Polymorphisms of Microsomal Epoxide Hydrolase and Glutathione S -transferase P1 in a Male Turkish Population

Abstract

Polymorphic genes encoding drug-metabolizing enzymes may account for interindividual differences in certain types of diseases especially cancer. In this study, microsomal epoxide hydrolase (EPHX1) and glutathione S-transferase P1 (GSTP1) gene polymorphisms were determined among 133 healthy males of a Turkish population. Frequencies of EPHX1 and GSTP1 gene polymorphisms were determined by using the polymerase chain reaction–restriction fragment length polymorphism (PCR/RFLP) method. The observed genotype frequencies of EPHX1 exon 3 were Tyr113Tyr:50.4%, Tyr113His: 42.1%, His113His: 7.5% and EPHX1 exon 4 were His139His: 69.2%, His139Arg: 28.6%, Arg133Arg: 2.2%. GSTP1 exon 5 genotype frequencies were Ile105Ile: 58.7%, Ile105Val: 35.3%, Val105Val: 6.0% and GSTP1 exon 6 genotype frequencies were Ala114Ala: 85.0%, Ala114Val: 14.3%, Val114Val: 0.7%. These results reveal that the frequencies of EPHX1 and GSTP1gene polymorphisms in a small sampling of males within a Turkish population are similar to European Caucasian populations.

Microsomal epoxide hydrolase (mEH) and glutathione S-transferase P1 (GSTP1) enzymes are known to be polymorphic and involved in the metabolism of xenobiotics (Board et al. 1990; Hasset et al. 1994)

The mEH cleaves a range of reactive epoxides to form trans-dihydrodiols (Guengerich 1982). Genetic polymorphisms have been described in the human gene encoding for microsomal epoxide hydrolase (EPHX1). Two of these polymorphisms, Tyr113His in exon 3 and His139Arg in exon 4, have been associated with a decrease or increase in enzyme activity, respectively (Hasset et al. 1994). Low-activity form is associated with susceptibility to aflatoxin B1–induced hepatocellular carcinoma (McGlynn et al. 1995) and decreased risk for ovarian cancer (Lancaster et al. 1996). High-activity isoform has been associated with chronic obstructive pulmonary disease (Benhamou et al. 1998).

Glutathione S-transferase P1 (GSTP1) is a member of the GST enzyme superfamily that is important for the detoxification of numerous carcinogenic compounds. Alterations in the structure, function, or expression levels of GSTP1 due to genetic polymorphisms could alter the ability to detoxify carcinogens and modulate lung cancer risk. Previous studies have suggested that genetic polymorphisms of GSTP1 exon 5 (Ile105Val) and exon 6 (Ala114Val) have a functional relevance on the GST gene product, resulting in reduced enzyme activity (Zimniak et al. 1994; Ali-Osman et al. 1997; Watson et al. 1998). Therefore, individuals with these variant GSTP1 genotypes that result in reduced GST enzymatic activity may be at greater risk for cancer such as lung cancer due to decreased detoxification of carcinogenic and mutagenic compounds (Wang et al. 2003).

Thus, associations between genetic polymorphism and development of environmental cancer clearly indicate the increased need to elucidate the effects of various polymorphic genes on cancer susceptibility and adverse health outcomes (Abdel-Rahman et al. 1996).

The frequencies of polymorphic genes in control populations have been reported to be different in various ethnic groups. In addition intraethnic differences have been well established (Garte et al. 2001). For example, the frequencies of the EPHX1 exon 3 Tyr113His and His113His mutations are 37.0% to 48.8% and 4.2% to 16.8% (see references in Table 2); EPHX1 exon 4His139Arg and Arg139Arg mutations are 26.1% to 39.0% and 0% to 6.5% (see references in Table 3), respectively, in European Caucasians. Furthermore, GSTP1 exon 5 Ile105Val and Val105Val mutations are 27.3% to 47.8% and 4.1% to 46.0% (see references in Table 5); GSTP1 exon 6 Ala114Val and Val114Val mutations are 7.3% to 14.3% and 0% to 1.5% (see references in Table 6), respectively, in European Caucasians. However, no differences in allele frequencies were seen by age or sex (Garte et al. 2001).

Studies regarding the GSTP1 exon 5 polymorphism in Turkish populations are rare and their results are contradictory (Toruner et al. 2001; Aynaciolu et al. 2004; Ates et al. 2005). In addition, besides the existence of rather scarce data for GSTP1 exon 6 polymorphism among Caucasian populations (Carstensen et al. 1999; Sorensen et al. 2004; Garcia-Closas et al. 2005), to our knowledge, there are no data for GSTP1 exon 6 and EPHX1 exon 3 and exon 4 polymorphisms in the Turkish population.

Thus, we aimed to clarify the frequencies of EPHX1 and GSTP1 gene polymorphisms in a small subset of Turkish males.

MATERIALS AND METHODS

Subjects

The subjects were 133 male unrelated individuals aged between 22 to 58 years 39 ± 8.58 (mean ± SD) living in Northwest Anatolia (Eregli-Karadeniz region) belonging to similar ethnic origin. All of the subjects were healthy volunteers and informed about the study. This study is approved by the Ethics Committee of Ankara University.

DNA Isolation

Venous blood samples (10 ml) were collected in heparin-containing vacutainers. DNA used for polymorphic analysis was isolated from lymphocytes of donors on the same day by using DNA isolation kit purchased from Promega (Madison WI, USA) following the manufacturer’s instructions. Isolated DNA was stored at −20°C until use.

Identification of EPHX1 Polymorphisms

Two separate polymerase chain reaction (PCR) assays were used to detect the two mutations located in exon 3 (Tyr113His) and exon 4 (His139Arg) of the EPHX1 gene. Genotyping for these polymorphisms was carried out by PCR-restriction fragment length polymorphism (RFLP) as previously described (Smith and Harrison 1997). Tyr/His polymorphism analysis was performed by amplifying a 162-bp fragment in a 30-μl reaction mixture containing 10 pmol of each of the following primers: (sense) 5′-GAT CGA TAA GTT CCG TTT CAC C and (anti-sense) 5′-ATC CTT AGT CTT GAA GTG AGG AT in the presence of 150 μmol dNTPs, 3 μl of 10× PCR buffer (100 mM Tris-HCl pH 9.0 25°C, 500 mM KCl), 2.2 mM MgCl₂, 0.8 μg DNA, and 0.75 U Taq DNA polymerase (Promega). PCR conditions were 40 cycles of 0.5 min at 94°C, 1 min at 56°C, and 1 min at 72°C. The PCR product was analyzed electrophoretically on an ethidium bromide–stained 2% Nusieve garose gel. The product was digested with EcoR V restriction enzyme (New England Biolabs; 10 units, 37°C for 16 h). After the Agarose gel electrophoresis, the band at 162 bp showed wild-type genotype, bands at 162, 140, and 20 bp showed heterozygous genotype, and the bands at 140 and 20 bp showed homozygous mutant genotype.

His/Arg polymorphism analysis was performed by amplifying a 210-bp fragment in a 30-μl reaction mixture containing 10 pmol of each of the following primers: (sense) 5′-ACA TCC ACT TCA TCC ACG T and (antisense) 5′-ATG CCT CTG AGA AGC CAT in the presence of 150 μmol dNTPs, 3 μl of 10× PCR buffer (100 mM Tris-HCl pH 9.0 25°C, 500 mM KCl), 2.2 mM MgCl₂, 0.8 μg DNA, and 0.75 U Taq DNA polymerase (Promega). PCR conditions were 40 cycles of 0.5 min at 94°C, 1 min at 56°C, and 1 min at 72°C. The PCR product was analyzed electrophoretically on an ethidium bromide–stained 2% Nusieve Agarose gel. The product was digested with Rsa I restriction enzyme (New England Biolabs; 10 units, 37°C for 16 h). After the Agarose gel electrophoresis, bands at 164 and 46 bp showed wild-type genotype, bands at 210, 164, and 46 bp showed heterozygous genotype, and the band at 210 bp showed homozygous mutant genotype.

Identification of GSTP1 Polymorphisms

GSTP1 exon 5 and exon 6 polymorphisms were determined by using the PCR-RFLP method described in Park et al. (1999). GSTP1 Ile/Val genetic polymorphism analysis was performed in a 30-μl reaction mixture containing 25 pmol of each of the following primers:(sense) 5′-AAT ACC ATC CTG CGT CAC CT and (antisense) 5′-TGA GGG CAC AAG AAG CCC CTT in the presence of 100 μmol dNTPs, 3 μl of 10× PCR buffer (100mM Tris-HCl pH 9.0 25°C, 500 mM KCl), 1.2 mM MgCl₂, 100 ng DNA, and 1 U Taq DNA polymerase (Promega). PCR conditions were 1 cycle of 2 min 95°C, 40 cycles of 0.5 min at 94°C, 0.5 min at 55°C, and 0.5 min at 72°C, followed by a final cycle of 10 min at 72°C. The PCR product was digested with BsmA1. After the Agarose gel electrophoresis, the bands at 305 and 138 bp showed wild-type genotype, bands at 305, 222, and 138 bp showed heterozygous genotype, and the bands at 222 and 138 bp showed homozygous mutant genotype.

GSTP1 Ala/Val genetic polymorphism analysis was performed in a 30-μl reaction mixture containing 25 pmol of each of the following primers:(sense) 5′-ACA GGA TTT GGT ACT AGC CT and (antisense) 5′-AGT GCC TTC ACA TAG TCA TCC TTG CGC in the presence of 100 μmol dNTPs, 3 μl of 10× PCR buffer (100 mM Tris-HCl pH 9.0 25°C, 500 mM KCl), 1.2 mM MgCl₂, 100 ng DNA and 1 U Taq DNA polymerase (Promega). PCR conditions were 1 cycle of 2 min 95°C, 40 cycles of 0.5 min at 94°C, 0.5 min at 48°C, and 0.5 min at 72°C, followed by a final cycle of 10 min at 72°C. The PCR product was digested with BstUI. After the Agarose gel electrophoresis, the bands at 144 and 26 bp showed wild-type genotype, bands at 170, 144, and 26 bp showed heterozygous genotype, and the band at 170 bp showed homozygous mutant genotype.

RESULTS AND DISCUSSION

This is the first study that provides the frequencies of EPHX1 exon 3, EPHX1 exon 4, and GSTP1 exon 6 gene polymorphisms in Turkish males. In the current study, the mutation frequencies of the four genes of the same individuals of a Turkish population have been studied. The observed frequencies of EPHX1 exon 3 were Tyr113Tyr: 50.4%, Tyr113His: 42.1%, and His113His: 7.5% (Table 1). Thus, for His113His (homozygous mutant) this particular population of Turkish males appears to have lower frequency than the populations of Czech Republic, Finland, France, and Italy but similar to the populations of Germany, Netherlands, Spain, Sweden, and UK (Table 2).

EPHX1 exon 4 genotype frequencies were found as His139His: 69.2%, His139Arg: 28.6%, and Arg133Arg: 2.2% (Table 1). The frequencies of EPHX1 exon 4 polymorphism observed in our study are in good agreement with the previous reports of other European countries (Table 3).

As shown in Table 4, genotype frequencies of GSTP1 exon 5 were Ile105Ile: 58.7%, Ile105Val: 35.3%, and Val105Val: 6.0%. Our results concerning GSTP1 exon 5 are consistent with the results of Toruner et al. (2001) but in contrast to those of Aynacioglu et al. (2004) and Ates g et al. (2005) in Turkish populations. At this stage we do not know the reason(s) behind these distinct observations among the Turkish populations in regard to GSTP1 exon 5 polymorphism. However, it could be due to several reasons such as the variability of the ethnic backgrounds of the study groups, the differences in the assays utilized, and/or the sample size of the studies. Although different assays were used in this study and that of Toruner et al. (2001), in which ethnical backgrounds of populations (predominantly from the nearby cities of Turkey) and sample sizes utilized were alike (133 and 121), similar findings were achieved (Table 5). On the other hand, although the similar assays were utilized in the studies of Toruner et al. (2001) and Aynacioglu et al. (2004), in which the sample sizes were different (121 versus 265) and the study populations were from distinct regions of Turkey, i.e., Central Anatolia and South East Anatolia, respectively, the findings were different. Furthermore, although the larger sample sizes were used in the studies of Aynacioglu et al. (2004) (n = 265) and Ates et al. (2005) (n = 204), in which the ethnic backgrounds of the populations and the assays utilized were not identical, the results observed were in agreement with each other. Thus, the picture appeared to be rather complicated. Further studies with larger sample size with the similar ethnic origin are needed to clarify the issue. Nevertheless, the current available data from the Turkish population, as other European Caucasian populations, has a lower Val105Val frequency than that reported for a subset of the Polish population (Table 5).

GSTP1 exon 6 genotype frequencies were Ala114Ala: 85.0%, Ala114Val: 14.3%, and Val114Val: 0.7% (Table 4). The polymorphism of GSTP1 exon 6 gene have been studied in only a few European countries, namely Denmark (Sorensen et al. 2004), Spain (Garcia-Closas et al. 2005), and Sweden (Carstensen et al. 1999). Hence, this study also provides additional data from South Eastern Europe and indicates that the frequencies of the gene in Turkish males studied here are not different from the above aforementioned populations of Europe (Table 6).

In line with the results obtained herein, recently the frequencies of some other xenobiotic metabolizing genes such as CYP1A1, GSTM1, and GSTT1 (Ada, Suzen, and Iscan 2004) and the DNA repair gene (e.g., XRCC1) (Kocabas and Karahalil 2006) polymorphisms in Turkish populations have also been reported to be similar to Caucasian populations.

In conclusion, this study shows that the general frequencies of EPHX1 and GSTP1 gene polymorphisms in a subset of Turkish males are similar to those previously published for other European Caucasian populations.