1 Department of Obstetrics and Gynecology and 2 Department of Biochemistry, Mersin University, School of Medicine, 33079, Mersin, Turkey
3 To whom correspondence should be addressed. Email: devrimertunc{at}hotmail.com
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Abstract |
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Key words: endometriosis/fluorophore/genital tract/glutathione-S-transferase P1/single nucleotide polymorphism
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Introduction |
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In recent years, the role of polymorphisms in glutathione-S-transferase (GST) system as a risk factor for endometriosis has been sought thoroughly. GST are key phase II enzymes that catalyse the conjugation of glutathione to numerous potentially genotoxic compounds, including aliphatic aromatic heterocyclic radicals, epoxides and arene oxides (Autrup, 2000). Although not entirely consistent, several studies suggest that there is a correlation between endometriosis and GSTM1 or GSTT1 genotype (Baranov et al., 1996
; Baranova et al., 1999
; Hsieh et al., 2004
; Hur et al., 2004
).
GSTP1, located at 11q13, is involved in the detoxification of electrophilic compounds by glutathione conjugation, like other members in GST family (Autrup, 2000). Two genetic polymorphisms have been found in exon 5 and exon 6, both resulting in amino acid substitutions. However, only the transition in exon 5 was linked to enzymatic activity since this is located within the region coding for the active site of the enzyme. The genetic change in exon 5 results in polymorphism at codon 105, where an adenosine-to-guanidine (A
G) transition causes an Ile-to-Val substitution (Zimniak et al., 1994
).
The cellular concentration and distribution of GST differ widely between tissues, and GST gene expression is differentially regulated by both endogenous and exogenous factors. Moreover, it has been demonstrated that different GST vary significantly in efficiency and enantio-selectivity for a number of toxic products (Hu et al., 1996a,b). In spite of the abundance of studies on GSTM1 and GSTT1 genotypes, and with respect to gaps in the current epidemiological knowledge base, there is insufficient data about the association of endometriosis with GSTP1 genotype. Furthermore, since GSTP1 is a key player in biotransformation and bioactivation of certain environmental pollutants (Harries et al., 1997
), GSTP1 may be even more important than other GST in the pathogenesis of endometriosis. For this reason, we investigated GSTP1 genotype distribution in patients with endometriosis and controls.
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Materials and methods |
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All patients were interviewed by researchers who were unaware of the indications and types and findings of surgery. Researchers asked the patients to complete a questionnaire regarding general characteristics (age at surgery, height, weight, waist and hip circumference, educational level and socioeconomic status), lifestyle factors (smoking, alcohol intake), reproductive history and menstrual history (age at menarche, average duration of bleeding, average cycle length). Body mass index (BMI) was calculated as weight (kg) divided by the height2 (m2). Waist:hip ratio (WHR) was calculated by dividing waist by hip measurements. Staging of endometriosis was performed according to the revised American Society for Reproductive Medicine (1997) classification. The women in this study had similar ethnic background and none of the women was consuming regular alcohol. For this reason, we did not consider race and alcohol consumption in the statistical analyses.
PCR analysis for GSTP1 single nucleotide polymorphisms
Heparinized venous blood samples were collected from women in the study and control groups. Immediately after collection, whole samples were stored at +4°C until use. Genomic DNA was extracted from whole blood using High Pure PCR Template Preparation kits (Roche Diagnostics GmbH, Mannheim, Germany).
Appropriate fragment of GST P1 is amplified with specific primers from human genomic DNA which were synthesized according to Ko et al. (2000). The amplicons are detected by fluorescence using specific pairs of hybridization probes which were synthesized by TIB MOLBIOL GmbH (Berlin, Germany). The hybridization probes consist of two different oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of PCR cycles. One probe is labelled at the 5'-end with a LightCycler-Red fluorophore (LightCycler-Red 640 or LightCycler-Red 705), and to avoid extension, modified at the 3'-end by phosphorylation. The other probe is labelled at the 3'-end with LightCycler-Fluorescein.
Only after hybridization to the template DNA do two probes come in close proximity, resulting in fluorescence resonance energy transfer (FRET) between two fluorophores. During FRET, LightCycler-Fluorescein, the donor fluorophore, is excited by the light source of the LightCycler Instrument, and part of the excitation energy is transferred to LightCycler-Red, the acceptor fluorophore. The emitted fluorescence of the LightCycler-Red fluorophore is measured by the LightCycler Instrument.
Genotype determination
The hybridization probes are also used to determine the genotype by performing a melting curve analysis after the amplification cycles are completed and the amplicons are formed. First, one oligonucleotide of each pair of hybridization probes hybridizes to a part of the target sequence. After that, the other oligonucleotide of each pair of hybridization probes spans the mutation site (mutation probe). The latter probe has a lower melting temperature (TM) than the anchor probe, thus ensuring that the fluorescent signal generated during the melting curve analysis is determined only by the mutation probe. The TM is dependent upon length and G + C content, but also upon the degree of homology between the mutation probe and the template DNA. If a mismatch between hybridization probe and target is present, the hybrid is destabilized, resulting in a lower TM. When the hybridization probe matches perfectly with the target, the hybrid has a higher TM.
Statistical analysis
Data were analysed by SPSS v10.0 (SPSS Inc., Chicago, IL, USA) for Windows. The characteristics of women in both groups were compared with Student's t-test or 2-test. Genotype distributions were evaluated by
2-test. MantelHaenszel analyses were used to estimate the odds ratios (OR) to test whether it was equal to one. Test for HardyWeinberg equilibrium was conducted by comparing observed versus expected genotype frequencies using a
2-test. P
0.05 was considered statistically significant.
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Results |
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Discussion |
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The results of the present study indicate that polymorphic GSTP1 may play an important role in endometriosis. However, GSTP1 gene does not have a major effect on the stage of the disease. We found that GSTP1 val/val was associated with significantly decreased risk of endometriosis and seems to be protective. Although the difference was not significant, patients with GSTP1 ile/ile are at 1.5-fold higher risk of developing endometriosis. The molecular mechanisms by which the expressing genotypes of GSTP1 loci may be linked to the increased or decreased risk of endometriosis remain to be elucidated, but most probably through involvement of environmental toxins and/or its metabolites (Rier and Foster, 2002; Johnson et al., 1997
; Yang et al., 1997
).
In the first report about GST polymorphisms and endometriosis, it has been suggested that GSTM1-null polymorphism is associated with increased risk of endometriosis (Baranov et al., 1996). GSTM1-null polymorphism was found to be significantly higher in endometriosis patients in a French population with 77% having this polymorphism (Baranova et al., 1999
). A tendency to endometriosis was also observed in GSTT1-negative subjects in the same study, but it did not reach statistical significance. These findings, however, were not supported by later studies (Hadfield et al., 2001
; Hur et al., 2004
). Furthermore, Hur et al. (2004)
found no association between endometriosis and GSTP1 polymorphism in Korean women, in contrast to our findings.
The differential effect of GSTP1 genotypes on the endometriosis risk might result from enantio-selectivity of these genotypes. A large inter-individual variation in GSTP1 activity has been shown using 1-chloro-2,4-dinitrobenzene (Hu et al., 1996b). GSPT1 ile/ile was found 3-fold more effective than GSTP1 val/val in detoxification of this product. In contrast, GSTP1 val/val had a 7-fold higher efficiency for polycyclic aromatic hydrocarbons. Since an association between environmental pollutants and endometriosis has been shown (Rier et al., 1993
; Johnson et al., 1997
; Seli et al., 2003
), it is probable that GSTP1 val/val might be involved in detoxification of endogenous or exogenous toxic substances related to endometriosis, and may decrease the risk of this disease by this mechanism. In contrast, GSTP1 ile/ile might inactivate other environmental or endogenous toxic substances that are not directly involved in the pathogenesis of endometriosis. Alternatively, although GST are generally recognized as detoxifying enzymes, they may also be involved in generation and activation of toxic compounds (Hayes and Pulford, 1995
).
Interestingly, similar results have also been attained about GSTP1 variants in benign disease processes of other systems. Juronen et al. (2000) defined GSTP1 val/val as a genetic risk factor for the development of cortical cataract, whereas GSTP1 ile/ile was protective. In smokers, GSTP1 val/val genotype was found to be associated not only with an increased risk of decline of lung function, but also with the acceleration of this process (He et al., 2002
).
Although GSTM1 and GSTT1 variants have been studied intensively, insufficient human data about the types and distribution of GST through genital system and peritoneum hinder our ability to draw exact conclusions on which type(s) of GST may be more important in possible pathogenesis of endometriosis. In the human ovary, GSTP isoenzymes were expressed at relatively high levels in stroma (Rahilly et al., 1991). The authors suggested that GSTP might be involved in proliferation and/or differentiation processes. Although only one study has looked for an association between ovarian carcinoma and GSTP genotype (Spurdle et al., 2001
), the results of epidemiological data do not confirm an association between ovarian cancer and any polymorphic GST isoenzymes (Coughlin and Hall, 2002). However, GSTP isoenzyme was almost consistently found to be related to drug resistance in ovarian carcinomas in many studies (Hamada et al., 1994
; Cheng et al., 1997
; Kigawa et al., 1998
; Ghalia et al., 2000
), indicating an important role of this isoenzyme in the genital system.
In conclusion, we found that GSTP1 polymorphism might modulate the risk of endometriosis with significantly decreased risk for GSTP1 val/val and a trend for increased risk for GSTP1 ile/ile. The involvement and the significance of different GST in endometriosis are controversial. On the other hand, the central role of GST in detoxification of endogenous and exogenous compound have been well established. Regarding the importance of these compounds in the pathogenesis of endometriosis, the changes in expression of GST may modulate the risk of this disease. Further studies on not only the disease processes but also normal distributions in female genital tract may provide better understanding about the role of GST types and their polymorphs in endometriosis.
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Submitted on January 14, 2005; resubmitted on April 4, 2005; accepted on April 7, 2005.
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