Associations of CYP 1 A 1 , GSTM 1 and GSTT 1 Polymorphisms with Lung Cancer Susceptibility in a Northern Indian Population

Carcinoma of the lung is the most common cancer and the most frequent cause of death in the patients with cancer around the world (Bethesda, 2001). Environmental carcinogens such as active and passive smoking, air pollution and environmental exposures have strong influences on individual factors (Perera, 1998). In humans, there are several genetic polymorphisms of the enzymes involved in metabolic activation and detoxification of pulmonary carcinogens Interindividual differences in ability to activate and detoxify carcinogens are expected to affect the risk of developing lung cancer (Raunio et al., 1995). Cytochrome P-450s, cytochrome P450 1A1 (CYP1A1), glutathione S-transferase M1 (GSTM1), and (GSTT1) phase II detoxifying enzymes are involved in the formation and elimination of carcinogens, have been extensively studied as possible modulators of risk for lung cancer that could explain varying susceptibilities to the disease (Taningher et al., 1999). CYP1A1 gene is involved in the activation step in the metabolism of polycyclic aromatic hydrocarbons (PAHs),


Introduction
Carcinoma of the lung is the most common cancer and the most frequent cause of death in the patients with cancer around the world (Bethesda, 2001).Environmental carcinogens such as active and passive smoking, air pollution and environmental exposures have strong influences on individual factors (Perera, 1998).In humans, there are several genetic polymorphisms of the enzymes involved in metabolic activation and detoxification of pulmonary carcinogens Interindividual differences in ability to activate and detoxify carcinogens are expected to affect the risk of developing lung cancer (Raunio et al., 1995).Cytochrome P-450s, cytochrome P450 1A1 (CYP1A1), glutathione S-transferase M1 (GSTM1), and (GSTT1) phase II detoxifying enzymes are involved in the formation and elimination of carcinogens, have been extensively studied as possible modulators of risk for lung cancer that could explain varying susceptibilities to the disease (Taningher et al., 1999).
CYP1A1 gene is involved in the activation step in the metabolism of polycyclic aromatic hydrocarbons (PAHs),

Associations of CYP1A1, GSTM1 and GSTT1 Polymorphisms with Lung Cancer Susceptibility in a Northern Indian Population
RK Shukla 1 , AR Tilak 2 , C Kumar 1,3 , S Kant 1,4 , A Kumar 2 , B Mittal 5 , *S Bhattacharya 1,3 such as those found in tobacco smoke, converting them to carcinogens (Gonzalez, 1990).Glutathione transferases (GSTs) comprise a multigene family encoding enzymes that catalyse the conjugation of glutathione to a wide variety of compounds with an electrophilic centre (Hayes and Pulford, 1995).GSTM1 is involved in the detoxification of tobacco-related carcinogens, such as epoxides and hydroxylated metabolites of benzo (α)pyrene (Ketterer et al., 1992), whereas GSTT1 is involved in the biotransformation of several low molecular weight toxins such as ethylene oxides, butadiene, etc. (Guengerich et al., 1995), which are constituents of tobacco smoke.
It is likely that several genetic polymorphisms cooperate in increasing individual risk.There may be specific genotypes or genotype combinations that greatly increase the risk of developing lung cancer.In view of the prevalence of tobacco smoking, and the incidence of lung cancer in India, we investigated the distribution and susceptibility of CYP1A1,GSTM1,and GSTT1 gene polymorphism in lung Cancer and healthy controls in Northern Indian Population or to determine whether any of the polymorphisms confer an increased risk.

Inclusion/exclusion criteria for case /control
A questionnaire was completed by both patient and control groups to provide relevant information regarding the risk factors for Lung cancer.The information collected included socio-demographic characteristics such as gender, age, lifetime occupational history (including exposure to known carcinogens), area of origin, family history of cancer among first degree relatives, smoking status, which included smoking duration and pack years smoked, medication history and pre-existence of respiratory or lung diseases.In order for the age and gender distributions of controls to match those of Lung cancer patients, most of the controls were age matched and the majority were males.Controls were also interviewed and asked about histories of cancer, occupation and smoking habits.Smoking information included past and/or present smoking status, amount smoked and duration of smoking.Smoking status of the subjects was calculated as the average tobacco consumption expressed in pack years.Pack years were computed as the number of cigarettes smoked per day multiplied by the duration of smoking in years.

Sample collection
Blood samples were collected from study subjects after obtaining their written informed consent.Peripheral blood (2 ml) collected from patients and all controls and was stored at -80 0 C until use.

Blood collection and DNA extraction
EDTA-buffered whole blood (5 ml) was drawn for subsequent DNA extraction by standard salting-out method (Miller et al., 1988).
Genotype analysis GSTM1 and GSTT1 null allele were determined by using multiplex polymerase chain reaction (PCR) with the CYP1A1 gene as an internal positive control (Setiawan et al., 2000).Briefly, a 215-bp region between exons 4 and 5 of the GSTM1 gene and 480-bp products for were amplified along with a 312-bp size product of CYP1A1.
The PCR products were electrophoresed on a 2% agrose gel.The absence of 480 and 215 bp bands indicated homozygous null genotypes of GSTM1 and GSTT1, respectively.
CYP1A1 -6235 T>C polymorphism involves the substitution of CTGG to CCGG in the Msp1 site at base 264 from the additional polyadenylation signal in the 3' flanking region.The region of interest was amplified by PCR using the primer sequences described by Kawajiri et al. (1990).CYP1A1 T and C alleles were determined by the presence or absence of the Msp1 restriction site through different band patterns on 2% agarose gel.The wild-type genotype (CYP1A1TT) showed a single band of 360 bp.The variant genotype (CYP1A1CC) resulted in two fragments of 220 and 140 bp, whereas the heterozygous genotype (CYP1A1TC) showed three bands of 360, 220, and 140 bp.

Statistical methods
Variables selected from the data set were age, gender, smoking status (non smokers, ex-smokers, and smokers), pack years of smoking, and polymorphisms in the CYP1A1, GSTM1 and GSTT1 genes.We estimated the study specific odds ratios (OR) of Lung cancer for each polymorphism using binary logistic regression modeling with 95% confidence intervals (CIs), and the difference in genotype prevalence and association between case and control group were assessed and adjusted for age, gender and smoking status.
To determine whether the genotype frequencies were significantly different between the patient and control population, a probability of P<0.05 was considered.Age, gender, smoking status and pack years were included as covariates as well as all the possible genotypes studied.GSTM1 and GSTT1 polymorphism was dichotomized into null genotype and wild type, while CYP1A1 MspI polymorphism was categorized into homozygous wild type and variant allele-containing genotypes.
Besides the main effect of CYP1A1, GSTM1 and GSTT1 polymorphism on Lung cancer.Wild type of CYP1A1 and non-null genotypes of GSTM1 and GSTT1 were used as reference groups to assess the combined effects of the two genes.To evaluate the possible interaction between genetic polymorphisms and smoking, a group of subjects with non-null genotype and no current smoking habits was used as a reference group.

Results
Mean age of healthy subjects (controls) and lung cancer patients was 56.15±7.84 and 56.14±11.91years, respectively (t test p value=ns).Lung cancer was highly prevalent in males (189 out of 218; 86.7%) than in females (29 out of 218; 13.3%).In patients with Lung cancer most of the cases were with squamous cell carcinoma (54.1%).Regarding smoking habit, 58.7% were smokers, 9.6% ex-smokers and 31.7%non-smokers among lung cancer patients with mean pack years of 13.95±7.93(years); in controls 72.3% were non-smoker, 13.4% ex-smoker and 14.3% smokers with mean pack year of 10.5±5.62 (years).

Association with susceptibility to lung cancer
There were no consistent patterns of elevated risk associated with the GSTM1 null genotype, but the frequency of the GSTT1 null genotype was 24.4% in controls and 37.6% in Lung cancer and showed significant association (OR=1.87,95%CI=1.25-2.80,P=0.002).However no significant association for lung cancer was found for CYP1A1 6235T>C polymorphism (TT, TC, and CC) in Lung cancer patients (64.2%, 31.7%,4.1%) vs healthy controls (55.0%,37.8%, 7.1%).

Discussion
Various form of CYP1A1, GSTM1 and GSTT1 gene have risk for development of Lung cancer in various population (Garte, 2001).The levels of expression and catalytic activities of cytochrome p450 and GSTM1 and GSTT1 enzymes in lungs, and their metabolic balance, may be an important determinant host factor underlying Lung cancer.In this study, we evaluate the effect of GSTT1, M1 and CYP1A1 Msp1 gene polymorphisms in Northern Indian Lung cancer patients and controls.
GSTM1 and GSTT1 members of the glutathione S-transferase multigene family are candidate cancer susceptibility genes because of their ability to regulate the conjugation of carcinogenic compounds to excretable hydrophilic metabolites.Individuals who are carriers of homozygous deletions in the GSTM1 and GSTT1 genes may have an impaired ability to eliminate carcinogenic compounds metabolically and may therefore be at an increased cancer risk.The frequencies of homozygous GSTM1 and GSTT1 deletion carriers are surprisingly high in most human populations, and noticeable differences between ethnic groups exist (Nelson et al., 1995).GSTM1 null genotype was present in about 50% of Caucasians, 33% of African Americans and 45% of Japanese (Persson et al., 1999;Roy et al., 2001;Chan-Yeung et al., 2004) and various studies have shown an increased Lung cancer risk for GSTM1 null genotype independent of ethnic background (Chen et al., 1996;Gao and Zhang, 1999).GSTT1 null genotype was present in 64% of Chinese, 60% of Koreans, 28% of Caucasians and 22% of African Americans (Nelson et al., 1995).
In our study, frequency of GSTT1 null genotype in Lung cancer patients is higher than healthy controls (37.6% vs 24.4%); while GSTM1 null genotype is similar to healthy controls (38.5% vs 37.8%).GSTT1 null genotype was significantly associated with lung cancer patients (p-value 0.001) but GSTM1 null genotype was

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not associated with it.This is consistent with some other similar findings in African Americans population (Taioli et al., 1998;Sorensen et al., 2004), but conflicts with certain other reports in China (Lan et al., 2000).Although intra ethnic as well as inter ethnic differences exist in the Indian population, the prevalence of in GSTM1 null genotype and GSTT1 null genotype in our population is comparable with that found in other Indian studies (Mishra et al., 2004;Naveen et al., 2004).CYP1A1 gene is important for the activation of pre carcinogens (Ingelman-Sundberg et al., 2001).It is located in the 3' flanking region of the CYP1A1 gene, which is originally found to be associated with Lung cancer in Asians (Kawajiri et al., 1990).In our study, there is a lower frequency of CYP1A1 genotype (TT, TC, and CC) in Lung cancer patients (64.2%, 31.7%,4.1%) vs healthy controls (55.0%,37.8%, 7.1%) no significant association was found, which is contradictive for other study (Sobti et al., 2004;Sreeja et al., 2005).
Tobacco smoke contains numerous carcinogens, including PAHs such as benzo[α]pyrene (B[α]P), which may play an important role in lung carcinogenesis and exposures in experimental animals have shown to induce squamous cell carcinoma (Deutsch-Wenzel, 1983).Deletions at one or both of GST loci and, with consequent, less detoxification of xenobiotic toxic substances, an individual may become susceptible to diseases produced by toxic substances present in the environment; hence, this positive association raises the possibility that the two enzymes are working in tandem rather than in a complementary way.
While cigarette smoking is the main attributable factor for lung cancer, environmental pollution and asbestos are considered the other risk factors.However, these risk factors cannot explain all Lung cancer cases and there is a substantial body of epidemiological evidence linking occupational exposures to dusts, gases/vapours, and fumes to chronic airflow destruction, with a substantial population attributable risk (15-20%) in non-smokers as well (Meldrum, 2005).In our study, GSTT1 null genotype was significantly associated with Lung cancer in smoker and GSTM1 null genotype was significantly associated with Lung cancer in non-smoker patients; CYP1A1 homozygote TC/CC genotype showed a protective role with non-smoker Lung cancer patient.
In conclusion, the limitation of this study is small sample sizes and the fact that only a few genes involved in the detoxification of smoke products were studied.In conclusion, we found association of GSTT1 null polymorphism with Lung cancer in a northern India population.Moreover, we also found association between GSTT1 null with smoker and GSTM1 null in non smokers with Lung cancer.