Clinical Biochemistry 43 (2010) 1124–1128

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Clinical Biochemistry

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Association of glutathione S-transferase M1 and T1 gene polymorphisms andoxidative stress markers in preterm labor

M.D. Mustafa a, Rahul Pathak a, Tanzeel Ahmed a, Rafat S. Ahmed a, A.K. Tripathi a,Kiran Guleria b, B.D. Banerjee a,⁎a Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, Dilshad Garden, Delhi 110 095, Indiab Department of Obstetrics and Gynaecology, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India

⁎ Corresponding author. Fax: +91 11 22590495.E-mail address: banerjeebd@hotmail.com (B.D. Bane

0009-9120/$ – see front matter © 2010 The Canadiandoi:10.1016/j.clinbiochem.2010.06.018

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 10 April 2010Received in revised form 10 June 2010Accepted 27 June 2010Available online 16 July 2010

Keywords:Preterm laborReactive oxygen speciesOxidative stressGlutathione S-transferaseGenetic polymorphism

Objective: Oxidative stress and related gene polymorphismmay be associated with the etiology of pretermlabor (PTL). The present studywas designed to investigate association ofGSTM1 and GSTT1 gene polymorphismswith PTL and their relationship with oxidative stress markers.

Design andmethods: Sixty cases of PTL and sixty three subjects of full term labor (FTL) were included in thestudy. Multiplex PCR was performed for GSTM1 and GSTT1 genes polymorphism and oxidative stress markerswere analyzed.

Result: MDA and 8-OHdG levels were increased, while GSH was decreased in PTL than FTL subjects.Frequency of GSTM1−/GSTT1−(null) was significantly higher in PTL in comparison to FTL (p=0.028, OR=3.4).Subjects with GSTM1−/GSTT1+, GSTM1+/GSTT1−, GSTM1−/GSTT1− have significant differences of oxidativestress markers as compared to GSTM1+/GSTT1+ genotype.

Conclusion: GSTM1−/GSTT1− (null) genotypemay be one of the associated genetic factor for the increased

risk of PTL.

© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

PTL (b37 weeks gestation) affects approximately 5–7% of live birthsin developed countries, but is significantly higher in developingcountries [1,2]. Incidence of preterm labor is 23.3% and that of pretermdelivery, 10–69% in India [3]. Worldwide, PTL is the single largest causeof infant mortality and morbidities and is regarded as syndrome withmultiple causes [4,5]. Despite substantial basic scientific and clinicalinvestigations, PTL continues to be a major clinical and public healthproblem.Mechanisms underlying onset of idiopathic PTL are still poorlyunderstood [1,6]. Cervical insufficiency, hormonal imbalance, geneticpredisposition, altered immune surveillance, inflammation and subse-quent oxidative stress may antedate PTL and contribute to itspathogenesis [4].

Recent studies from our laboratory have shown that the higherlevels of some organochlorine pesticides residues may be associatedwith PTL and increased oxidative stress [5,7]. Present study is incontinuation of previous study and was designed to investigate theassociation of metabolic detoxifying gene deletion, oxidative stressand risk of PTL.

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Society of Clinical Chemists. Publish

Oxidative stress is characterized by imbalance between the levelsof reactive oxygen species (ROS) and antioxidant mechanisms of theorganism. Hypoxic events during labor, transition from low to highoxygen environment and activation of inflammatory responses andinfections results in generation of ROS [8]. There is circumstantialevidence that perinatal free radical generation and oxidative stresscan contribute in PTL [9,10].

Glutathione S-transferases (GSTs), a family of phase-II isoenzymes,play a critical role in providing protection against electrophiles andproducts of oxidative stress [11]Oxidative stress-related genes involvedin metabolic detoxification process have been associated with prema-ture delivery [12,13]. GlutathionS-transferaseμ1 (GSTM1) and θ1(GSTT1), the major phase II enzymes, are good candidate pretermdelivery susceptibility genes [14]. The polymorphism in GSTT1 andGSTM1 gene loci is caused by a deletion which results in the absence ofenzyme activity, especially in individuals with null genotypes [15].GSTM1 null genotypewas found to increase the risk for preterm birth inKorean women [14]. Further, it has been reported that the oxidativestress injury is modified by GSTM1 polymorphism in term pregnantwomen [16]. Despite the importance of oxidative stress in thepathogenesis of PTL, the available data are not conclusive and onlyfew studies appear tohave addressed the issue of association of PTLwithGST genes polymorphism [12,14,16]. Hence, the present study wasdesigned to investigate GSTM1 and GSTT1 gene polymorphisms,oxidative stress and association with PTL.

ed by Elsevier Inc. All rights reserved.

Fig. 1. (a) Gel picture showing GSTM1 gene deletion. M-100 bp marker, Lane 1 and3: GSTM1 deletion. (b) Gel picture showing GSTT1 gene deletion. M-100 bp marker,Lanes 2, 3, 4, 5 and 7: GSTT1 deletion.

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Materials and methods

Study population

Sixty women who had b37 weeks of gestation (PTL) and sixty threewomen having gestation period of N37 weeks (FTL) were included inthis age matched case-control study conducted at Guru Teg BahadurHospital associated with University College of Medical Sciences, Delhifrom August 2008 to December 2009. All the women went into laborspontaneously with intact membrane. Women with anemia, hyperten-sion, toxemia of pregnancy, renal disease, heart disease, diabetes,urinary tract infections, metabolic disorders, tuberculosis, smoking,alcohol consumption or chronic drug intake and having complicationsduring pregnancy and/or labor were excluded from both the groups. Alife style survey of the women was done to collect general demograph-ical information in order to define the inclusion/exclusion criteria. Thewomen who participated in this study were of relatively homogenousgroup, similar in terms of demographical characteristics such as age,weight, food habits, socioeconomic status, drinking water supply andlife style. None of the womenwere occupationally and/or environmen-tally exposed to agricultural pollutant. Women confirmed theirparticipation by signing an informed consent form. This study wasapproved by the institutional ethics committee for human research.Fourml ofmaternal bloodwas collected at the time of labor and equallydivided into two vials- a plain vial and an EDTA containing vial. Serumwas separated from plain vial and stored at -20 °C until further analysisof oxidative stress parameters. Blood in EDTA coated vial was stored at4 °C and used for estimation of GSH and extraction of genomic DNA.Isolated genomic DNA was then stored at -20 °C until further analysis.

Genomic analysis

Genomic DNA for genotyping was isolated from whole blood byusing commercially available Himedia Hipura blood genomic DNAisolation kit (HiMedia Laboratories Pvt. Ltd.23, Vadhani Ind. Est.,Mumbai, India) as per manufacturer's protocol. Multiplex PCR wasperformed for GST M1and GSTT1genes. Briefly, 50 ng of genomic DNAwas amplified in a 25-μl multiplex reaction mixture containing 30 pmolof the GSTM1 and GSTT1 primers in a medium consisting of 1.5 mMMgCl2, 200 μmol dNTPs, 2.5 μl 10× PCR buffer (10×500 mM KCl,100 mM Tris–HCl, pH 9.0), and 1 U TaqDNA polymerase (Promega,Madison,WI, USA). Primers used are shown in Table 1. The PCR protocolincluded an initial melting temperature of 94 °C (5 min) followed by 35cycles of amplification (2 min at 94 °C, 1 min at 59 °C, and extensionfor 1 min at 72 °C). A final 10-min extension step (72 °C) terminated theprocess. Thefinal PCRproducts fromco-amplification ofGSTM1 (215 bp)and CYP1A1 (312 bp) as well as GSTT1 (480 bp) and CYP1A1 (312 bp)were visualized after electrophoresis in ethidium bromide-stained 2%agarose gel. The absence of amplifiable GSTM1 and GSTT1 (in thepresence of CYP1A1 PCR product) indicates a GSTM1−/GSTT1−(null)genotype in Fig. 1a and b.

Estimation of oxidative stress parameters in FTL and PTL subjects:Measurement of lipid peroxidation

The lipid peroxide levels in plasma of maternal blood weremeasured using a thiobarbituric acid reactive substances (TBARS)

Table 1Oligonucleotide primers used for amplification.

GSTM1 G1—5' GAA CTC CCT GAA AAG CTA AAG C 3'G2—5' GTT GGG CTC AAA TAT ACG GTG G 3'

GSTT1 T1—5' TTC CTT ACT GGT CCT CAC ATC TC 3'T2—5' TCA CCG GAT CAT GGC CAG CA 3'

CYP1A1 F—5' GAA CTG CCA CTT CAG CTG TCT 3'R—5' CAG CTG CAT TTG GAA GTG CTC 3'

assay, which monitors MDA production based on the method of Satohet al [17]. The MDA-TBA adduct formation was measured spectro-photometrically at 532 nm. The concentration of MDA was expressedas nmol/ml.

Measurement of reduced glutathione

Total reduced glutathione (GSH) content in blood was measuredby the method of Tietze [18] using 5, 5' dithiobis-2 nitrobenzoic acid(DTNB). In this method, GSH was oxidized by DTNB and then reducedby GSH reductase, with NADPH as hydrogen donor. The oxidation ofGSH by DTNBwas detected photometrically by a change of absorptionat 412 nm. The concentration of blood GSH was expressed as μmol/dl.

Measurement of Ferric reducing ability of plasma

Ferric-reducing ability of plasma (FRAP) was determined bymeasuring the ability of plasma to reduce Fe3+ to Fe2+ by the methodof Benzie et al. [19]. The complex between Fe2+ and 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) gives a blue color with absorbance at593 nm. Concentration of FRAP was expressed in μmol/l serum

Measurement of glutathione S-transferase activity

GST activity in serum was determined according to the methoddescribed by Habig et al [20] using 1-chloro-2,4-dinitrobenzene(CDNB) as substrate. The formation of adduct of GSH-CDNB (2, 4,

Table 2Demographical profile of FTL and PTL subjects.

Characteristics Mean±SD Mean±SDFTL PTL

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dinitro phenyl glutathione) was monitored by estimating its increaseat 340 nm by using a spectrophotometer. The specific activity wasexpressed as U/mg protein. Protein concentration was estimated bythe method of Lowry et al. [21].

Maternal Age (years) 25.17±3.28 24.16±2.93Maternal weight (kg) 51.42±10.88 48.34±9.89Gestational Age (weeks) 37.1±1.43 32.12±1.23Newborn weight (kg) 2.47±0.28 2.90±2.23

EducationIlliterate 14 16Intermediate (10+2) pass or less 40 36Graduate 09 08

Drinking water supplyGovernment source 42 40Private source 21 20

Living conditionSlum 20 21Market area 07 05Colony 36 34

Dietary habitsVegetarian 44 40Non-vegetarian 19 20

Age group19–24 36 3725–29 27 23

Measurement of DNA damage

Quantification of DNA damagewas performed using 8-OHdG ELISAkit by diluting the samples (Cayman Chemical Company, Ann Arbor,MI, US) according to manufacturer's instructions. The kit can measure8-OHdGvalues ranging from 33 to 3000 pg/ml. Briefly, platewells weremarked as blank, non-specific binding (NSB), maximum binding (B0),total activity (TA), standard and samples. 100 μl of EIA bufferwas addedtoNSB and 50 μl of EIA bufferwas added to B0wells respectively. 50 μl ofstandards and samples in duplicateswere added to their respectivewell.50 μl of 8-OHdG AChE tracer was added to each wells except TA andblank wells. 8-OHdG monoclonal antibody (50 μl) was added to eachwell except TA, NSB and blank wells. Plate then incubated for 18 h at4 °C. Following incubation, the plate was washed to remove anyunbound reagents and then Ellman's reagent (which contains thesubstrate to AChE)was added to thewell. The product of this enzymaticreaction has a distinct yellow colour and absorbs strongly at 412 nm.Because the concentration of the 8-OHdG tracer is held constant whileconcentration of 8-OHdG varies, the amount of 8-OHdG Tracer that isable to bind to the 8-OHdG monoclonal antibody, is inversely propor-tional to the concentration of 8-OHdG.

Statistical analysis

The Student's t-test was used to identify differences between thesubjects of PTL (cases) and FTL (controls). Group genotype distributionand allele frequencies were compared using the χ2-test. Crude oddsratios and 95% confidence intervals were compared. Pearson correla-tion coefficient between the levels of oxidative stress biomarkers(both enzymatic and non-enzymatic) and genotypes was determinedby Pearson test. The value of pb0.05 was considered to denotesignificance.

Table 3Comparison of oxidative stress markers in FTL and PTL subjects.

Oxidative stress parameters FTL PTL

MDA (nmol/ml) 1.17±0.33 2.50±1.18*GSH (μmol/dl) 241.34±37.04 190.01±48.3*FRAP (μmol/l) 255.92±66.46 238.02±57.92GST (U/mg protein) 0.944±.183 0.907±.2938-OHDG (pg/ml) 55.39±5.88 72.85±7.59*

*pb0.05.

Results

The women who participated in this study had similar demo-graphical characteristics such as age, education, parity, drinking watersupply, dietary habit and area of residence (Table 2). The levels ofMDA and 8-OHdG were significantly higher (pb0.05) and GSH wassignificantly lower (pb0.05), also FRAP, GST were lower in maternalblood of cases with PTL in comparison to FTL mothers (Table 3).

Genetic polymorphism in GSTM1 and GSTT1 genes were investigatedand four possible genetic combinations and their genotypic distributionwere observed (Table 4). In Fig. 1a and b the presence of DNA bands at215 bp and 480 bp corresponds to an individual with at least one intactGSTM1 and GSTT1 allele respectively. The absence of either of thesebands (null genotype) corresponds to individuals who are homozygousfor the null allele. In Table 4 the frequency of GSTM1+/GSTT1+34/63(53.9%)washigher in FTL thanPTL20/60(33.3%)whileGSTM1−/GSTT1+,GSTM1+/GSTT1− in mothers with PTL were 16/60 (26.6%) and 12/60(20%) which were higher than FTL. GSTM1−/GSTT1−(null) wassignificantly higher in subjects with PTL compared to FTL (p=0.028).Fig. 2a and b shows that subjects with GSTM1−/GSTT1+, GSTM1+/GSTT1−, GSTM1−/GSTT1− have significantly higher MDA and 8-OHdG(pb0.05) and significantly lower GSH and FRAP (pb0.05) compared toGSTM1+/GSTT1+ genotype. Enzymatic marker of oxidative stress, GSTwas lower in GSTM1−/GSTT1+, GSTM1+/GSTT1−, GSTM1−/GSTT1−compared to GSTM1+/GSTT1+ in maternal blood at the time of labor.

Discussion

Changes in ROS levels during pregnancy are associated withnormal development of the embryo and foetus. During early post-implantation period when the embryo is most sensitive to oxidativestress, the environment in the uterus is relatively hypoxic that mayinduces cell proliferation. [22]. Later, increased oxidative stress leadsto differentiation and ROS act as secondary messengers by regulatingtranscription factors affecting gene expression in the embryo[23]. Theimportance of oxidative stress in PTL has recently received attention.Studies in animal models have demonstrated that maternal systemicinflammation causes preterm birth and is associated with free radical(FR) formation and oxidative stress in both mother and foetus [24].Oxidative stress may lead to cellular damage andmalfunction throughthe free radical-mediated decomposition of vital molecules, such asDNA, proteins and lipids [25,26].

Excessive oxidative stress during pregnancy has been associatedwith various pregnancy complications such as PTL, intrauterinegrowth retardation, pre-eclampsia, diabetes etc. [27,28]. We observedsignificantly higher levels of MDA and 8-OHdG (pb0.05) andsignificantly decreased levels of GSH (pb0.05) in maternal blood ofPTL compared to FTL indicating diminished ability to resist oxidativedamage (Table 3). FRAP assay measures the total ability of a person toresist oxidative challenge. The FRAP as well as GST activities were alsofound to be decreased slightly. This is in accordance with previousstudies [5,16,29]. This increased oxidative stress and decreased non-enzymatic antioxidant reserve in mother would enhance thevulnerability to free radical damage by ROS to mother as well as child.

Table 4Genotypic distribution of glutathione S-transferase genotypes in FTL and PTL subjects.

GST genotypes FTL (N=63) PTL (N=60) Odds ratio (OR) 95% Confidence interval (CI) Chi-square value(χ2) p-value

GSTM1+/GSTT1+ 34 (53.9%) 20 (33.3%) 1.00 (reference) – – –

GSTM1−/GSTT1+ 14 (22.2%) 16 (26.6%) 1.94 0.78–5.495 4.80 0.14GSTM1+/ GSTT1− 09 (14.2%) 12 (20%) 2.26 0.81–6.32 2.50 0.11GSTM1−/GSTT1− 06 (9.52%) 12 (20%) 3.4 1.10–10.47 4.8 0.028*

*pb0.05.

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Polymorphic large deletions causing inactivation of two oxidativestress genes, GSTM1 and GSTT1 have previously been associated withincreased oxidative stress [14,16,30,31]. Studies on healthy popula-tion, including a previous study from this laboratory have reportedsignificant presence of GSTM1 and GSTT1 null genotype amongstIndians [32,33]. Frequency of GSTM1 and GSTT1 in normal healthynorth-Indian Individuals was reported to be 33% and 18.4% respec-tively with the concomitant lack of both genes was observed in 7% ofpopulation [33]. Singh et al. have reported the prevalence ofGSTM1andGSTT1 null genotype to be 21% and 27.4% respectively, withsimultaneous double deletion in 0.7% in Delhi population. The GST1null phenotype has a frequency of greater than 50% among Caucasians,Chinese and Indians populations [34]. About 60% of Asians, 40% of

Fig. 2. (a) Histogram showing the comparison of 8-OHdG, GSH and FRAP levels inGSTM1+/GSTT1+ with GSTM1−/GSTT1+, GSTM1+/GSTT1− and GSTM1−/GSTT1−(null) in all subjects (FTL+PTL). *pb0.05. (b) Histogram showing the comparison ofMDA and GST levels in GSTM1+/GSTT1+ with GSTM1−/GSTT1+, GSTM1+/GSTT1−and GSTM1−/GSTT1−(null) in all subjects (FTL+PTL). *pb0.05.

Africans and 20% of Caucasians don't express GSTT1 enzyme [35].Recent studies have investigated the role of genetic susceptibility andgene-environment interactions in PTL [1,12–14,36]. These studieshave been motivated by growing evidence indicating that familial orintergenerational factors influence the risk of PTL. This influence maybe due to shared environmental factors or different genetic factors, orboth.

Our study has investigated GSTM1 and GSTT1 gene polymorphismsin subjects with PTL and FTL and demonstrated that the frequency ofGSTM1+/GSTT1+ was lower in PTL cases compared to FTL whereasthe frequency of GSTM1−/GSTT1− (null genotype) in PTL wassignificantly higher compared to FTL (p=0.028,OR=3.4) in Table 4.Fig. 2a and b shows significantly higher levels of MDA and 8-OHdG(pb0.05) and significantly lower levels of FRAP and GSH (pb0.05) inGSTM1+/GSTT1−, GSTM1−/GSTT1+ and GSTM1−/GSTT1− comparedto GSTM1+/GSTT1+. Our findings corroborates with the other studieswhere it is shown that individuals with either or both GSTM1, GSTT1gene deletion have higher oxidative stress [14,16,26,31]. Takentogether, these observations suggest that a genetic predisposition(GST gene polymorphisms) may contribute to the development of PTL.The most probable explanation is based on the antioxidant activity ofGST enzyme. It is worth noting that deleted polymorphisms in the GSTgenes may also influence the susceptibility to PTL by modulatingdetoxification of genotoxic secondary metabolites.

To the best of our knowledge this study provides evidence for thefirst time that leukocyte DNA damage in terms of 8-OHdG, lipidperoxidation in terms of MDA and antioxidant markers like GSH andFRAP is more intense in PTL cases having the GST M1−/GSTT1−(null),GSTM1−/GSTT1+, GSTM1+/GSTT1− genotypes. Our study supports arecent study on GSTT1 genotypes which showed that null (non-functional) genotype in both mothers and foetus increased the risk ofPTL during third trimester of pregnancy regardless of maternal smokingstatus [1]. GSTM1 null genotype was found to increase the risk forpreterm birth in Korean women [14]. This study also provides possibleeffects of gene-gene or gene-environment interaction in oxidativedamage toDNA, lipid aswell as loweringof antioxidants suchasGSHandFRAP in PTL patients. The results indicate that GSTM1−/GSTT1−(null)genotypes were found significantly higher in PTL and seem to be hostfactors that canmodulate 8-OHdG andMDA levels and antioxidants likeGSH and FRAP. The significant differences of oxidative stress markersbetween PTL and FTL indicate the pathogenic role of oxidative stress inPTL and subjects with GSTM1−/GSTT1− (null) genotype are morevulnerable to oxidative damage. Hence, it may be said that GSTM1−/GSTT1− (null) genotype may be associated with increased risk of PTL.However, a definitive conclusion cannot be drawn, since the sample sizeis low in our study. Further studies with larger sample size are neededand are in progress in our laboratory.

Themechanismof oxidative stressmediated PTL and associationwithmetabolic gene polymorphism are extremely complex; however, thisapproach may be one way of investigating the genes’ modulating effecton such incidences of preterm labor. Gene therapy targeting oxidantsystems are also being developed, but their role in PTL remains to beelucidated. Further studieswith large sample sizeswhich identify the roleof other oxidative related gene polymorphisms, their gene expressionprofiles in PTL along with assessment of hormonal, inflammatory andimmunological factors are clearly needed.

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Acknowledgments

Authors are thankful to the Ministry of Environment and Forests(Govt. of India) for sanction of research project vide no. RE-19-10-2007.The authors are grateful toDr. Sudip SenMD, PhD for critically reviewingthe manuscript.

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