Introduction

Studies have indicated that gastric cancer has heritability [1, 2] it means that several environmental and genetic predisposing factors are involved in its pathogenesis [3, 4]. Although the pathogenesis of gastric cancer has not been understood completely, it is well established that oxidative stress implicated in its development [5]. Reactive oxygen species (ROSs) are unstable metabolite of oxygen and leads to oxidation of many macromolecules, including DNA [6]. Oxidative stress can occur due to an increased production of ROSs and/or a reduction in cellular antioxidant capacity [7].

Enzymatic and non-enzymatic antioxidant systems protect cells and body from the ROSs toxicity [8]. The enzymatic system contains several antioxidant enzyme families such as superoxide dismutases family (SODs; EC 1.15.1.1) [8, 9]. The SOD converts superoxide into hydrogen peroxide and is the most important defense system against ROS. It is classified into three distinct isoforms in mammals. The SOD1 (OMIM: 147450), SOD2 (OMIM: 147460), and SOD3 (OMIM: 185490) are cytosolic, mitochondrial and extracellular enzymes, respectively. The SOD1 and SOD3 enzymes contain copper and zinc and the SOD2 contains manganese in their active sites [9, 10]. Several polymorphisms were reported in the SOD1, SOD2, and SOD3. A 50 bp Insertion/Deletion (Ins/Del) genetic polymorphism has been reported in the promoter region of SOD1 (1684 bp upstream of the ATG start codon) [11]. This polymorphism alters the SOD1 expression levels; the Del allele associated with the reduced SOD1 mRNA level [12]. The polymorphisms of rs5746136 and rs2758339 have been reported for the SOD2. These polymorphisms are located in the vicinity of SP1 and NF-κB transcription element sequences [13] and glucocorticoid receptor binding site [14]. The rs2536512 polymorphism of SOD3 results in substitution of alanine by threonine [15]. Therefore, it seems that the above-mentioned polymorphisms are functional.

Losses of 4p and 21q [16,17,18,19,20] and gains of 6q are non-randomly reported in gastric cancer [21, 22]. Genome scan showed that the human 21q chromosome segment is associated with the risk of gastric cancer [23]. The high expression levels of the SOD2 have been reported in gastric cancer [24]. On the other hand, the genes encoding SOD1, SOD2, and SOD3 were assigned to human chromosomes 21q22, 6q25.3 and 4p15.2, respectively [25,26,27]. Taken together, it is suggested that genetic polymorphisms of the SOD family might be associated with the risk of gastric cancer.

In a few studies, the association of SOD1, SOD2, and SOD3 polymorphisms with several types of cancers have been studied [14, 29,30,31]. Considering that there is no published data on the association between the SOD1 (Ins/Del), SOD2 (rs5746136, rs2758339), and SOD3 (rs2536512) polymorphisms and the risk of gastric cancer, the present study was carried out.

Materials and Methods

Study Subjects

The present case-control study included a total of 159 (103 males, 56 females) patients with gastric cancer who were referred to chemotherapy department of Namazi hospital (Shiraz, south-west Iran) and 242 (167 males, 75 females) normal control subjects. The mean age (SD) of the patients and the controls were 57.3 (12.8) and 56.7 (9.8) years, respectively. There was no significant difference between case and control groups. Iranian population is one of the most heterogeneous populations [32,33,34]. To control for such variation and have a more homogeneous groups, we selected our patients and controls from the same ethnical religious group (Persian Muslims living in Fars province, south-west Iran).

This study was approved by the Shiraz University ethics committee. Informed consent was obtained from all participants before the study. This work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for Ethical Principles for Medical Research Involving Human Subjects.

DNA Extraction and Genotyping Analysis

Blood samples with EDTA anticoagulant were obtained from patient and control groups and stored at −20 °C until use. Genomic DNA from whole blood was isolated by standard procedure. Genotypes of the study polymorphisms were detected using PCR based methods, as described previously [11, 13,14,15]. It should be noted that we failed to successfully determine the rs2758339 polymorphism in 4 participants, explaining the variation in the total number of samples listed in Table 1.

Table 1 Distributions of SOD1 (Ins/Del), SOD2 (rs2758339, rs5746136) and SOD3 (rs2536512) polymorphisms with the risk of gastric cancer
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Statistical Analysis

A Chi-square test was performed for each polymorphism to determine if the control participants demonstrated Hardy–Weinberg equilibrium. The associations between the genotypes of study polymorphisms and the risk of gastric cancer were expressed as odds ratios (ORs). Ninety-five percent confidence intervals for the ORs (95% CI) were reported.

Smoking habit is one of the important risk factor for gastric cancer [4, 28]. Therefore, the participants were stratified by their smoking habit and the data were reanalyzed. Data on smoking status in the control and gastric cancer subjects were missed for 21 and 6 participants, respectively. In order to study the potential influence of the smoking on gastric cancer risk as well as the risk associated with the SOD2 polymorphisms, the “sensitivity analysis” was used. For this analysis we assumed that 50% of the missing cases were smokers.

The software SNPAlyze(TM) ver. 6 Standard (Dynacom Co, Ltd. Kanagawa, Japan) was used to evaluate the status of pair wise linkage disequilibrium for the studied polymorphisms. Statistical analyses were performed with SPSS for Windows (version 17.0; SPSS Inc., Chicago, IL). The P-values of less than 0.05 were considered statistically significant.

Data Availability

No additional data are available for this study.

Results and Discussion

Table 1 presents the genotypic and allelic frequencies of the genes encoding SOD family among gastric cancer patients and healthy controls. The observed genotypic frequencies of the study polymorphisms among control subjects were consistent with the expected values based on Hardy-Weinberg equilibrium (for the SOD1 Ins/Del polymorphism: χ2 = 2.36, df = 1, P = 0.123; for the SOD2 rs2758339 polymorphism: χ2 = 0.28, df = 1, P = 0.594; for the SOD2 rs5746136 polymorphism: χ2 = 1.22, df = 1, P = 0.268; for the SOD3 rs2536512 polymorphism: χ2 = 3.00, df = 1, P = 0.082).

The SOD1 Ins/Del and SOD3 rs2536512 polymorphisms did not appear to have relationship with the risk of gastric cancer (Table 1). There is no study on relationship between SOD1 Ins/Del polymorphism and cancer risk. However, there is only one study on the association between the SOD3 rs2536512 polymorphism and breast cancer risk, which is inconsistent with our present findings [31]. Both SOD2 polymorphisms showed significant association with the risk of gastric cancer, under assumption that the variant alleles act as dominant alleles (Table 1). A few studies were published in relation to association between these polymorphisms and risk of other types of cancer revealed consistent results with our present findings [14, 29, 30].

A significant linkage disequilibrium was observed between the SOD2 polymorphisms (for control group: D’ = −0.8523, r2 = 0.2988, χ2 = 141.0, P < 0.001; for gastric cancer group: D’ = −0.7418, r2 = 0.2690, χ2 = 85.31, P < 0.001). Considering that the CC and GG genotypes of the rs2758339 and rs5746136 polymorphisms, respectively, showed the lower risks for gastric cancer (Table 1), we used the CG haplotype as a reference. Statistical analysis showed that there was no significant association between the study haplotypes and the risk of gastric cancer (Table 2). This finding confirms the fact that both variant alleles of the SOD2 polymorphisms act as dominant alleles.

Table 2 Comparison of the haplotypes of the rs2758339 and rs5746136 SOD2 polymorphisms in gastric cancer patients and healthy controls
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In further analysis, we stratified the participants based on the recessive and dominant genotypes of the SOD2 polymorphisms. The numbers of putative high-risk genotypes of the SOD2 polymorphisms in gastric cancer and control groups were shown in Table 3. There was a significant linear trend for the numbers of putative high-risk genotypes and the risk of gastric cancer (χ2 = 6.06, P = 0.014).

Table 3 Association between numbers of putative high risk genotypes of the SOD2 (rs2758339, rs5746136) polymorphisms stratified by the smoking status of the participants
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The prevalence of smoker subjects among control and patient groups were 21.1% (out of 217 participants) and 40.5% (out of 153 participants), respectively. There was significant association between smoking habit and risk of gastric cancer (OR = 2.54, 95% CI = 1.61–4.02, P < 0.001). Tobacco smoke is one of the well known risk factor for development of gastric cancer [4, 28]. It has been reported that cigarette smoking condensate influenced the expression of SOD2 [35, 36]. We know that many of risk factors may act additively. In order to investigate the additive effects of smoking and the SOD2 genotypes, participants were stratified by their smoking habit (Table 3). Smoker individuals having 2 putative high-risk genotypes showed higher-risk of gastric cancer compared with nonsmokers with no high-risk genotypes (OR = 5.75, 95% CI = 1.59–20.6, P = 0.007). Assuming that smoking habit and the genotypes are risk factors, there was a significant linear trend for the numbers of risk factors and gastric cancer risk (χ2 = 22.9, P < 0.001), indicating that smoking and the dominant genotypes of the SOD2 polymorphisms act in an additive model. Considering that data on smoking status in 27 participants were missed, we carried out the “sensitivity analysis” under assumption that 50% of them were smokers. After sensitivity analysis the above-mentioned associations did not change.

The gene encoding SOD2 was located on human chromosome 6q25 [27]. Interestingly, gain of 6q chromosome segment is non-randomly has been reported in gastric cancer [21, 22]. On the other hand, SOD2 was highly expressed in gastric cancer [24]. Taken together, it is suggested that SOD2 might be involved in the risk of gastric cancer. Both polymorphisms of the SOD2 (rs2758339 and rs5746136) are located in the vicinity of SP1 and NF-κB transcription element sequences [13] and glucocorticoid receptor binding site [14]. Our present findings support the possible involvement of SOD2 in the development of gastric cancer. It has been reported that cigarette smoke contains oxidant compounds able to generate superoxide and alter the expression of SOD2 [35, 36], which confirmed by our present findings (additive effect of smoking and the dominant genotypes of the SOD2 polymorphisms, Table 3).

Small sample size is the major limitation of the present study. Therefore, the present findings should be confirmed by other studies with larger sample size in other populations.