797ISSN 1752-036310.2217/BMM.12.61 © 2012 Future Medicine Ltd Biomarkers Med. (2012) 6(6), 797–803

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Association between polymorphisms of glutathione S-transferase genes (GSTM1, GSTP1 and GSTT1) and breast cancer risk in a sample Iranian population

Molecular epidemiological studies have verified that the majority of malignancies are caused by both genetic and environmental factors. Breast cancer is the most common malignancy and cause of cancer-related death worldwide. Breast cancer is a serious public health risk in both developed and developing countries and is responsible for 18% of all female malignancies [1,2]. Genetic poly-morphisms in enzymes involved in carcinogen metabolism have been discovered to increase the risk of cancer development [3–5]. The carcinogen-metabolizing enzymes play important roles in the activation/deactivation of various chemical agents, including xenobiotics and sex hormones [6]. Glutathione S-transferases (GSTs), known as a superfamily of phase II metabolic enzymes, are responsible for the detoxification of many carcino-gens through catalysis of the conjugation between glutathione and chemotherapeutic drugs, envi-ronmental pollutants, carcinogens and wide-rang-ing xeno biotics. Inherited GST variants, which modify GST-mediated detoxification of carci-nogenic chemicals, have been broadly examined as potential genes for breast cancer susceptibility [7–10]. Most recently, GSTs are classified into eight distinct groups, including a, µ, k, w, p, s, t and z, which are encoded by GSTA, GSTM, GSTK, GSTO, GSTP, GSTS, GSTT and GSTZ genes, respectively. Three of the GST genes, GSTP1, GSTM1 and GSTT1, have been demonstrated to have functional polymorphisms that occur more frequently in the general population [11]. GSTM1,

GSTP1 and GSTT1 genes are mapped on chromosome 1 (1p13.3), chromosome 11 (11q13) and chromosome 22 (22q11.2), respectively [12]. In humans, GSTM1 is expressed in a variety of tissues including liver, stomach, brain and breast, while the GSTT1 enzyme is chiefly expressed in liver and erythrocytes [13]. GSTM1 detoxifies carcinogenic polycyclic aromatic hydro carbons, such as the smoke carcinogen benzopyrene, whereas GSTT1 detoxifies smaller reactive hydro-carbons, such as ethylene oxide. Deletions in two GST genes, GSTM1 and GSTT1, occur at differ-ent frequencies in various populations. Individuals who are deletion homo zygotes, categorized as GSTM1 null or GSTT1 null, display a lack of enzymatic activity and are hypothesized to be at increased risk for the carcinogenic effects of a diverse spectrum of environmental exposures [14]. The GSTM1 null genotype was significantly associated with breast cancer risk in postmeno-pausal women [12]; however, an increased risk for premenopausal women was also reported [15]. GSTP1 is primarily found in spleen, heart and lung tissue, and is the major GST expressed in breast tissue [8,13,16]. For the GSTP1 gene, one point mutation in the coding region results in substitution of Ile for Val at codon 105, which modifies enzyme activity [4,13].

Although, there are several studies concerning the association between GST polymorphisms and susceptibility to breast cancer, the findings are controversial [7,8,12–14,17–19]. To the best of

Aim: Genetic and environmental factors are risk factors for breast cancer. Our aim was to investigate the associations between genetic polymorphism of GST genes (GSTM1, GSTT1 and GSTP1) and susceptibility to breast cancer in an Iranian population. Materials & methods: This case–control study was carried out on 134 patients with breast cancer and 152 healthy, cancer-free women. GSTP1 polymorphism was determined using tetra-primer amplification refractory mutation system PCR assay and GSTM1 and GSTT1 were genotyped by a multiplex PCR. Results: We found that the GSTM1 null genotype is a risk factor for predisposition to breast cancer (odds ratio [OR] = 2.01; 95% CI = 1.78–3.45; p = 0.010). No significant difference was found between the groups regarding GSTT1 null genotype (p > 0.05). The GSTP1 Ile/Val and Val/Val genotypes were associated with breast cancer risk (OR = 3.29; 95% CI = 1.84–5.91; p < 0.0001 and OR = 20.68; 95% CI = 5.66–75.60; p < 0.0001, respectively). Conclusion: In summary, GSTM1 and GSTP1, but not GSTT1 genetic polymorphisms are associated with increased risk of breast cancer in our population.

KeywoRds: breast cancer n glutathione S-transferase n polymorphism Mohammad Hashemi*‡1,2, Ebrahim Eskandari-Nasab‡2, Aliakbar Fazaeli2, Mohsen Taheri3, Hamzeh Rezaei2, Mohammadali Mashhadi4, Farshid Arbabi5, Mahmoud-Ali Kaykhaei4, Mehdi Jahantigh6 & Gholamreza Bahari2

1Cellular & Molecular Research Center, Zahedan University of Medical Sciences, Zahedan, Iran 2Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 3Genetic of Non-Communicable Disease Research Center, Zahedan University of Medical Sciences, Zahedan, Iran 4Department of Internal Medicine, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 5Brain & Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran 6Department of Pathology, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran *Author for correspondence: mhd.hashemi@gmail.com ‡Authors contributed equally

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our knowledge, no study has yet investigated the role of GST polymorphisms and breast cancer risk in an Iranian population. Therefore, the present study’s aim was to evaluate the impact of GST polymorphisms on the susceptibil-ity of breast cancer in a sample of the Iranian population.

Materials & methodsThis case–control study was performed on 134 pathologically confirmed patients with breast cancer, referred consecutively to Ali Ebneh Abitaleb hospital (Zahedan, Iran; age: 47.9 ± 13.3 y), and 152 population-based healthy women (age: 34.5 ± 13.5 y), unrelated to the patients, who participated in the metabolic syndrome project [20]. The local ethics commit-tee of Zahedan University of Medical Sciences (Zahedan, Iran) approved the project, and informed consent was obtained from all patients and healthy individuals. Blood samples were col-lected in EDTA-containing tubes and DNA was extracted as previously described [21].

The GSTM1 and GSTT1 gene polymorphism screening was performed using multiplex PCR, using the TLR2 gene as an internal control, as previously described by Hashemi et al. [22].

The primers used were 5´-GCTGCC-CTACTTGATTGATG-3´ (sense) and 5́ -CC- CCAAATCCAAACTCT GTC-3´ (antisense) for the GSTM1 gene, resulting in a 325-base pair (bp) fragment; 5´-TTCTGCTT-TATGGTGGGGTC-3´ (sense) and 5´-GT- GATGTTCCCTG TTTTCCT-3´ (antisense) for the GSTT1 gene, resulting in a 542 bp fragment, and 5́ -GATGCATTTGTT-TCTTACAGTGAGCG-3´ (sense) and 5́ -GTG-ATGTTCCCTGTTTTCCT-3´ (anti sense) for the TLR2 gene, resulting in a 259-bp fragment. The GSTM and GSTT1 null variant forms were defined by the absence of the 325- and 542-bp fragments, respectively.

GSTP1 polymorphism was determined by tetra-primer amplification refractory mutation system PCR as previously described [22]. Two outer primers (forward outer: 5́ -CAGGTGTCAGGTGAG-CTCTGAGCACC-3 ,́ reverse outer: 5́ -ATAA- GGGTGCAGGTTGTGTCTTGTCCCA-3´) and the two inner primers were (forward inner [A allele or Ile allele]: 5́ -CGTGGAGGACC- TCCGCTGCAAATCCA-3 ,́ reverse inner [G allele or Val allele]: 5́ -GCTCACATAGTT- GGTGTAGATGAGGGATAC-3´). The amplicon size was 233 bp for the A allele, 290 bp for the G allele and 467 bp for the outer primers (control band).

Statistical analysis of the data was performed using statistical software package, SPSS 18 soft-ware. The associations between the genotype of GST gene and breast cancer were estimated by computing the odds ratio (OR) and 95% CI from logistic regression analyses.

ResultsThe frequency of the GSTM1, GSTT1 and GSTP1 genotypes was compared between 134 breast cancer cases and 152 controls (Table 1). The null genotype of GSTM1 was found to be prevalent in the patients (64.2%) compared with the cases (46.7%) resulting in significant association between incidence of breast cancer and GSTM1 null genotype (OR: 2.01; 95% CI: 1.78–3.45; p = 0.010). However, no significant difference in genotype frequencies of GSTT1 was observed between controls and breast cancer cases. The GSTT1 null geno-type did not increase the risk of breast cancer (OR = 1.82; 95% CI = 0.75–4.41; p = 0.182).

The differences in the GSTP1 genotype distribution between the breast cancer patients and the control subjects were extremely statistically significant (c2 = 48.5; p < 0.0001). The IleVal and ValVal genotypes were found to be associated with breast cancer risk (OR = 3.29; 95% CI = 1.84–5.91 and OR = 20.68; 95% CI = 5.66–75.60; p < 0.0001, respectively).

Allele frequencies for Ile and Val were found to be 0.53 and 0.47 in the breast cancer patients group, and 0.81 and 0.19 in the control group, respectively. The results showed that the Val allele is a risk factor for predisposition to breast cancer (OR = 3.65; 95% CI = 2.51–5.31; p < 0.0001).

Table 2 shows the association between GST polymorphisms and menopausal status. The results showed that the GSTM1 null genotype was a risk factor for predisposition to breast cancer in postmenopausal women (OR = 3.68; 95% CI = 1.54–8.81; p = 0.003), but not in pre-menopausal (OR = 1.71; 95% CI = 0.91–3.21; p = 0.117). No significant difference was observed regarding the GSTT1 null genotype and risk of breast cancer in premenopausal or postmenopausal women. The GSTP1 Val allele was found to be a risk factor for breast cancer in premenopausal and postmenopausal women (Table 2).

We also determined gene–gene interaction. In comparison to the reference GSTT1 present, GSTM1 present and GSTP1 AA genotype, the GSTT1 null, GSTM1 null and GSTP1AG/GG genotype as well as GSTT1 present, GSTM1 present and GSTP1AG/GG genotype, and GSTT1 present, GSTM1 null and GSTP1AG/GG

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genotypes are risk factors for predisposition to breast cancer (Table 3).

The genotype frequencies of GSTP1 were tested for Hardy–Weinberg equilibrium separately in patients and control subjects. The genotype fre-quencies in case and control groups were found

to be in Hardy–Weinberg equilibrium (c2 = 0.87; p = 0.350 and c2 = 1.77; p = 0.183, respectively).

discussionIn the present study, we examined the association of GSTM1, GSTT1 and GSTP1 genotypes and

Table 2. Frequency distribution of the GSTM1, GSTT1 and GSTP1 genotypes in women with and without breast cancer according to menopausal status.

GST genotypes Premenopausal women Postmenopausal women

Patientsn (%)

Controln (%)

oR (95% CI) p-value Patientsn (%)

Controln (%)

oR (95% CI) p-value

GSTM1

Wild 23 (36.5) 59 (49.6) 1.0 (reference) – 25 (35.2) 22 (66.7) 1.0 (reference) –

Null 40 (63.5) 60 (50.4) 1.71 (0.91–3.21) 0.117 46 (64.7) 11 (33.3) 3.68 (1.54–8.81) 0.003

GSTT1

Wild 54 (85.7) 111 (93.3) 1.0 (reference) – 62 (87.3) 29 (87.9) 1.0 (reference) –

Null 9 (14.3) 8 (6.7) 2.31 (0.85–6.33) 0.112 9 (12.7) 4 (12.1) 1.05 (0.23–3.70) 0.937

GSTP1

AA (Ile/Ile) 19 (30.2) 75 (63.0) 1.0 (reference) – 17 (24.0) 22 (66.7) 1.0 (reference) –

AG (Ile/Val) 31 (49.2) 41 (34.5) 2.99 (1.50–5.93) 0.002 41 (57.7) 11 (33.3) 4.82 (1.93–12.09) <0.001

GG (Val/Val) 13 (20.6) 3 (2.5) 17.11 (4.42–66.17) <0.0001 13 (18.3) 0 (0.0) – –

AG + GG 44 (69.8) 44 (37.0) 3.95 (2.05–7.60) <0.0001 54 (76.0) 11 (3.3) 5.18 (2.07–12.93) <0.001

GSTP1 allele

A allele (Ile allele) 69 (54.8) 191 (80.3) 1.0 (reference) – 75 (52.8) 55 (83.3) 1.0 (reference) –

G allele (Val allele) 57 (45.2) 47 (19.7) 3.36 (2.11–5.40) <0.0001 67 (47.2) 11 (16.7) 4.48 (2.16–9.24) <0.0001

OR: Odds ratio.

Table 1. Frequency distribution of the GSTM1, GSTT1 and GSTP1 genotypes in women with and without breast cancer.

GST genotypes Patients,n (%)

Control,n (%)

oR (95% CI) p-value oR (95% CI)† p-value

GSTM1

Wild 48 (35.8) 81 (53.3) 1.0 (reference) – 1.0 (reference) –

Null 86 (64.2) 71 (46.7) 2.04 (1.27–3.29) 0.003 2.01 (1.78–3.45) 0.010

GSTT1

Wild 116 (86.6) 140 (92.1) 1.0 (reference) – 1.0 (reference) –

Null 18 (13.4) 12 (7.9) 1.81 (0.84–3.91) 0.131 1.82 (0.75–4.41) 0.182

GSTP1

AA (Ile/Ile) 36 (26.9) 97 (63.8) 1.0 (reference) – 1.0 (reference) –

AG (Ile/Val) 72 (53.7) 52 (34.2) 3.73 (2.21–6.29) <0.0001 3.29 (1.84–5.91) <0.0001

GG (Val/Val) 26 (19.4) 3 (2.0) 23.35 (6.69–81.91) <0.0001 20.68 (5.66–75.60) <0.0001

GSTP1 allele

A allele (Ile allele) 144 (53.3) 246 (80.9) 1.0 (reference) – 1.0 (reference) –

G allele (Val allele) 124 (46.7) 58 (19.1) 3.65 (2.51–5.31) <0.0001 3.65 (2.51–5.31) <0.0001†Adjusted for age and menopausal status. OR: Odds ratio.

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breast cancer risk in a sample of the Iranian population. GSTM1 null genotype was found to be significantly associated with the risk of breast cancer. However, no association was found between GSTT1 null genotype and susceptibility to breast cancer. In addition, the GSTP1 Ile105Val geno types frequencies were significantly higher in breast cancer than normal subjects.

It has been reported that GSTM1 and GSTT1 null genotypes are related to the lack of GSTM1 and GSTT1 enzymatic activity [23,24], and GSTP1 Ile105Val polymorphism is correlated with decreased catalytic function [25,26]. Individuals who harbor GSTM1 and GSTT1 null genotypes or GSTP1 Ile105Val polymorphism, are thought to represent less GST detoxification and, thus, potential increase in the levels of toxic metabolites.

Contrary to our findings, Unlu et al. did not find a connection between the GSTM1 or the GSTT1 null genotypes and elevated risk for breast cancer [7]. They stated that the Val allele compared with the Ile allele in codon 105 in GSTP1 did not increase the risk of breast cancer develop-ment. However, the combination of GSTT1 null, GSTM1 null and GSTP1 Val was correlated with increased risk for breast cancer. Helzlsouer et al. [27] and Mitrunen et al. [12] have also reported a higher risk of breast cancer associated with combinatory effects of GST genotypes.

Contrary to our findings, there are also some studies that showed that the patients with only one polymorphic gene did not have a considerable increased risk of developing of breast cancer, and that the GSTM1 null or the GSTT1 null geno-types were not associated with higher risk for breast cancer [7,14,18,19]. Our findings revealed that the GSTM1 null genotype polymorphism was significantly associated with increased breast cancer risk. Our finding was in agreement with

other studies that showed the null GSTM1 geno-type was associated with breast cancer risk [27,28]. Garcia-Closas et al. have found that the GSTM1 and GSTT1 null genotypes were not associated with an increased risk of breast cancer; however they showed that the GSTT1 null genotype was associated with decreased risk of breast can-cer among premenopausal women [19]. Kelsey et al. have found no significant association between GSTM1 null genotype and increased risk of breast cancer [29].

Concerning GSTP1, we found a statistically signif icant association between GSTP1 Ile105Val polymorphisms and breast cancer risk. Furthermore, women with Val allele were found to be at higher risk for breast cancer. In contrast to our finding, Unlu et al. [7], Gudmundsdottir et al. [17], Millikan et al. [14], Cerne et al. [30] and Mitrunen et al. [12] have found no association between GSTP1 Ile105Val polymorphisms and breast cancer risk. In a meta-analysis performed by Sergentanis et al., no statistically significant associations were noticed in non-Chinese populations (25 studies) regarding GSTP1 Ile105Val polymorphism [31]. Whereas, the Val/Val genotype was associated with increased breast cancer risk in Chinese populations (five studies) [31]. Antognelli et al. have found the GSTP1 mutant Val allele was associated with a decreased risk of developing breast cancer, which is contrary to the previous studies and our findings [32].

Genes involved in the detoxification process have long been considered plausible candidate genes for susceptibility to breast and other cancers. The GSTs are a superfamily of enzymes responsible for detoxification of a broad spectrum of xenobiotics, such as environmental carcinogens, steroids and reactive oxygen species. Interindividual disparities in the metabolisms of carcinogens could possibly

Table 3. Interaction of GSTT1, GSTM1 and GSTP1 on breast cancer risk.

GSTT1 GSTM1 GSTP1 Casen (%)

Control,n (%)

oR (95% CI)† p-value

Present Present AA 18 (14.3) 53 (34.9) 1.0 (reference) –

Null Present AA – – – –

Present Null AA 15 (11.2) 37 (24.3) 1.28 (0.52–3.14) 0.591

Null Null AA 1 (0.7) 6 (3.9) 0.55 (0.04–6.92) 0.643

Present Present AG/GG 30 (22.4) 28 (18.4) 3.28 (1.45–7.45) 0.005

Null Present AG/GG – – – –

Present Null AG/GG 56 (41.8) 22 (14.5) 6.93 (3.13–15.38) <0.0001

Null Null AG/GG 14 (10.4) 6 (3.9) 5.67 (1.68–19.18) 0.005†Adjusted for age and menopausal status. OR: Odds ratio.

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be the result of diverse functions of metaboliz-ing enzymes in the liver and target tissues. These disparities may cause dissimilar vulnerabilities to the breast cancer progress. Elevated levels of DNA adducts have been revealed in breast cancer tissues of patients with polymorphic GSTM1 and NAT2 [33]. It is well known that increased oxidative stress and inflammation are associated with elevated risk of cancer [34]. GSTs catalyze the detoxification of many toxic xenobiotics by conjugation with glutathione [35]. Most GST substrates are thought to be electrophiles and products of oxidative stress (i.e., reactive oxygen species) [36].

Lack of GSTP1 activity potentially makes the cells vulnerable to oxidative DNA damage and, owing to the accumulation of DNA base adducts, tumors are prone to relevant genetic alterations during breast carcinogenesis [13,37]. In addition, because of the vital role of GSTM1 in detoxification of carcinogenic poly cyclic aromatic hydrocarbons, any malfunction, such as observations made in the null geno type, will reduce its capacity to detoxify certain carcinogens [38].

Although, some studies have examined the association of GST polymorphism and breast cancer risk, the association still remains equivocal and much debated. The dissimilarity in the results of these studies may be due to differences in the populations studied and in their exposures to the agents related to the progress of breast cancer. Genes that interact with GSTs or the presence of a family history may also cause disparities in the results of these studies. To the best of our

knowledge, this is the first study evaluating the association of GSTM1, GSTT1 and GSTP1 poly-morphisms and breast cancer risk in a sample of the Iranian population. Further study with larger samples is required to validate our findings.

The limitations of the present study are: we have no data on known risk factors (e.g., family history, previous benign conditions, parity, oral contraceptive or hormone therapy use, breast-feeding and obesity) and the statistical difference between the age of patients and control groups. Although, this difference probably has not had a significant impact on the results, since we used age as a covariate in logistic regression analysis.

ConclusionIn conclusion, we found an association between GSTM1 null genotype and GSTP1 Ile105Val polymorphisms and increased risk for breast cancer in a sample of the Iranian population.

Future perspectiveFurther research on various cohorts is needed to clarify the impact of GST polymorphisms, as well as gene–gene interactions on breast cancer risk. The main limitation of current studies worldwide is the technique used for detection of genetic polymorphisms, since they are costly and are able to genotype only a few polymor-phic sites. New techniques and cutting-edge methodologies in the future will help to evalu-ate the impact of multiple GST polymorphisms on various diseases more rapidly and accurately.

executive summary

Glutathione s-transferases as a superfamily of phase II metabolizing enzymes

� Glutathione S-transferases (GSTs) are responsible for the detoxification of many carcinogens.

� They catalyze the conjugation of glutathione with xenobiotics and carcinogens.

� Most GST substrates are electrophiles and products of oxidative stress (i.e., reactive oxygen species).

Individuals who are deletion homozygotes are known as GsTM1 null or GsTT1 null

� Null genotypes lack enzymatic activities.

� Null genotypes are at increased risk of the effects of carcinogens.

� GSTM1 null genotype was found to be associated with risk of breast cancer.

� GSTM1 is expressed in a variety of tissues including liver, stomach, brain and breast.

� GSTT1 enzyme is chiefly expressed in liver and erythrocytes.

� No association was found between GSTT1 null genotype and susceptibility to breast cancer.

One point mutation in the coding regions of GsTP1 modifies the enzyme activity

� We found a significant association between GSTP1 Ile105Val polymorphism and breast cancer risk.

� Women with the valine allele were at higher risk for breast cancer.

Conclusion

� The pace for detection of genetic alterations and their impacts of cancer risk is accelerating.

� GST genes are found to be of significant importance in predisposing individuals to breast cancer.

� Future studies will provide us with new methodologies that will enable us to detect multiple genetic alterations in the GST genes quickly and accurately.

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Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manu-script. This includes employment, consultancies, honoraria, stock ownership or options, expert testi-mony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

ethical conduct of research The authors state that they have obtained appropri-ate insti tutional review board approval or have fol-lowed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involv-ing human subjects, informed consent has been obtained from the participants involved.

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