DOI:http://dx.doi.org/10.7314/APJCP.2015.16.3.1145 GSTM1, GSTT1 and CYP1A1 Gene Variants and Oral Cancer Risk in Pashtun Population of Pakistan. RESEARCH ARTICLE Genetic Susceptibility to Oral Cancer due to Combined Effects of GSTT1, GSTM1 and CYP1A1 Gene Variants in Tobacco Addicted Patients of Pashtun Ethnicity of Khyber Pakhtunkhwa Province of Pakistan Zakiullah1&*, Ahmadullah2&, Muhammad Khisroon2, Muhammad Saeed1*, Ajmal Khan2, Fazli Khuda1, Sajid Ali3, Nabila Javed4, Muhammad Ovais6, Nosheen Masood5, Nasir Khan Khalil1, Mohammad Ismail1 Abstract Associations of GSTT1, GSTM1 and CYP1A1 gene variants with risk of developing oral cancer were evaluated in this study. A case-control study was conducted in Pashtun population of Khyber Pakhtunkhwa province of Pakistan in which 200 hospital based oral cancer cases and 151 population based healthy controls exposed to similar environmental conditions were included. Sociodemographic data were obtained and blood samples were collected with informed consent for analysis. GSTM1 and GSTT1 were analysed through conventional PCR method while specific RT-PCR method was used to detect CYP1A1 polymorphisms. Results were analyzed for conditional logistic regression model by SPSS version 20. The study shows that patients with either GSTM1 or GSTT1 null genotypes have significantly higher risk of oral cancer (adjusted odds (OR): (3.019 (1.861-4.898) and 3.011(1.865-4.862), respectively), which further increased when either one or both null genes were present in combination (adjusted odds (OR): (3.627 (1.981-6.642 and 9.261 (4.495-19.079), respectively). CYP1A1 rs4646903 gene variants individually showed weak association OR: 1.121 (0.717-1.752); however, in the presence of GSTM1 and/or GSTT1 null genotypes further increasing the association (adjusted odds (ORs): 4.576 (2.038-10.273), 5.593 (2.530-12.362) and 16.10 (3.854-67.260 for GSTM/GSTT null and CYP1A1 wild type, GSTM/GSTT either null and CYP1A1 variant alleles, and all 3 gene polymorphisms combinations, respectively). Our findings suggest that presence of GSTM1 and/or GSTT1 null genotypes along with variant alleles of CYP1A1 may be the risk alleles for oral cancer susceptibility in Pashtun population. Keywords: Oral cancer risk - GSTT1 - GSTM1 and CYP1A1 gene variants - Pashtun population - Pakistan Asian Pac J Cancer Prev, 16 (3), 1145-1150 Introduction and neck area cancers, especially that of the oral cavity (Warnakulasuriya et al., 2005; Amtha et al., 2009). In Oral cancer is the fourth most common cancer in the Pakistan, about 50% tumors in males and 25% those of world (Amtha et al., 2009; Gupta and Johnson, 2014). in females are associated with consumption of tobacco It is estimated that over 400,000 cases occur annually products (Warnakulasuriya et al., 2005; Bhurgri et al., with a wide variation in global burden. Its incidence in 2006). In Bangladesh and India, about one-third of all South and South East Asia is amongst the highest in the cancers are tobacco-related. High incidence of oral and world. Incidence is also on the rise in Western and Eastern pharyngeal cancers has been reported in South Asia, even Europe, Latin America and Pacific regions (Ariyawardana among female population, for which smokeless tobacco and Johnson, 2013). In Pakistan oral cancer is the second products are considered to be the most prominent risk most common malignancy after breast cancer and is factor (Moore et al., 2000; Gupta and Johnson, 2014). significantly higher than other member states of the World Association of oropharyngeal cancer with smokeless Health Organization’s Eastern Mediterranean (WHO- tobacco products is reportedly four times higher relative to EMRO) Region (Bile et al., 2010). no history of tobacco use after adjusting for confounding Tobacco use and alcohol drinking are the main factors (Merchant 2000; Bile et al., 2010). Similarly pan independent risk factors for the development of head with or without tobacco has been associated with oral 1Department of Pharmacy, 2Department of Zoology, 4Institute of Radiotherapy & Nuclear Medicine,6Centre of Biotechnology & Microbiology, University of Peshawar, Peshawar, 3Department of Biotechnology, Abdul Wali Khan, University Mardan, 5Fatima Jinnah Women University, Rawalpindi, Pakistan *For correspondence: [email protected]; [email protected] Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 1145 Zakiullah et al cancer (Merchant et al., 2000). Several studies from null genotype reportedly occurs in about 50% of Asians India and Pakistan have provided sufficient evidence that and Caucasians (Amtha et al., 2009, Nosheen et al., use of tobacco mixed with lime and chewing betel quid 2014). Previous studies on the association of oral cancer containing tobacco were carcinogenic to humans with with polymorphism in these genes have shown varying significant dose-response relationships for frequency of results. Some studies have shown the association of chewing per day and for duration of tobacco consumption polymorphisms in CYP1A1 and GSTM1 genes with oral (Rao and Desai, 1998; Nandakumar et al., 1990; Dikshit cancer (Tanimoto et al., 1999; Wogan et al., 2004), while and Kanhere, 2000; Balaram et al., 2002; Znaor et al., some studies have reported a lack of association (Park 2003). et al., 2000; Olshan et al., 2000; Sreelekha et al., 2001; Tobacco products contain more than 30 carcinogens. Sharma et al., 2006; Cha et al., 2007). These seemingly However, studies on the mechanism of their carcinogenesis conflicting observations are due to geographic and ethnic suggest that three classes among them are of major variations in the distribution of genotype frequencies of importance i.e., Tobacco Specific Nitrosamines (TSNAs), both CYP and GST alleles along with other environmental Poly Aromatic Hydrocarbons (PAHs) and aromatic amines factors. A recent study has emphasized on the inclusion of (Rickert et al., 2009). These compounds are actually country of study, source of population, specific ethnicity procarcinogens and are metabolized by Xenobiotic and characteristics of general demographic variables in metabolizing enzymes, namely Cytochrome P450 (CYPs) future studies to deduce conclusive and more reliable and Glutathione-S-transferases (GSTs). The former is results (Nosheen et al., 2014). involved in their activation to carcinogenic species; while Association of genetic polymorphisms of the above the latter is involved in their detoxification and excretion mentioned genes with oral cancer has not been so far from the body (Zakiullah et al., 2014). Various isoforms reported in Pashtun population of Khyber Pakhtunkhwa of CYPs including CYP1A1 are reportedly involved in province of Pakistan. Therefore, a case-control study was activation process (D’Errico et al., 1996; Olshan et al., carried out to evaluate the potential role of CYP1A1 (T>C, 2000). Similarly among GSTs, M1 (GSTM1) and T1 rs4646903), GSTM1 and GSTT1 gene polymorphisms (GSTT1) enzymes are more important in detoxification in the susceptibility to oral cancer in Pashtun population (Nair et al., 1999). of Khyber Pakhtunkhwa province of Pakistan. This will However, the expression of these enzymes differs help to adopt pro-active approaches for early detection and in individuals resulting in differences in the metabolic preventive life style modification strategies to decrease the processing of carcinogens. Certain individuals are incidence of the disease in the target population. genetically more susceptible to cancer when exposed to carcinogens owing to their genotype for enzymes Materials and Methods responsible for their activation or detoxification (Abbas et al., 2014). This is evident from the general observation that Sample collection only a small number of individuals among those exposed Study sample comprised of 200 oral cancer patients to tobacco or alcohol under the same environmental and 151 healthy control subjects between 30 and 70 years conditions develop cancer in their life time. Allelic of age as per exclusion/inclusion criteria. Patients were variants influencing the enzyme activity of CYP1A1 registered at the Institute of Radiotherapy and Nuclear along with environmental factors such as tobacco use play Medicine (IRNUM), Peshawar, Khyber Pakhtunkhwa; key roles in making individuals susceptible to different while eligible control samples were collected from various types of cancers (Xia et al., 2013). The CYP1A1 gene districts of the same province. The study period was from located on chromosome 15q22-q24 encodes an enzyme July, 2012- July, 2013. All the patients were having histo- with aryl hydrocarbon hydroxylase activity that plays pathologically confirmed oral cancer. important role in the metabolism of polycyclic aromatic Inclusion criteria (patients): Histo-pathologically hydrocarbons (PAH) and nitrosamines from tobacco, and proven oral cancer patients having age between 30 and inherited differences in metabolic capacity are considered 70 years with Pashtun ethnicity, and not less than 20 to contribute significantly in carcinogenesis (Sabitha et years of tobacco exposure in any form. Exclusion criteria al., 2010). Certain allelic variations in the CYP1A1 gene (patients): Patients with non-Pashtun ethnicity and/or and prolonged exposure to tobacco products could lead having more than 70 years of age. Criteria for selection of to higher levels of reactive metabolites, thereby causing control subjects: Normal healthy age-matched subjects of DNA damage in addition to other contributing factors similar