individuals with some NAT2 rapid acetylator phenotypes and a diet rich in fried red meat, but others infirm these results (28–30). For effective disease prevention, knowledge of the pattern of inheritance of these susceptible genes in different ethnic groups is very important. Aim: To establish the role of the GSTM1-null as an increased susceptibility risk factor for sporadic CRC in both male and female Romanian patients; to evaluate the gene–gene interaction, the combined effect of the GSTM1 genotype/NAT2 acetylator phenotype on the risk for sporadic CRC, as well as the interaction with cigarette smoking and high fried red meat intake. MATERIALS AND METHODS Patient selection The present study included 150 patients with histologically confirmed sporadic CRC (80—53.33% females, mean age 62.99 ± 10.82 years, and 70—46.67% males, mean age 64.66 ± 9.24 years), randomly selected, who were admitted in the Surgical Clinic III, University of Medicine of ClujNapoca, between 2004 and 2008. The inclusion criteria were: ages ranging 25 to 85 years and histologically confirmed sporadic CRC. According to the above criteria, 96 (64%) patients had sporadic colon cancer and 54 (36%) had sporadic rectal cancer. Patients with familial adenomatous polyposis or HNPCC were excluded from the study. The control group comprised 162 subjects matched for age (100—61.73% females, mean age 63.5 ± 11.69 years, and 62—38.27% males, mean age 64.67 ± 8.29 years). They
were hospitalized patients, admitted for symptoms which required colonoscopy (rectorrhagia, transit disorders) but in which the investigation did not reveal colorectal lesions except diverticula and hemorrhoids. The exclusion criteria for the control group were a personal or family history of familial adenomatous polyposis or HNPCC, a history of inflammatory bowel disease, a history of colorectal surgery; nonperformance of total colonoscopy. All patients and controls were interviewed using a questionnaire regarding socio-demographic characteristics (age, sex), dietary habits (fried red meat intake more than 4 days a week), and lifestyle factors including cigarette smoking (yes or no). We considered as smokers the individuals who smoked more than 20 cigarettes/day over the past 5 years. We considered high fried red meat intake a diet including fried red meat more than 5 days a week. The indications for colonoscopy were: rectorrhagia, transit disorders, tenesmus, anemia, subocclusion, and occlusion. Colonoscopies were performed in the Department of Digestive Endoscopy, and the diagnosis of CRC was confirmed histopathologically in the Department of Pathology of the “Prof. Dr. Octavian Fodor” Emergency Hospital of ClujNapoca. For genotype identification, we collected 5 mL of blood (in tubes containing anticoagulant) from all patients and controls included in the study. Identification of the NAT2 and GSTM1 genotypes was performed in the Department of Biology, University College London and in the Department of Biochemistry of the “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca. The study was approved by the local Ethics Committee of our university. All patients and controls gave their informed consent to participate in the study. The demographic data and the clinical characteristics of the participants in the study are shown in Table 1. METHODS PCR-RFLP analysis Total genomic DNA was extracted from 50 μL peripheral blood drawn with EDTA (etilen-diamino-tetra-acetate) as an anticoagulant agent, using a ZYMO research kit. The amplification of the region of interest of the GSTM1 gene containing the null genotype was carried out in an Eppendorf mastercycler using the following primers: forward (5 -CTGCCCTACTTGATTGATGGG-3 ) and reverse (5 -CTGGATTGTAGCAGATCATGC-3 ) primer (31). The 273bp product was generated for the GSTM1-ins genotype. In the case of the GSTM1-null genotype, this fragment was not present. Amplification for NAT2∗ 5A and NAT2∗ 6B was carried out using the following primers: forward (5 AAGGATCAGCCTCAGGTGCCTT-3 /5 -GGACCAAATC AGGAGAGAGCAG-3 ) and reverse (5 -CTGCTCTCTC CTGATTTGGTCC-3 /5 -GTTGGAGACGTCTGCAGGTAT G-3 ) primer (25). PCRs products were then digested with the restriction enzymes Kpn (NAT2∗ 5A) and TaqI (NAT2∗ 6B), respectively. Regarding the NAT2∗ 5A polymorphism, the negative allele C481 gave two fragments of 60 bp and 24 bp, Cancer Investigation
C 2014 Informa Healthcare USA, Inc. Copyright
20 (66.67%) 16 (53.33% 12 (40% 4 (13.33%) 4 (13.33%) – 4 (13.33%) 26 (86.67%) 1.43 [.72–6.46] .72 26 (86.67%) 4 (13.33%) 7.89 [2.24–30.46]
ORIGINAL ARTICLE
GSTM1-null Genotype as a Risk Factor for Sporadic Colorectal Cancer in a Romanian Population. Association with the NAT2-rapid-acetylator Phenotype and Exposure to Environmental Factors Lucia Maria Procopciuc1 and Gelu Osian2 Department of Medical Biochemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, 2 3rd Surgical Clinic, Cluj-Napoca, Romania
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1
of CRC occur as part of hereditary disorders: familial adenomatous polyposis 1% and hereditary nonpolyposis cancer (HNPCC; Lynch syndromes) 3–4%. In these situations, the genetic mutations involved are known, the APC gene in the case of familial adenomatous polyposis, and mismatch repair genes in HNPCC (2). The rest 95% of CRC are sporadic cancers (3). Unlike familial CRC that occurs with a high frequency in the relatives of the affected patient and has hereditary transmission, sporadic CRC involves interactions between external chemical factors and the genetic susceptibility to CRC of the individuals concerned. Thus, sporadic CRC is considered a multifactorial disease that appears due to the exposure of individuals with specific genetic deficiencies to compounds with a carcinogenic effect (4). Environmental risk factors include dietary habits consisting of high fried red meat intake and low fiber, vegetable and fruit consumption, smoking behavior, and alcohol use. There are a lot of carcinogenic compounds in meat processed by pyrolysis and in tobacco smoke, such as polycyclic aromatic hydrocarbons [PAH], heterocyclic amines or nitrosamines (5–7). The role of smoking in CRC was confirmed by many studies (8). Other studies failed to find any association (9, 10). One of the protective mechanisms against cancer includes the efficient metabolization of carcinogens that enter our bodies from diet and tobacco smoke. Thus, the polymorphic genes that encode enzymes involved in the metabolism of these carcinogens are very important susceptibility genes in cancer, including CRC (7, 11, 12). One of these enzymes is glutathione S-transferase (GST, chromosome 1p13.3), a phase II enzyme that catalyzes the metabolic transformation of carcinogens in the reaction with glutathione into inactive products. These products are incapable of interacting with the DNA, with the possibility to be excreted in urine (13–15). There are seven cytosolic GST isoenzymes, the most important being GSTM, GSTP, and GSTT(1, 13). The deletion of the GSTM1 gene in both alleles (homozygotes or GSTM1-null genotype) results in the loss of enzyme activity
We evaluated the association between the presence of the GSTM1-null genotype and the combined presence of the GSTM1-null genotype/NAT2 rapid acetylator phenotype and the risk of developing sporadic colorectal cancer (CRC), as well as their interaction with environmental risk factors. One hundred and fifty patients with sporadic CRC and 162 controls were genotyped using PCR-RFLP analysis. For testing and quantification of the simple effect (main effect) and of the gene–gene and gene–environment interaction (modification effect), univariate and multivariate logistic regression was used. In the multiplicative model, from the genetic factors, GSTM1-null and NAT2∗ 6B had a statistically significant influence on the risk for CRC, while from the environmental factors, smoking and diet had similar effects. The combination of GSTM1-null/NAT2 rapid acetylator phenotype/smoking behavior or GSTM1-null/NAT2 rapid acetylator phenotype/diet rich in fried red meat was not found to influence the sporadic CRC risk in Romanians, but the GSTM1-null genotype, NAT2 rapid acetylator phenotype influenced the sporadic CRC risk differently depending on the gender of the patient. Keywords: Sporadic colorectal cancer, GSTM1-null, NAT2-rapid acetylator phenotype, Smoking, Diet rich in fried red meat, Multiplicative model, Romania
INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in both women and men, with a higher frequency in European countries, representing 13% of all cancers. In Romanians, CRC is the most frequent gastrointestinal cancer, with an incidence of 41.2% in males and 27.9% in females and a mortality rate of 25.2% in males and 14.7% in females [data refers to the Estimated ASRs (European standard) of cancer incidence and mortality by sex, cancer site, and country, 2008]. In 2008, 45.900 new cases of CRC in males and 41.400 in females were estimated(1). Three to 5%
Correspondence to: Procopciuc Lucia Maria, Department of Medical Biochemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy, Padis Street no. 8/14, Cluj-Napoca 400452, Romania. email: [email protected] Received 1 April 2012; revised 26 October 2013; accepted 15 November 2013.
Cancer Invest Downloaded from informahealthcare.com by McMaster University on 10/24/14 For personal use only.
L. M. Procopciuc and G. Osian
and thus in a possibly higher risk to develop CRC (12, 16). The frequency of the GSTM1-null genotype in the African population is 23–48% and increases to 33–63% and 39–62%, respectively, in Asian and European populations (17–19). Some studies evidenced a strong association between the GSTM1-null genotype and CRC, but other studies failed to find any association (20–24). The study performed by Wang et al (2011) in a Indian population showed a 1.55 risk for CRC in patients with the GSTM1-null genotype(11). In one meta-analysis conducted in 2010 by Economopoulos et al on 11.998 Caucasian individuals, the risk for CRC associated with this genotype was 1.15 (14). The relation between CRC and the GSTM1-null genotype is not very well established, possibly because of the gene–environment interaction. Another phase II enzyme involved in the chemical detoxification is N-acetyl transferase (NAT2, chr 8p22). It acts by the O-acetylation of heterocyclic aromatic amines (HAA) from tobacco smoke and cooked meals (25–27). Two of the genetic variations that influence acetylation activity are NAT2∗ 5A (C481T, P161L) and NAT2∗ 6B (G590A, R197Q). Individuals heterozygous or homozygous for the negative allele are rapid acetylators, while individuals with two positive alleles are slow acetylators (25). Some studies, such as the study carried out by Hein et al (2002), have shown that rapid acetylators have an increased risk for developing CRC (26). Other studies do not confirm these results (28, 29). There are studies confirming the risk of sporadic CRC in individuals with some NAT2 rapid acetylator phenotypes and a diet rich in fried red meat, but others infirm these results (28–30). For effective disease prevention, knowledge of the pattern of inheritance of these susceptible genes in different ethnic groups is very important. Aim: To establish the role of the GSTM1-null as an increased susceptibility risk factor for sporadic CRC in both male and female Romanian patients; to evaluate the gene–gene interaction, the combined effect of the GSTM1 genotype/NAT2 acetylator phenotype on the risk for sporadic CRC, as well as the interaction with cigarette smoking and high fried red meat intake. MATERIALS AND METHODS Patient selection The present study included 150 patients with histologically confirmed sporadic CRC (80—53.33% females, mean age 62.99 ± 10.82 years, and 70—46.67% males, mean age 64.66 ± 9.24 years), randomly selected, who were admitted in the Surgical Clinic III, University of Medicine of ClujNapoca, between 2004 and 2008. The inclusion criteria were: ages ranging 25 to 85 years and histologically confirmed sporadic CRC. According to the above criteria, 96 (64%) patients had sporadic colon cancer and 54 (36%) had sporadic rectal cancer. Patients with familial adenomatous polyposis or HNPCC were excluded from the study. The control group comprised 162 subjects matched for age (100—61.73% females, mean age 63.5 ± 11.69 years, and 62—38.27% males, mean age 64.67 ± 8.29 years). They
were hospitalized patients, admitted for symptoms which required colonoscopy (rectorrhagia, transit disorders) but in which the investigation did not reveal colorectal lesions except diverticula and hemorrhoids. The exclusion criteria for the control group were a personal or family history of familial adenomatous polyposis or HNPCC, a history of inflammatory bowel disease, a history of colorectal surgery; nonperformance of total colonoscopy. All patients and controls were interviewed using a questionnaire regarding socio-demographic characteristics (age, sex), dietary habits (fried red meat intake more than 4 days a week), and lifestyle factors including cigarette smoking (yes or no). We considered as smokers the individuals who smoked more than 20 cigarettes/day over the past 5 years. We considered high fried red meat intake a diet including fried red meat more than 5 days a week. The indications for colonoscopy were: rectorrhagia, transit disorders, tenesmus, anemia, subocclusion, and occlusion. Colonoscopies were performed in the Department of Digestive Endoscopy, and the diagnosis of CRC was confirmed histopathologically in the Department of Pathology of the “Prof. Dr. Octavian Fodor” Emergency Hospital of ClujNapoca. For genotype identification, we collected 5 mL of blood (in tubes containing anticoagulant) from all patients and controls included in the study. Identification of the NAT2 and GSTM1 genotypes was performed in the Department of Biology, University College London and in the Department of Biochemistry of the “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca. The study was approved by the local Ethics Committee of our university. All patients and controls gave their informed consent to participate in the study. The demographic data and the clinical characteristics of the participants in the study are shown in Table 1. METHODS PCR-RFLP analysis Total genomic DNA was extracted from 50 μL peripheral blood drawn with EDTA (etilen-diamino-tetra-acetate) as an anticoagulant agent, using a ZYMO research kit. The amplification of the region of interest of the GSTM1 gene containing the null genotype was carried out in an Eppendorf mastercycler using the following primers: forward (5 -CTGCCCTACTTGATTGATGGG-3 ) and reverse (5 -CTGGATTGTAGCAGATCATGC-3 ) primer (31). The 273bp product was generated for the GSTM1-ins genotype. In the case of the GSTM1-null genotype, this fragment was not present. Amplification for NAT2∗ 5A and NAT2∗ 6B was carried out using the following primers: forward (5 AAGGATCAGCCTCAGGTGCCTT-3 /5 -GGACCAAATC AGGAGAGAGCAG-3 ) and reverse (5 -CTGCTCTCTC CTGATTTGGTCC-3 /5 -GTTGGAGACGTCTGCAGGTAT G-3 ) primer (25). PCRs products were then digested with the restriction enzymes Kpn (NAT2∗ 5A) and TaqI (NAT2∗ 6B), respectively. Regarding the NAT2∗ 5A polymorphism, the negative allele C481 gave two fragments of 60 bp and 24 bp, Cancer Investigation
C 2014 Informa Healthcare USA, Inc. Copyright
20 (66.67%) 16 (53.33% 12 (40% 4 (13.33%) 4 (13.33%) – 4 (13.33%) 26 (86.67%) 1.43 [.72–6.46] .72 26 (86.67%) 4 (13.33%) 7.89 [2.24–30.46]
