Molecular and Cellular Biochemistry 266: 1–9, 2004. c 2004 Kluwer Academic Publishers. Printed in the Netherlands.
Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population R.C. Sobti,1,∗ S. Sharma,1 A. Joshi,1 S.K. Jindal2 and A. Janmeja3 1
Department of Biotechnology, Punjab University, Chandigarh, India; 2 Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India; 3 Department of Chest and Infectious Diseases, Government Medical College, Chandigarh, India Received 16 October 2003; accepted 16 January 2004
Abstract CYP1A1, CYP2E1, GSTM1 and GSTT1 polymorphisms were evaluated in north Indian lung cancer patients and controls. The estimated relative risk for lung cancer associated with the CYP1A1*2C allele was 2.68. Apart from the CYP1A1*2C genotype, there was no attributable risk in relation to other genotypes when analyzed singly. However, in the presence of a single copy of the variant CYP1A1 (CYP1A1*1/2A) and null GSTT1 genes, there was a three-fold increased risk for lung cancer; when stratified histologically the relative risk increased to 3.7 in case of SQCC. Similarly individuals carrying the mutant CYP1A1*2C genotype and single copy of the variant CYP1A1 Msp1 allele, had a relative risk of 2.85 for lung cancer. In case of the GSTM1 and CYP1A1 genotypes, null GSTM1 and variant Msp1 alleles had two-fold elevated risk for SQCC. On the other hand CYP1A1*2C and null GSTM1 genotype had a 3.5-fold elevated risk for SCLC. Stratified analysis indicated a multiplicative interaction between tobacco smoking and variant CYP1A1 genotypes on the risk for SQCC and SCLC. The heavy smokers (BI > 400) with CYP1A1*2C genotype were at a very high risk to develop SCLC with an OR of 29.30 (95% CI = 2.42–355, p = 0.008). Taken together, these findings, the first to be analyzed in north Indian population, suggest that combined GSTT1, GSTM1 and CYP1A1 polymorphisms could be susceptible to lung cancer induced by bidi (an Indian cigarette) smoking. (Mol Cell Biochem 266: 1–9, 2004) Key words: polymorphism, genotype, lung cancer, risk, smoking, individuals, histology Abbreviations: SQCC, squamous cell carcinoma; SCLC, small cell carcinoma; PAH, polycyclic aromatic hydrocarbons; NNN, nitrosonornicotine; NNK, 4-methyl nitroso-amino-1,3-pyridyl-1-butanone; OR, odds ratio; CI, confidence interval; BI, Brinkman index; Msp1, Moraxella species; Pst1, Providencia stuartii; Rsa1, Rhodopseudomonas sphaeroides; Dra1, Deinococcus radiophilus
Introduction Lung cancer accounts for 19% of all types of cancer and a mortality rate of 29% for all cancer related deaths particularly in men and is second only to breast cancer in women [1, 2]. The major cause of lung cancer is tobacco smoking, which is also associated with the risk of cancer of larynx,
mouth, esophagus, urinary bladder and kidney [3]. Tobacco smoke contains many carcinogens such as benzo(a)pyrene, PAHs, NNN and NNK [4]. In order to manifest their carcinogenic effects, most pro-carcinogens in tobacco smoke require metabolic activation by phase I enzymes represented by cytochrome P450 [5]. The activated intermediates thus produced are further converted into soluble moieties with
Address for offprints: Dr. R.C. Sobti, Department of Biotechnology, Punjab University, Chandigarh, India (E-mail:
[email protected])
2 the help of phase II enzymes represented by glutathione-Stransferases [6]. The genes expressing phase I and phase II enzymes are polymorphic and therefore, it is possible that individual variations in metabolic activities of each phase or in tandem coordination of both phases may regulate the clearance of toxic DNA intermediates and may be partially responsible for individual host susceptibility to lung cancer [7]. Cytochrome P450 is a large family of genes and has 72 different members with a large number of polymorphic forms [8]. CYP1A1 is key to the metabolic activation of PAHs found in tobacco smoke and is expressed in human lung cells [9]. This gene has several polymorphic forms [10]. These include a T → C transition in the 3 non-coding region (Msp1 polymorphism m1), which affects the transcriptional control elements involved in enzyme inducibility [11, 12]. The second polymorphism (m2) is an A → G transition in exon 7 of CYP1A1 locus results substitution of isoleucine to valine in the heme-binding region which increases the activity of microsomal enzyme [9, 13]. There are differences in the frequencies of different genotypes of CYP1A1 in different ethnic groups. Significantly higher frequencies of CYP1A1 m1 and m2 alleles have been reported among Asians as compared to Caucasians and African-Americans [14, 15]. Most of the reports from Japan and China indicate that the CYP1A1 m1 and m2 polymorphisms have indicated strong association with risk towards lung cancer [16–19]. These findings have not been confirmed in studies on Caucasian populations, where the frequency of m1 and m2 alleles is reportedly low [20–22]. However, larger prospective studies in mixed American populations do point out an elevated risk towards lung cancer in relation to m1 allele [4, 5, 23]. In Brazilian populations it is related to m2 allele [24, 25]. Similarly the CYP1A1 polymorphisms (Msp1 and CYP1A1*1/*2C) have also been associated with an elevated risk towards lung cancer in South-American populations such as Chilean [26]. Cytochrome P4502E1 polymorphisms may be important in human carcinogenesis as it has been demonstrated that CYP2E1 expression is dramatically enhanced by ethanol and benzene [27] and is also important in the metabolic activation of various nitosoamines, including the potent tobacco specific pro-carcinogen 4-(methylnitrosoamine)-1-(3-pyridyl)1-butanone [28, 29]. A CYP2E1 Dra1 restriction fragment length polymorphism (RFLP) located in intron 6 has been associated with lung cancer in a Japanese case–control study [27]. However subsequent studies failed to implicate the CYP2E1 Dra1 RFLP as a cancer risk factor in American whites and African-Americans [30]. Two other linked polymorphisms detectable with Pst1 and Rsa1 in the 5 flanking region affect the CYP2E1 transcription levels [31]. The association between CYP2E1 Pst1 or Rsa1 genotypes and lung cancer susceptibility has been evaluated in Finnish, US and Swedish populations but the results are inconsistent [32, 33].
Phase II enzymes are represented by glutathione-Stransferase and reveal several polymorphic forms such as GSTM1, GSTT1 and GSTP1 [34]. Both GSTM1 and GSTT1 are involved in the detoxification of tobacco related carcinogens such as epoxides, hydroxylated metabolites of benzo(a)pyrene, methyl-halogenoids and ethylene oxide [34]. Lung carcinogenesis could be related to the absence of the functional GSTM1 and GSTT1 genes, because of which tobacco related carcinogens fail to get detoxified [35]. Both the GSTM1 and GSTT1 genes are polymorphic in nature and with around 50% of Caucasians and Japanese have the null GSTM1 genotype [36]. The null genotype of GSTT1 ranges from 9 to 64% in different populations [8]. Several studies have shown that GSTM1 polymorphism is associated with an increased risk towards lung cancer [37–39]. Lack of the GSTT1 has been associated with bladder cancer [40], but not with lung cancer [41–43]. Little is known about the impact of CYP1A1, CYP2E1, GSTM1, and GSTT1 polymorphisms towards the risk for lung cancer in an ethnic Indian population. In view of the prevalence of bidi smoking (type of Indian cigarette) and increasing lung cancer incidence in India and a lack of data on the second biggest population of the world, we were prompted to carry out a case–control study to evaluate the role of these polymorphic genes as a genetic risk modifier in the etiology of lung cancer. To our knowledge this is the first such study on lung cancer on an Indian population.
Materials and methods Study subjects This case–control study consisted of 100 patients suffering from lung cancer and 76 general population-selected healthy controls. Lung cancer cases were recruited from patients undergoing bronchoscopy at the Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, and from the Department of Chest and Infectious Diseases, Government Medical College, Chandigarh. Informed consent was obtained from all the cases and controls before aspirating 4 ml of blood. At recruitment, each participant was personally interviewed to obtain a detailed information about smoking, dietary habits, alcohol consumption and demographic characteristics. The subjects selected in this study represented five out of the eight regions of north India. These include the states of Himachal Pradesh, Haryana, Uttar Pradesh, Punjab and Chandigarh. Unrelated healthy controls were recruited from blood donors who accompanied patients seeking treatment and from members of different community centers educational institutes and employee groups. People from all parts of northern India migrate to Chandigarh for employment but continue to maintain close
3 regional association through respective community centers. In this study only healthy subjects with no known case history of cancer and other major illness were recruited. The controls in this study were recruited in such a way that they were matched in concordance to the patient’s ethnic background. The controls selected in this study were matched by region in concordance to the lung cancer cases. In order to achieve an approximate balance of age, ethnicity and gender between cases and controls, we sampled potential controls based on the distribution of the above said factors among cases diagnosed.
CYP1A1 genotyping Genomic DNA was isolated from peripheral blood samples of cases and controls according to the protocol of Field et al. [44]. CYP1A1 genotyping for CYP1A1*1/*2A (Msp1) and CYP1A1*1/*2C (Ile-Val) polymorphisms were analyzed by PCR-based restriction fragment length polymorphism and allele-specific PCR methods [45]. The primers for the CYP1A1 (T3801C) mutation were P79 5-AAG AGG TGT AGC CGC TGC ACT and P80 5-TAG GAG TCT CTC ATG CCT which amplified a 335 bp fragment. Briefly genomic DNA was amplified in 1× PCR buffer (Sigma, St. Louis, MI, USA), 2 mM MgCl2 , 100 µg BSA, 200 µm dNTPs, 1.5 U Taq (MBI, Fermentas, Lithuania), 0.5 µM of each primer. Initial denaturation was carried out at 95 ◦ C for 5 min, followed by 30 cycles of denaturation for 1 min, annealing at 56 ◦ C for 1 min and extension at 72 ◦ C for 1 min in thermal cycler (Minicycler, MJ Research, USA). The PCR products were digested with 15 units of Msp1 restriction enzyme (Sigma, St. Louis, MI, USA) at 37 ◦ C for 3 h, and then subjected to electrophoresis in a 2.5% agarose gel (USB, Cleveland, OH, USA) and stained with ethidium bromide. The wildtype allele (CYP1A1*1) revealed a single band of 335 bps. The variant mutant allele (CYP1A1*2A) resulted in two fragments of 206 and 129 bps, whereas the heterozygous allele (CYP1A1*1/2A) showed three bands of 335, 206 and 129 bps. The A2455G (Ile-Val) polymorphism at exon 7 of the CYP1A1 gene was assessed by allele-specific PCR. For this, genomic DNA was amplified with primers (0.5 µM of each) IA-5 GAA AGG CTG GGT CCA TCT; 2A-5 AAG ACC TCC CAG CGG GCA AT and primer 2G-5 AAG ACC TCC CAG GCA AC (primer IA was commonly used each time) in the presence of 1× PCR buffer (Sigma, St. Louis, MI, USA), 2 mM MgCl2 , 100 µg BSA, 200 µm dNTPs, 1.5 U Taq (MBI, Fermentas, Lithuania). Initial denaturation was performed at 95 ◦ C for 5 min, followed by 25 cycles of denaturation at 95 ◦ C for 1 min, annealing at 63 ◦ C for 1 min and an extension of 72 ◦ C for 1 min. PCR products were analyzed in a 2% agarose gel (USB, Cleveland, USA) and stained with ethidium bromide. The PCR analysis resulted in a 322 bp
fragment with the classification wild allele (CYP1A1*1), heterozygote (CYP1A1*1/2C) and mutant (CYP1A1*2C). The CYP1A1*1/2C polymorphism analysis was repeated in duplicate.
PCR-RFLP analysis of CYP2E1 gene polymorphism The 5 flanking polymorphic site of the CYP2E1 gene was analyzed according to protocol of Hayashi et al. [31]. Briefly genomic DNA was amplified using the primers 5-CCA GTC GAG TCT ACA TTG TCA and 5-TTC ATT CTG TCT TCT AAC TGG in 1× reaction buffer (Sigma, St. Louis, MI, USA), 1.5 mM MgCl2 , 100 µg BSA, 200 µm dNTPs, 1.5 U Taq (MBI, Fermentas, Lithuania), 0.5 µM of each primer. Initial denaturation was performed at 95 ◦ C for 5 min, followed by 30 cycles of denaturation at 95 ◦ C for 1 min, annealing at 55 ◦ C for 1 min and extension at 72 ◦ C for 1 min in thermal cycler (Minicycler, MJ Research). PCR products were digested with 10 units of Pst1 restriction enzyme (Boheringer Manhiem, Germany) at 37 ◦ C overnight and then subjected to electrophoresis on a 2.5% agarose gel (USB, Cleveland, OH, USA) and stained with ethidium bromide. The genotypes of CYP2E1 were classified as homozygous wild (c1/c1) 410 bp, heterozygote (c1/c2) 410, 290, 120 bps and homozygous mutant (c2/c2) 290, 120 bps.
Multiplex PCR for GSTM1 and GSTT1 genotyping The GSTM1 and GSTT1 genotypes were analyzed by a multiplex PCR according to the protocol of Arand et al. [46]. Briefly genomic DNA was amplified by using six sets of primers GSTM1 (F) 5 -GAA CTC CCT GAA AAG CTA AAG C, GSTM1 (R) 5 -GTTGGG CTC AAA TAT ACG GTG G, GSTT1 (F) 5-TTC CTT ACT GGT CCT CAG ATC TC, GSTT1 (R) 5-TCA CCG GAT CAT GGC CAG CA albumin (F) 5 -GCC CTC TGC TAA CAA GTC CTA and albumin (R) CTA AAA AGA AAA TCG CCA ATC in 1× reaction buffer (Sigma, St. Louis, MI, USA), 2.5 mM MgCl2 , 100 µg BSA, 5% DMSO, 200 µm dNTPs, 2.5 U Taq (MBI, Fermentas, Lithuania), 0.5 µM of GSTM1, 0.3 µM of GSTT1 and 0.15 µM of albumin primers. Initial denaturation was carried out at 95 ◦ C for 5 min, followed by 30 cycles of denaturation at 95 ◦ C for 1 min, annealing at 59 ◦ C for 1 min, extension at 72 ◦ C for 1 min and a final extension at 72 ◦ C for 5 min in thermal cycler (Minicycler, MJ Research). The PCR products were then subjected to electrophoresis on a 2.5% agarose gel (USB, Cleveland). The presence of a 480 and 215 bps was indicative of the GSTT1 and GSTM1 genotypes; whereas the absence of the product indicated the null genotype. Albumin was used as an internal control.
4 Table 1. Distribution of demographic variables for patients and controls Patients (n = 100)%
Controls (n = 76)%
Male (%)
95 (95%)
73 (96.1%)
Female (%)
5 (5%)
3 (3.9%)
Mean age (±S.D.)
55.5 ± 11.3
50.9 ± 8.1
Range
27–80
28–65
Variables Gender
Age (years)
Smoking status Non-smoker (%)
14 (14%)
17 (22.4%)
Smoker (%)
86 (86%)
59a (77.6%)
Light smokers
14 (16.3%)
16 (27.1%)
Heavy smokers
72 (83.7%)
43 (72.9%)
Histology Squamous cell carcinoma
71 (71%)
Small cell carcinoma
24 (24%)
Adenocarcinoma
4 (4%)
Large cell carcinoma
1 (1%)
a Two-sided
χ 2 -test, p < 0.0001.
Statistical analysis SPSS and Epical 2000 (version 1.02) were used for statistical analysis. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. The OR was adjusted for age, sex and smoking by using logistic regression analysis. All data were considered significant when p < 0.05.
Results The relevant characteristics of the subjects studied are shown in Table 1. The average age of cases was 55.5 (±11.3) and for controls it was 50.9 (±8.1). No significant difference in the gender distribution was observed between cases and controls. Although an effort was made to obtain a frequency match on smoking status between cases and controls, more smokers
were presenting the cancer group as compared to controls (86 and 77.6%, two-sided χ 2 -test, p < 0.0001), respectively. Moreover, the cancer cases had a higher value of smoking index (heavy smokers BI > 400) than controls: 83.7% of cases in comparison to 72.9% controls. The detailed analyses of the CYP1A1, GSTM1, and GSTT1 polymorphisms are given in Table 2. Among cases and controls, the frequency of both the heterozygous and homozygous Msp1 variant allele (CYP1A1*1/2A and CYP1A1*2A) was 51 and 44.8%, respectively, thus the lung cancer group had a higher frequency of the variant Msp1 alleles. The frequency of the CYP1A1*1 (Ile-Ile) allele was 4% in cases and 10.5% in controls and for the CYP1A1*2C (Val-Val) allele it was 29 and 19.7%. The CYP2E1 genotype revealed variant allele neither in cases nor in controls. The frequencies of GSTM1 and GSTT1 null genotypes were 38 and 18%, respectively, in cancer patients. As is evident in Table 2 the proportion of both GSTM1 and GSTT1 null genotypes increased in lung cancer group as compared to controls. Table 3 presents the ORs and 95% CI for all lung cancer and specific histological cell types by CYP1A1, GSTM1 and GSTT1 genotypes. Since the frequency of the CYP1A1*2A allele was low, on combining the heterozygous (CYP1A1*1/2A) and mutant (CYP1A1*2A) alleles as a single genotype, no significant association was found between CYP1A1 Msp1 genotype and lung cancer. For the CYP1A1 (Ile-Val) genotypes, the individuals who carried both the mutant alleles, i.e. (CYP1A1*2C) were at a 2.5-fold elevated risk for lung cancer (OR = 2.68; 95% CI; 0.64–11.27, p = 0.0451). A statistically significant association was found between CYP1A1*2C allele and SCLC (OR = 4.06; 95% CI; 1.53–10.83, p = 0.008), but not for SQCC (OR = 1.18; 95% CI; 0.54–2.61, p = 0.831). The presence of at least one copy of the Msp1 variant (CYP1A1*1/2A) and CYP1A1*2C allele of the CYP1A1 genotype was associated with an elevated risk with an OR of 2.85 (95% CI = 0.22–2.33) (data not shown). When stratified according to histology, there was a strong association for SCLC with an OR of 3.24 (95% CI = 0.87–12.08); even for SQCC
Table 2. Distribution of cases and controls with CYP1A1*1/*2A, CYP1A1*1/*2C, GSTM1 and GSTT1 genotypes CYP1A1 (Msp1)
Controls
CYP1A1 (CYP1A1*1/*2C) GSTM1 and GSTT1
CYP1A1 *1/*1
CYP1A1 *1/*2A
CYP1A1 *2A/*2A
CYP1A1 *1/*1
CYP1A1 *1/*2C
CYP1A1 *2C/*2C
GSTM1 (+)
GSTM1 (−)
GSTT1 (+)
GSTT1 (−)
42 (55.3%)
29 (38.2%)
5 (6.6%)
8 (10.5%)
53 (69.7%)
15 (19.7%)
52 (68.4%)
24 (31.6%)
65 (85.5%)
11 (14.5%)
Lung cancer
49 (49%)
45 (45%)
6 (6%)
4 (4%)
67 (67%)
29 (29%)
62 (62%)
38 (38%)
82 (82%)
18 (18%)
SQCC
32 (45%)
36 (50.8%)
3 (4.22%)
4 (5.6%)
50 (70.4%)
17 (23.9%)
42 (59.15%)
29 (40.48%)
56 (78.8%)
15 (21%)
SCLC
14 (58.3%)
8 (33.3%)
2 (8.33%)
–
12 (50%)
12 (50%)
15 (62.5%)
9 (37.5%)
22 (91.6%)
Others
3 (60%)
1 (20%)
1 (20%)
–
5 (100%)
–
4 (80%)
4 (100%)
–
2 (8.33%) 1 (20%)
5 Table 3. ORs and (95% CI) for lung cancer by CYP1A1, GSTM1 and GSTT1 genotypes All (100/76)
SQCC
SCC
CYP1A1 (Msp1)
OR1 (95% CI)1
OR1 (95% CI)2
OR1 (95% CI)2
CYP1A1*1/*1
1.0
1.0
1.0
CYP1A1*1/2A
1.16 (0.60–2.22)
1.39 (0.67–2.86)
0.79 (0.28–2.22)
CYP1A1*2A
1.17 (0.32–4.23
0.85 (0.18–4.05)
1.27 (0.22–7.52)
CYP1A11/2A and
1.16 (0.62–2.15)
1.30 (0.65–2.62)
0.86 (0.33–2.25)
CYP1A1*2A (combined)
p = 0.5
p = 0.28
p = 0.98
CYP1A1*1/*1
1.0
1.0
1.0
CYP1A1*1/2C
2.29 (0.64–11.27)
1.52 (0.41–5.62)
CYP1A1*2C
2.68 (0.64–11.27)
1.01 (0.54–2.61)
4.06 (1.53–10.83)
p = 0.004
p = 0.83
p = 0.008
GSTM1 (+)
1.0
1.0
1.0
GSTM1 (−)
1.22 (0.64–2.36)
1.5 (0.76–2.94)
1.1 (0.50–3.39)
p = 0.46
p = 0.32
p = 0.67
GSTT1 (+)
1.0
1.0
1.0
GSTT1 (−)
1.23 (0.52–2.87)
1.6 (0.67–3.73)
0.54 (0.11–2.64)
p = 0.67
p = 0.40
p = 0.66
CYP1A1 (Ile-Val)
1 OR,
–
odds ratio was adjusted for age, gender and smoking habits. CI), confidence interval.
2 (95%
there was a two-fold risk with an OR of 1.86 (95% CI = 0.61– 5.67). On the whole, the risk factor was more pronounced for SCLC than SQCC (data not shown). The combined effects of GSTT1 and CYP1A1 genotypes for all lung cancer and those of specific cell type were also analyzed (data not shown). The presence of at least one copy of the Msp1 variant CYP1A1*1/2A allele and null GSTT1 genotype had a three-fold increased risk for all lung cancer (OR = 2.91, 95% CI; 0.74–11.4, p = 0.14) and 3.7-fold risk towards SQCC which was found to be significant (OR = 3.7, 95% CI; 0.88–15.6, p = 0.04), but was not associated with SCLC (OR = 0.80, 95% CI; 0.07–8.70). The CYP1A1*2C allele and null GSTT1 genotype had an OR of 1.64 (95% CI = 0.39–6.90) for all lung cancer. A combination of CYP1A1*2C allele and GSTT1 (+) genotype was found to have an approximately four-fold risk towards SCLC (OR = 3.78, 95% CI; 1.31– 10.91, p = 0.01) which was statistically significant but on the other hand such a combination was not found to be strongly associated with SQCC. Table 4 shows the risk of developing lung cancer in relation to CYP1A1, GSTM1 and GSTT1 genotypes and smoking. In India, bidi (type of Indian cigarette) is the main mode of smoking. Bidi takes the leading position followed by cigarette, hookah and cigar. In this study all the subjects were bidi smokers. Individuals who were heavy smokers and
carrying the CYP1A1*2C alleles were at a 29-fold increase in risk towards lung cancer, which reached statistical significance ( p = 0.008). When the subjects were stratified according to lifetime smoking history, the GSTM1 null genotype in heavy smokers had slight elevation in risk of lung cancer (OR = 2.01; 95% CI, 0.63–6.35). The GSTT1 null genotype had a higher risk of lung cancer both in heavy (OR = 4.34, 95% CI; 0.70–27.03) and non (OR = 3.87; 95% CI, 0.56–26.8) smokers. The risk of developing SQCC in relation to smoking and its interaction with the various genotypes has also been analyzed (data not shown). Individuals with the CYP1A1*2C alleles and heavy smokers (BI > 400) had a 1.5-fold risk over nonsmokers (OR = 1.54, 95% CI; 0.37–6.43). For the GSTT1 null genotype, it was found that the non-smokers carrying the null gene were at a higher risk for SQCC as compared to light and heavy smokers with an OR of 6.38 (95% CI = 0.73–55.9). On stratifying the subjects according to lifetime smoking history, a slight elevation in risk to SQCC (OR = 1.63; 95% CI, 0.49–5.44) in relation to GSTM1 null genotype was observed in heavy smokers (BI > 400) but this did not reach statistical significance. The risk of developing SCLC in relation to smoking and its interaction with the various genotypes has also been analyzed (data not shown). Individuals with the CYP1A1*2C
6 Table 4. Odds ratio of developing lung cancer with CYP1A1*1/*2C, CYP1A1*1/*2A, GSTM1 and GSTT1 genotypes stratified by smoking status
CYP1A1 (Ile-Val)
CYP1A1 (Msp1)
GSTM1
GSTT1
Genotypes
Non-smoker; OR1 (95% CI)2
Light smoker; OR1 (95% CI)2
Heavy smoker; OR1 (95% CI)2
CYP1A1*1 and CYP1A1*2C
1
1.41 (0.44–4.59)
1.17 (0.44–3.07)
CYP1A1*2C
0.64 (0.02–7.2)
0.77 (0.18–3.31)
3.75 (1.06–13.26)∗
CYP1A1*1
1
0.61 (0.14–2.68)
1.09 (0.29–4.06)
CYP1A1*1/2A and CYP1A1*2A
0.53 (0.10–2.74)
1.55 (0.36–6.66)
1.74 (0.48–6.35)
Present
1
1.28 (0.36–4.53)
1.34 (0.47–3.88)
Null
1.01 (0.14–7.47)
0.94 (0.23–3.94)
2.01 (0.63–6.35)
Present
1
1.56 (0.48–5.12)
2.05 (0.73–5.77)
Null
3.87 (0.56–26.8)
1.19 (0.20–7.26)
4.34 (0.70–27.03)
1 (OR),
odds ratio was adjusted for age, gender and smoking habits. CI), confidence interval. ∗ p = 0.048. 2 (95%
alleles and heavy smokers (BI > 400) had a very high risk for developing SCLC with an OR of 29.30 (95% CI = 2.42– 355, p = 0.008). On the whole due to fewer subjects no clear-cut relationship could be advocated between smoking and risk towards different histological cell types especially for SCLC and lung cancer.
Discussion In the present study the prevalence of genetic polymorphism in the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and their association with risk to lung cancer in a north Indian population have been investigated. It has been found that the CYP1A1*2C allele is strongly associated with a risk to lung cancer with an OR of 2.68. There is a four-fold increased risk for SCLC, but not for SQCC for the CYP1A1*2C allele. Sugimura et al. [18] have also observed an elevated risk for SCLC and CYP1A1*2C allele. The proportion of individuals with CYP1A1*1/2C polymorphism varies with ethnicity. The frequency of CYP1A1*2C allele is considerably lower in Caucasians than Japanese [47]. In contrast, the present study, however, has observed a higher frequency of the CYP1A1*2C and CYP1A1*1/2C alleles. The results obtained here for the CYP1A1*1/2C are not in agreement with the Hardy-Weinberg equilibrium. Similarly, Hong et al. [48] had also observed a higher frequency of CYP1A1*1/2C allele in Korean population, and their results did not obey the Hardy-Weinberg equilibrium equation. A study carried out on oral cancer patients in south India reported a high frequency of CYP1A1*1/2C and CYP1A1*2C alleles as compared to the controls [49]. A higher frequency of both the heterozygous and mutant genotypes has also been observed in the Chilean lung cancer cases
by Quinones et al. [26]. However, as far our knowledge goes, this is the first study reporting a high representation of the CYP1A1*2C allele both in cases and controls (29 and 19.7%) in an Indian population. In this study but for a moderate increase towards SQCC, no significant association was found between lung cancer and combined variant alleles of CYP1A1 Msp1 genotype. This association of the mutant CYP1A1 (CYP1A1*2A) genotype with increased risk of SQCC has been observed in Japanese, Caucasian and Hawaiian populations [4, 50]. The results from this study point out an elevated risk among individuals with combined CYP1A1*2C and variant Msp1 alleles (CYP1A1*1/2A). The risk was more pronounced in SCLC than SQCC. In Japanese, the association was clearly stronger for SQCC [47, 51, 52]. In contrast, studies in CYP1A1 and lung cancer conducted on Caucasians have mostly been inconsistent with some early studies finding no association [20, 21, 51] and more recent ones reporting an increased risk with the variant alleles [23, 54]. The prevalence of Msp1 genotype is increased in lung cancer as a consequence of linkage by Ile-Val genotypes [55]. Zhang et al. [56] have reported that the isoleucine → valine substitution alone does not increase the enzymatic activity, but suggested that the Ile-Val genotype in linkage with Msp1 genotype might result in an increased inducibility risk of lung cancer. This implies that these polymorphisms might cause higher concentration of enzymes in vivo, due to enhanced inducibility. Thus it might be possible that in an Indian population, the presence of at least a single copy of variant Msp1 allele in combination with a homozygous mutant Val-Val genotype (CYP1A1*2C) might increase the metabolic activation of pro-carcinogens to ultimate carcinogens. Hence CYP1A1 polymorphisms may not only be playing an important role
7 for lung cancer risk in south east Asian populations, but to a great extent in Indian population too. Subjects with the null genotype for GSTM1 and GSTT1 had no significant statistical lung cancer risk. However, there was a slightly higher risk for null GSTT1 gene and SQCC development but this did not reach statistical significance. It has been hypothesized that the combination of increased metabolic activation by the phase I and decreased detoxification by phase II enzymes might lead to an elevated risk of lung carcinogenesis [57]. Thus, we further analyzed the combined effects of CYP1A1*1/2A and CYP1A1*2A alleles and GSTT1 null genotype. A statistically strong association was found. As for the CYP1A1*2C allele and GSTT1 genotypes, the risk was more pronounced for SCLC than SQCC. Very few studies have seen the relation between GSTT1 and CYP1A1 genotypes. A Japanese study has pointed out a higher risk for lung cancer with the null GSTT1 and CYP1A1 variant alleles [17]. The null GSTM1 genotype and CYP1A1*1/2A allele pointed a trend towards a slight risk for lung cancer however, such an association was more pronounced for SQCC. Similar observations have been made in Japanese [16, 17]. Even in the Caucasians, there was a two–three-fold risk towards lung cancer with such a genotype [4, 5]. The null GSTM1 genotype and CYP1A1*2C allele were slightly associated with risk of lung cancer. Such an association was found to be 3.5-fold in SCLC. Similar observations have been made in Japanese [16, 17]. Bidi (type of cigarette) is the most prevalent form of smoking. Unlike cigarettes, these are non-filtered. Manufactured in India, bidis consist of tobacco wrapped in a tendu or temburni leaf. Being cruder form of tobacco, it has a higher concentration of tar and nicotine and is thus more carcinogenic than cigarette. The present data clearly indicates that individuals who were heavy smokers (BI > 400) and had CYP1A1*2C allele, were at 29-fold risk for lung cancer. The data are consistent with the studies by Song et al. [10] who had found a strong tendency of increased risk of lung cancer for heavy smokers with the CYP1A1*2C allele. Other reports however have demonstrated a stronger tendency for light smokers [17, 18]. This risk factor was found to be highly associative in SCLC than SQCC. When the GSTM1 null gene was considered with lifetime smoking exposure, a slight elevation in risk of lung cancer in more casual smokers (BI > 400) was observed. Several epidemiological studies, have evaluated the interaction between GSTM1 genotype and cumulative smoking, but have reported contradictory results [58]. Stronger associations for more casual smokers were found in four studies [37], whereas other studies found a strong association with a low smoking exposure [17] and one failed to document such a difference [36]. With respect to lifetime tobacco exposure and lung cancer risk in relation to null GSTT1 gene, an increased risk at a heavy smoking dose was observed. This association was not
only restricted to heavy smoking group, but to non-smokers also. In the case of SQCC, non-smokers had an approximately 6.5-fold risk towards lung cancer, whereas, for SCLC it was difficult to have a clear-cut relation between smoking and its interaction with null GSTT1 genotype since no non- and light smokers were found in this study. Jourenkova et al. [59] reported a high risk towards laryngeal cancer at a low level of tobacco exposure and null GSTT1 gene. A few studies, carried out in this regard, have failed to provide clear-cut explanations. The present study clearly demonstrates the lack of association of CYP2E1 Pst1 polymorphism with lung cancer risk in north Indian population. Both in cases and controls, not a single variant allele was observed. Several studies have reported that the variant alleles are associated with enhanced enzyme activity among Asians and with lower activity among Caucasians, thus susceptibility to cancer is different [30]. There is a possibility that in our study the Pst1 polymorphism is not linked to lung cancer. It is however possible that other genetic polymorphic sites such as Dra1 and Taq1 of CYP2E1 Pst1 might have an association with risk of lung cancer. Further work on these polymorphic sites of CYP2E1 and their phenotypes is warranted in Indian population.
Conclusions The present study, the first one to be carried out in an ethnic north Indian population, indicates that apart from the CYP1A1*1/2C polymorphism the role of the other polymorphic genes such as CYP1A1 (Msp1) CYP2E1, GSTT1 and GSTM1 when analyzed as a single genotype has no significant association with lung cancer. But when these genotypes, like both the CYP1A1 polymorphic alleles are combined, they do point out to an increased risk towards lung cancer, especially SCLC and SQCC. Combined genotyping of susceptible CYP1A1, GSTT1 and GSTM1 genes revealed a higher risk than that ascribed to a single susceptible gene with the association being strongest for CYP1A1 Msp1 variant alleles and null GSTT1 gene than the GSTM1 null gene and similar CYP1A1 genotype. Further these data, as given, provide additional evidence that these polymorphic genes are an important determinant in susceptibility to bidi smoking induced lung carcinogenesis. These data may also support the hypothesis that susceptibility to certain cancer may depend upon ethnic-specific gene polymorphisms. As India has a rich diversity of ethnic population with different life styles, such a study might provide an impetus to conduct larger prospective studies involving different ethnic Indian populations and help in generating information about the factors for which particular sub-population is at a larger risk for lung cancer. The inconsistency between our results and other Asian populations, such as Japanese, Chinese, Korean and Taiwanese,
8 might be ascribed to different environmental factors. There are however certain limitations in our study, firstly the small number of cases and controls and thus the statistical power of the study is not very high so to confirm whether some genes can act as true susceptibility genes for lung cancer. Secondly our study did not have a sufficient number of subjects to allow the precise estimation of genotype–smoking interaction. This is especially important because it is likely that these polymorphic genes do not act in isolation, and evaluation of multiple genes interacting with the exposure may be further required to understand the phenomenon of gene–smoking interaction. However since this is the first study to be conducted in north Indian population, a possible trend towards lung cancer risk and role of polymorphic genes has emerged which needs further investigation with more case–control studies.
Acknowledgements We thank Dr. T. Katoh, Miyazaki Medical School, Miyazaki University, Miyazaki, Japan, for the statistical analysis.
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