The effect of CYP1A1,GSTT1 and GSTM1 polymorphisms on the risk of lung cancer

Abstract

Lung cancer, which is mainly affected by environmental factors, is a lethal malignancy. It is also important to investigate the effect of genetic factors on lung cancer aetiology. In this study, we aimed to investigate the distribution of CYP1A1*2C, GSTT1 and GSTM1 polymorphisms in Turkish lung cancer patients to determine whether any promoting effect of polymorphisms could cause development of lung cancer. For this purpose, genomic DNA samples obtained from peripheral blood of 128 patients with lung cancer and 122 healthy subjects were analyzed. Genotyping of polymorphic enzymes were carried out by polymerase chain reaction–restriction fragment length polymorphism methods. Although there were no significant differences between groups in terms of CYP1A1 polymorphism, the carriers of CYP1A1 Ile/Val genotype (odds ratio [OR] = 1.224, 95% confidence interval [CI]: 0.585–2.564) or CYP1A1 Val/Val genotype (OR = 3.058, 95% CI: 0.312–30.303) had an increased risk of lung cancer development. There was no statistical difference between groups in terms of both GSTT1 null genotype (OR = 1.114, 95% CI: 0.590–2.105) and GSTM1 null genotype (OR = 0.776, 95% CI: 0.466–1.290). This is the first case–control study investigating CYP1A1 Ile/Val, GSTT1 and GSTM1 polymorphisms in Turkish lung cancer patients. Although we suggest that other genes in addition to the proposed genes could play a role in lung cancer development, the results of our study will contribute to the possible associations between CYP1A1 Ile/Val, GSTT1 and GSTM1 gene polymorphism on the risk of lung cancer.

Keywords

lung cancer

Introduction

The incidence of cancer has increased in recent years.¹ The observed difficulties in conventional therapy models of cancers lead not only to develop the new therapy protocols but also to determine the specific risk factors for each cancer type. It is known that the general risk factors for cancer are environmental components as well as genetic and epigenetic modulators.¹ Lung cancer, which is mainly affected by environmental factors, is a lethal malignancy.² Although the major risk factor for lung cancer is smoking,^3,4 it is known that nonsmokers and fewer than 20% of smokers are exposed to lung cancer.^3,5 For this reason, it is considered that genetic factors may also be the risk factors in lung cancer aetiology.⁶

Phase I and phase II enzymatic pathways play an important role at carcinogen metabolism in cell. Phase I enzymes convert precarcinogens to carcinogens, and Phase II enzymes detoxify the activated intermediates that are formed from Phase I reactions. Thus, activated intermediates are converted into soluble moieties and are easily exerted from the membrane of cell. The level of activated intermediates is dependent on the metabolic activity of these enzymes. It is suggested that increased phase I activity and decreased phase II activity could cause an increase in cancer risk.^7,8 Genetic polymorphisms could change the enzymatic activity of enzymes. Polymorphic genes encoding enzymes that are responsible for activating and detoxifying of carcinogenic molecules could contribute to explain the genetic susceptibility to cancer individually.

Cytochrome 1A1 (CYP1A1), a member of human phase I CYP P450 enzymes, has an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) and aromatic amine, which are included in tobacco.⁹ The intermediate products generated by Phase I enzymes could form DNA adducts by covalently binding to DNA and thus promote the beginning of carcinogenic pathways.¹⁰ Functional genetic polymorphisms have been reported in CYP1A1 gene locus, including CYP1A1*2C polymorphism at codon 462 in exon 7. CYP1A1*2C polymorphism is an A-G transition resulting in Ile462Val exchange in enzyme structure and increased metabolic activation.¹¹

Enzymes of Glutathione S-transferase (GST) family are phase II enzymes, which detoxify the carcinogenic metabolites by conjugation reactions. GSTT1 and GSTM1 are polymorphic forms of GST enzymes, and these play an important role in detoxification of carcinogenic metabolites derived from tobacco smoke. Although it has been indicated that GSTT1 and GSTMI null polymorphisms could increase the cancer risk in most of the studies, the association between GSTT1, GSTM1 polymorphism and lung cancer susceptibility is still contradictory.¹²

As both the environmental toxic and carcinogenic factors (especially tobacco smoking) and genetic factors are important for lung cancer development, we aimed to investigate the distribution of CYP1A1*2C, GSTT1 and GSTM1 genotypes in Turkish lung cancer patients to determine whether any promoting effect of polymorphisms could cause the development of lung cancer.

Materials and methods

Study population

This case–control study was approved by Review Boards of two different local ethical committees, the Institutional Ethical Committee of Kırıkkale University, Faculty of Medicine and Atatürk Chest Diseases and Chest Surgery Training and Research Hospital, Ankara, Turkey. The ethnicity of both patients and controls were Turkish. While all patients with primary lung cancer were diagnosed by pathologists in two hospitals, controls were chosen from the subjects who have no other diseases and cancer history. A total of 128 patients and 122 controls were recruited in this study. All subjects were informed about the study and signed the informed consent to participate in the study.

DNA extraction

Genomic DNA of peripheral blood was isolated with phenol–chloroform extraction and ethanol precipitation.¹³ These DNA samples were stored at −20°C for genotype analysis.

Genotyping of CYP1A1

CYP1A1 genotype was analyzed by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) method as described previously.¹⁴ The primers for the CYP1A1 are F5′-CTG TCT CCC TCT GGT TAC AGG AAG C -3′ and R5′- TTC CAC CCG TTG CAG CAG GAT AGC C -3′, which generate a 204-base pair (bp) fragment. In the thermocyling procedure, initial denaturation was performed at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 1 minute, extension at 72°C for 1 minute and a final extension at 72°C for 10 minutes in thermal cycler (Biometra, Fermentas, Germany). The PCR products were digested overnight with BsrDI (Fermentas) for RFLP analysis. The RFLP products were analyzed in 2% agarose gel with ethidium bromide. The size of the products and their assigned genotyping were 149 and 55 bp, AA (homozygous wild type); 204, 149 and 55 bp products, AG (heterozygous) and only 204 bp products, GG (homozygous mutant).

Genotyping of GSTT1 and GSTM1

GSTT1 and GSTM1 genotypes were analyzed by a multiplex PCR. Genomic DNA was amplified using different primers.¹⁵ The primers of GSTT1 and GSTM1 are F5′- TTC CTT ACT GGT CCT CAC ACT TC-3′ and R5′-TCA CCG GAT CAT GGC CAG CA-3′, which generate a 480-bp fragment product, and F5′-GAA CTC CCT GAA AAG CTA AAG C-3′ and R5′-GTT GGG CTC AAA TAT ACG GTG G-3′, which generate a 215-bp product. β-Globulin gene was used as an internal control. The primers for the β-globulin are F5′-CAA CTT CAT CCA CGT TCA CC-3′ and R5′-GAA GAG CCA AGG ACA GGT AC-3′, which generate a 268-bp fragment product. Initial denaturation was carried out at 94°C for 4 minutes and then followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 45 seconds, extension at 72°C for 1 minute and a final extension at 72°C for 10 minutes in thermal cycler (Biometra). The PCR products were then subjected to electrophoresis on 2% agarose gel with ethidium bromide. The presence of 480 and 215 bps was indicative of the presence of GSTT1 and GSTM1 genotypes, respectively. Absence of these products indicates the null genotype for GSTT1 and GSTM1 genes. In order to be sure about GSTs gene polymorphism, GST polymorphisms were studied three times.

Statistical analysis

CYP1A1 genotype distribution was examined for a significant departure from the Hardy–Weinberg equilibrium by the likelihood ratio test (G statistics). In order to compare two independent groups for categorical variables, chi-square test was used. Patient and control groups were compared in terms of age by Student’s t test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression analysis, where adjustment was made for age, gender and smoking status. A multiple logistic regression was performed to identify the independent variables (CYP1A1, GSTT1 and GSTM1) in terms of lung cancer. SPSS for Windows 11.5 (SPSS Inc, Chicago, IL, USA) was used for statistical analysis; p < 0.05 was considered statistically significant.

Results

Although the study was carried out on 128 patients and 122 controls, we could not determine the CYP1A1, GSTT1 or GSTM1 polymorphisms for some of the patients or controls. Thus, there could be some discrepancies between the numbers in tables.

For CYP1A1*2C polymorphism, the genotype distributions in the controls and patients were in agreement with the Hardy–Weinberg equilibrium (p = 0.100 and p = 0.584, respectively). Demographic characteristics of the subjects are shown in Table 1. Although there was no statistically significant difference between groups in terms of age (p > 0.05), gender distribution and the proportion of smoking were different (p < 0.001). While 93.0% of the patients were male, 61.3% of controls were female. Also, the proportions of smoking subjects were 89.5% and 19.8% for the patients and controls, respectively. The risk of lung cancer increased by 21-fold in males compared to females and by 34-fold in smokers compared to nonsmokers.

Table 1.

Main characteristics of the study groups

Variable	Cases	Controls	OR	95% CI	p Value
Age (in years)	58.86 ± 8.02^a; 58.5 (31–76)^b	57.22 ± 8.44^a; 56 (40–84)^b
Gender
Female, No. (%)	9 (7.0)	73 (61.3)	1
Male, No. (%)	119 (93.0)	46 (38.7)	20.983	9.701–45.387	0.000*
Smoking status
Non smoker, No. (%)	12 (10.5)	97 (80.2)	1
Smoker, No. (%)	102 (89.5)	24 (19.8)	34.483	16.393–71.429	0.000*

CI: confidence interval, OR: odds ratio.

^a Mean ± standard deviation.

^b Median (minimum–maximum).

^* p<0.001

The genotype distribution (CYP1A1, GSTT1 and GSTM1) among cases and controls were shown in Table 2. The frequency for CYP1A1 Ile/Ile, Ile/Val and Val/Val genotypes were similar between groups (86.5%, 12.7% and 0.8% in control group and 82.6%, 14.9% and 2.5% in patient group, respectively). Although there were no statistically significant differences between the groups in terms of CYP1A1, the carriers of CYP1A1 Ile/Val (AG) genotype (OR = 1.224, 95% CI: 0.585–2.564) or CYP1A1 Val/Val (GG) genotype (OR = 3.058, 95% CI: 0.312–30.303) had an increased risk of lung cancer development. Also, the frequency of CYP1A1 Ile allele was 90.1% in patients and 92.8% for controls, which indicates that persons carrying Val allele had an increased risk of lung cancer (OR = 1.418, 95% CI: 0.741–2.717), but this difference was not statistically significant.

Table 2.

Genotype distribution among cases and controls

Genotype (CYP1A1)	Cases, No. (%)	Controls, No. (%)	OR	95% CI	p Value
Ile/Ile	100 (82.6)	102 (86.5)	1
Ile/Val	18 (14.9)	15 (12.7)	1.224	0.585–2.564	0.591
Ile/Val	18 (14.9)	15 (12.7)	1.783	0.485–6.536^a	0.383^a
Val/Val	3 (2.5)	1 (0.8)	3.058	0.312–30.303	0.621
Val/Val	3 (2.5)	1 (0.8)	1.748	0.058–52.632^a	0.749^a
Ile/Val + Val/Val	21 (17.4)	16 (13.5)	1.339	0.661–2.710	0.417
Ile/Val + Val/Val	21 (17.4)	16 (13.5)	1.779	0.516–6.135^a	0.361^a
Allele
Ile	218 (90.1)	219 (92.8)	1
Val	24 (9.9)	17 (7.2)	1.418	0.741–2.717	0.289
Genotype (GSTT1)
Present	102 (80.3)	100 (82.0)	1
Null	25 (19.7)	22 (18.0)	1.114	0.590–2.105	0.739
Null	25 (19.7)	22 (18.0)	1.112	0.409–3.021^a	0.835^a
Genotype (GSTM1)
Present	72 (57.6)	59 (51.3)	1
Null	53 (42.4)	56 (48.7)	0.776	0.466–1.290	0.328
Null	53 (42.4)	56 (48.7)	0.794	0.322–1.957^a	0.617^a

CI: confidence interval, OR: odds ratio.

^aOR adjusted for age, gender and smoking status.

The frequency of subjects carrying the GSTT1 null genotype was slightly higher in the patient group (19.7%) compared with controls (18.0%). However, there was no statistically significant difference between groups in terms of GSTT1 null genotype (OR = 1.114, 95% CI: 0.590–2.105). GSTM1 genotype frequency was similar in patient and control groups (42.4% and 48.7%, respectively) and this difference was not statistically significant (OR = 0.776, 95% CI: 0.466–1.290; Table 2).

When the multiple logistic regression was performed to identify the independent variables (CYP1A1, GSTT1 and GSTM1) in terms of lung cancer, neither the main effects of genotypes nor their interactions could be found as statistically significant. Thus, we did not give the risks of combined effects of these genotypes in detail.

Discussion

Lifestyle, diet, environmental carcinogens and toxic molecules play an important role in the pathophysiology of lung cancer. As tobacco includes highly carcinogenic molecules that was detoxified by phase I and phase II enzymes in cell, smoking is one of the most important factors in lung cancer.¹⁶ Also, there is an evidence that the mutations and/or polymorphisms in the genes that code these enzymes could change the activity of these enzymes. The metabolic balance between activating phase I and detoxifying phase II enzymes could be used to determine the lung cancer susceptibility of individuals.¹⁷ Several genes, such as CYP1A1, GSTT1 and GSTM1, which are related to lung cancer susceptibility, were suggested by many researchers.^3,18–24

CYP1A1 is a phase I enzyme and activates PAH that is included in tobacco.²⁵ CYP1A1 polymorphic Val allele increases enzymatic activity of enzyme. The production of carcinogenic molecules could increase in the presence of polymorphic genotypes of CYP1A1 gene and therefore an increased risk of lung cancer could be expected. It was reported in several researches and meta-analysis studies that there was an association between CYP1A1 variant genotype and lung cancer risk in different populations.^{20,22,26–28} While the results of our study are consistent with the literature, we could not find any statistical differences.

The frequency of CYP1A1 variant alleles is higher in Far Eastern populations than Caucasians in both healthy controls^{6,16,23,29–33} and patients with lung cancer.^{3,11,21,24,34} The frequency of CYP1A1 variant genotypes in present study is found to be between Far Eastern and Caucasian populations, and this result is consistent with that of another study carried on same population.³⁵ As the Anatolian region is acted as a bridge during the development of mankind and as it got lots of immigrants from both Fareast Asian and Europe regions,³⁶ it is expected to find a frequency of CYP1A1 Ile/Val for Anatolian region, which is between Europe and Fareast regions.

The free radical scavenging enzymes, GSTT1 and GSTM1, have an important role in detoxifying systems due to their ability to convert the carcinogenic compounds to excretable metabolites. There is evidence that GSTs are candidate genes on cancer susceptibility. As GST enzymes malfunction in the case of GST homozygous null polymorphism, the risk of cancer may increase in an individual who have GSTs null polymorphisms.³⁷ Although we found that GSTT1 null genotype in patients was slightly higher than controls, this difference was not statistically significant. However, the frequency of GSTM1 was a bit higher in controls compared to patients. While the results were similar to those of Ye et al. in terms of GSTT1, the frequency of GSTM1 null genotype in cases was higher than controls.³⁸ Although the frequency of GSTT1 was higher for controls and that of GSTM1 was higher for patients, no statistically significant results were found in the study by Yang et al.⁶ In other meta-analyses, it was stated that GSTM1 null variant is associated with increased risk of lung cancer.^34,39 These different results imply that various detoxifying enzymes and other factors except gene structure could play a role in carcinogen metabolism and lung cancer development.

In the literature, there are limited number of researches studying the effect of polymorphisms and risk of lung cancer in Turkish population. An article by Oztürk et al. studied the CYP1A1 polymorphism and GSTM1 on 55 patients and 60 controls.⁸ Pinarbasi et al. examined only the GSTM1 polymorphism on 101 patients with lung cancer and 206 controls.⁴⁰ A study performed by Altinisik et al. investigated the GSTT1 and GSTM1 on 75 patients with lung cancer and 55 controls.⁹ Ada et al. studied only 138 patients with nonsmall cell carcinoma for examination of CYP1A1, GSTs and CYP1B1.⁴¹ Gonlugur et al. in a case study investigated the GSTT1 and GSTM1 effects on lung cancer.⁴² An article by Demir et al. studied CYP1A1 Msp1 polymorphism on 31 patients with lung cancer and 37 controls.⁴³ However, articles mentioned above discusses the association between GSTs and CYP1A1 (separately) polymorphisms and lung cancer in Turkish population with small sample sizes. As the CYP1A1 and GSTs are important in the detoxification of toxic molecules in tobacco, their variant genotypes should be investigated together in susceptibility to lung cancer. Therefore, our study is the first case–control study that examines the relationship between GSTs and CYP1A1 polymorphisms (together) and lung cancer in Turkish population. However, the main limitation of this study is the small sample size. Thus, more comprehensive studies with larger sample sizes should be performed.

There are lots of studies in the literature that analyze the effects of CYP1A1, GSTT1 and GSTM1 polymorphisms on the risk of lung cancer. When the findings of them are examined in detail, it can be seen that different results exist. The underlying reasons of these differences were the sample size of study, ethnic differences, the smoking habits (heavy/mild/light smoker), the type of endogenous and exogenous genotoxicants and exposure time to them as well as genetic structures of enzymes having role for the detoxification. Also, the genetic structure (in terms of mutation and polymorphisms) of other enzymes having a role for the detoxification, diet and the exposure time to the endogenous and exogenous genotoxicants may affect the age of onset and type of lung cancer among different populations. Because of the uncertain effects of these factors, the association of these polymorphisms and lung cancer susceptibility are still contradictory. Also, based on the results of this study, we suggest that other genes in addition to the proposed genes such as CYP1A1 and GSTs could play a role in lung cancer development. Thus, further studies should be designed to find out the real associations independent from the factors mentioned above.

Footnotes

Declaration of Conflicting Interests

The authors declared no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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