Free Radical Biology & Medicine 49 (2010) 1746–1754

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Original Contribution

Polymorphism in glutathione S-transferase P1 is associated with susceptibility toPlasmodium vivax malaria compared to P. falciparum and upregulates the GST levelduring malarial infection

Mohammad Sohail a,1, Ritesh Kumar b, Asha Kaul a, Ehtesham Arif b, Sanjit Kumar c, Tridibes Adak a,⁎a Molecular Parasitology Laboratory, National Institute of Malaria Research, Dwarka, New Delhi 77, Indiab Faculty of Science, Hamdard University, New Delhi 62, Indiac Department of Biophysics, All India Institute of Medical Sciences, New Delhi 29, India

Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene;benzoic acid); GPX, glutathione peroxidase; GSH, reduceS-transferase; JNK, c-Jun N-terminal kinase lipid peroxspecies; SNP, single-nucleotide polymorphism.⁎ Corresponding author.

E-mail address: adak.mrc@gmail.com (T. Adak).1 Present address: Institute for Integrative Geno

Entomology, University of California at Riverside, Rivers

0891-5849/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.freeradbiomed.2010.09.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 March 2010Revised 2 August 2010Accepted 5 September 2010Available online 16 September 2010

Keywords:GSTP1P. vivax malariaP. falciparum malariaPolymorphismGSTFree radicals

Glutathione S-transferase P1 (GSTP1) is a member of the GST superfamily, which has well-establishedmultiple roles in various infectious and parasitic diseases. The genetic regulation of GSTP1 has beenextensively studied. Thus, its biological significance and disease association prompted us to investigate therole of GSTP1 polymorphisms in Plasmodium-mediated pathogenesis in infected humans. The genotypicdistribution of Ile105Val in Plasmodium vivax infection was observed to be significant and strongly associated(OR=4.5) with the progression of pathology, whereas in P. falciparum infection no significant association wasobserved compared to healthy subjects. Interestingly, we observed significant elevation of GST in vivaxinfection, with both genotypes Ile105Val and Val105Val, compared to healthy subjects, whereas in P.falciparum infection we found marginally elevated GST levels of mutated genotypes but significantly depletedcompared to healthy subjects. Further, during vivax and falciparum infection overall significant elevations ofglutathione, glutathione peroxidase, and GST levels were observed. Expression of both GSTP1 mRNA andprotein was significantly upregulated during vivax infection compared to falciparum infection and both weresignificantly upregulated compared to the levels in healthy subjects as well. These studies suggest that GSTP1polymorphism is involved in the pathogenesis of malaria and it may serve as a valuable molecular marker,possessing a promising rationale for diagnostic potential in assessing disease progression during clinicalmalaria.

DTNB, 5,5′-dithiobis-(2-nitro-d glutathione; GST, glutathioneidation; ROS, reactive oxygen

me Biology, Department ofide, CA 92521, USA.

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

Glutathione S-transferases (GSTs) play an important role in themetabolism of endobiotics and xenobiotics by virtue of theirconjugation reactions with glutathione. GSTs are also helpful in thetransportation of hemin generated during the metabolism of varioushemoproteins [1] and involved in the acquisition of drug resistance invarious diseases including malaria [2]. Glutathione S-transferase P1(GSTP1) is a member of the cytosolic GST superfamily [3–5]. Becausemalaria infection results in increased ROS (reactive oxygen species) aswell as reduction of antioxidants, we defined oxidative stress as theimbalance between the two, resulting from either excess ROS orantioxidant reduction. The action of oxidative stress during malariainfection is unclear; some researchers have demonstrated a protectiverole, whereas others have confirmed its relation with malarial

pathology [5,6]. Tropical malaria, which is caused by the protozoanparasites Plasmodium falciparum and Plasmodium vivax, is responsiblefor 515 million clinical cases [7] and 1 to 3 million deaths annually [8].The emergence and spread of drug resistance to commonly usedchemotherapeutics are major factors contributing to this increasingburden. Thus, the characterization of alternative drug targets isurgently required [9–11]. GST activity has been detected in allPlasmodium species studied so far as well as in all intraerythrocyticstages of the parasite [12]. Human GSTs (cytosolic) comprise a familyof proteins classified into five major genes, GST-Alpha (GSTA), GST-Mu (GSTM), GST-Theta (GSTT), GST-Pi (GSTP), and GST-Zeta (GSTZ),with one or more genes in each class. The enzymes have different, butsometimes overlapping, substrate affinities. Among the most inten-sively studied GSTs are GSTM1, GSTT1, and GSTP1. Many studies haveshown strong associations between impaired or deficient GST activityand diseases such as lung cancer, acute leukemia, and many others inboth human and animal models [13]. Kavishe et al. [14] has shownthat GSTM1 enzyme is significantly overrepresented in the malariawith minor complications and severe malaria groups compared withthe uncomplicated falciparum malaria group, substantiating ourhypothesis of the involvement of GST polymorphisms in malarial

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pathogenesis [14]. Recently the same group of investigators hasshown an association of GSTP1 I105V and a trend, but not significantassociation, of GSTM1 with severe malaria anemia [15]. GSTP1 andGSTM1 are expressed in all blood cells, with higher expression inlymphoid than in erythroid cell types, whereas GSTT1 and GSTA areexpressed at higher levels in erythrocytes than in lymphoid cells [16].In conclusion, GSTP1, and possibly GSTM1, may have a significant rolein malarial complications and disease progression. Furthermore, theGSTs, and the Pi class in particular, have attracted considerableattention because of their ability to modulate other biochemicalpathways, such as intracellular signaling processes, including the Junkinase pathway [12], and the activation of the essential antioxidantprotein peroxiredoxin [17]. Therefore, the GSTs are critical multi-functional proteins and have probably evolved as an importantdefense against cellular damage due to chemical and oxidative stress.

Malaria infection induces oxidative stress in the host cells.Antioxidant enzymes such as GSTs are responsible for fightingreactive oxygen species and reduction of oxidative stress. CommonGST polymorphisms have been associated with susceptibility tovarious diseases whose pathologies involve oxidative stress. Individ-ual variations in response to a drug originate from different causes,such as genetic polymorphisms and altered expression levels of drugtarget molecules (e.g., membrane receptors, nuclear receptors, andenzymes), as well as those of drug-metabolizing enzymes and drugtransporters [18,19].

Specifically, the human Pi class GST, GSTP1, is a polymorphicgene localized to chromosome 11, with a number of single-nucleotide polymorphisms (SNPs) that yield an amino acid changein the encoded protein, i.e., Ile105Val, Ala114Val, Asp147Tyr, andPhe151Leu [19]. The Ile105Val and Ala114Val polymorphisms havebeen the most widely studied to date; although both are within theelectrophile-binding active site of the protein, only the former yieldsa significant reduction in activity of the protein [12,17]. Because ofthe functional importance and widespread nature of GSTP1-1, anumber of studies have been carried out to investigate the role ofthese polymorphisms in disease susceptibility, particularly infec-tious diseases. Although GSTP1 is an important gene, a systematicstudy of common genetic variations in this gene and characteriza-tion of the functional significance of those variations has not beenreported in malaria.

Mutated genotypes of GSTP1 exhibit elevation of enzymaticactivity and are hypothesized to modulate an increased risk in thecase of vivax infection and pathology. However, our knowledge of themalaria-induced oxidative stress, GST levels, and GSTP1 polymorph-isms and their relation to pathology, diagnosis, and immunomodu-latory response in malarial patients is very limited and notsystematically elucidated. The aim of this study was to identify thepotential association between GSTP1 deleted polymorphisms andmalarial risk and pathology in themalarial population. Thus, to furtherstudy the interaction between Plasmodium infection and GSTP1polymorphism, we investigated the impact of the content of lipidperoxidation (LPO), reduced glutathione (GSH), glutathione peroxi-dase (GPX), and GST in serum on clinical malaria pathogenesis anddiagnosis in adults who visited the malaria clinic located at theNational Institute of Malaria Research at 2-Nanak Enclave, New Delhi,India, as outpatients. GST and other enzymes were estimated becauseof their biological relevance to alterations in redox metabolism andtheir paramount roles in other metabolic and oxidative changes. Toobtain a global measure of the patients’ actual reactivity towardPlasmodium challenge, the study of GST and other enzymes becomesinevitable as it also forms a central aspect of the host response tomalaria and immunomodulation. Apart from this the other objectiveof this investigation was to explore the polymorphic expression of theGSTP1 gene, which is important for regulating gene expression andthe secretion of GST. However, there has been no published studyexamining the association between vivax malaria and the Ile105Val

polymorphism in exon 5 of this GST gene. In this study, weinvestigated whether these polymorphisms in the exon region ofthe GST gene are associated with vivax infection and circulating GSTlevels in various clinical groups and the possible correlations withclinical symptoms in the Indian population. We investigated theimpact of this allele on clinical malaria pathogenesis and diagnosis inadults.

The results of these studies provide comprehensive informationwith regard to common sequence variation in GSTP1, as well aspathological association resulting from variation. These data alsoprovide a foundation for future genotype–phenotype associationstudies involving both malarial risk and variation in antimalarial drugresponse.

Materials and methods

Human blood samples

All human blood samples used in this study were collected afterobtaining consent from the study participants under protocolsapproved by the Ethical Review Board of the National Institute ofMalaria Research, New Delhi, and human ethical guidelines asreflected in the guidelines of the Medical Ethics Committee, Ministryof Health, Government of India. Peripheral venous blood (2 ml in eachtube) was collected from each subject, before administration ofantimalarial therapy, aseptically by dripping from the syringe withoutanticoagulant into a sterile microfuge tube for assessment of serumGST activity, lipid peroxidation assay, and GSH and GPX activity andwith anticoagulant (heparin or EDTA) for DNA, RNA, and proteinisolation. P. vivax or P. falciparum infection was confirmed by thick-and thin-smear blood films using standard JSB staining techniquesand light microscopy [20]. Co-infection with other malaria specieswas ruled out by light microscopy and the use of First ResponseMalaria pLDH/HR2 combo test kits (Premier Medical Corporation,Mumbai, India) as per the manufacturer's guideline. Blood wasallowed to coagulate in a refrigerator for 4 to 6 h at 4 °C before beingprocessed by centrifugation. Sera were preserved in three to fivealiquots at −70 °C until measurements were performed.

Biochemical parameters

Malondialdehyde, an LPO product, was estimated by a standardmethod [21]. GSH was estimated by the method of Jollow et al. [22].Serum (0.2 ml) was added to 2 ml of 4% 5-sulfosalicylic acid andkept for 1 h. The assay mixture further contained phosphate bufferand 0.4 ml of DTNB in a total reaction mixture of 3 ml. The yellowcolor that developed was read at 412 nm. GPX activity wasmeasured by the method of McFarland et al. [23]. To 0.2 ml ofbuffer, 0.2 ml of EDTA, 0.1 ml of sodium azide, and 0.2 ml of serumwere added. To that mixture, 0.2 ml of glutathione solution and0.1 ml of hydrogen peroxide were added. The contents were mixedwell and incubated at 37 °C for 10 min along with the control tubesand the color that developed was read at 340 nm for 3 min. GSTactivity in serum was assayed and the reaction was initiated byusing the electrophilic substrate 1-chloro-2,4-dinitrobenzene(CDNB) according to the procedure described by Habig et al. [24].GST was estimated in 1 ml of incubation mixture containing 850 μlof 0.1 M phosphate buffer (pH 6.5), 50 μl of 20 mM CDNB reagent,and preincubated at 37 °C for 10 min. Reaction was started byadding 50 μl of 20 mM glutathione and 50 μl of serum. Enzymeactivity was determined by continually monitoring the change inabsorbance at 340 nm followed at 1-min intervals for 5 min. Thebiochemical levels were estimated on a high-throughput Spectra-Max Plus 384 spectrophotometer (Molecular Devices, Sunnyvale,CA, USA). The coefficients of variation for inter- and intrabatchassessment of all biochemical parameters were b5%.

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Glutathione S-transferase P1 genotyping

DNA was extracted from blood spots dried on filter paper using aDNA isolation kit (QIAmp Blood Kit; Qiagen, Krefeld, Germany)according to the manufacturer's instructions. To detect the variants ofcodon 105 of GSTP1 at exon 5 the isolated DNA (100 ng)was amplifiedas described in [25] in a total reaction volume of 25 μl. The cyclingcondition for GSTP1was an elevated annealing temperature applied inthe first 15 cycles of PCR to prevent nonspecific priming. After aninitial denaturation step of 12 min at 95 °C, 15 cycles of PCR wereperformed (denaturation 30 s at 95 °C, annealing 30 s at 58 °C,elongation 60 s at 72 °C) followed by 25 cycles of amplification(denaturation 30 s at 95 °C, annealing 30 s at 55 °C, elongation 60 s at72 °C) and 1 cycle of elongation, 5 min at 72 °C, yielding a PCR productof 433 bp. The GSTP1 polymorphism was screened by restrictionfragment length polymorphism using 2.5 U of BsmA1 (New EnglandBio Labs, Cambridge, UK) restriction endonuclease in 10 μl of the PCRproduct in a total reaction volume of 20 μl, and the digested PCRfragments were separated on a 2% agarose gel.

Cloning and sequence analysis of GSTP1

The amplified PCR products were gel-purified using a gelextraction kit (Qiagen), ligated into the pGEMT-Easy vector (Promega,Madison, WI, USA), and transformed into DH5α Escherichia coli.Sequencing reactions were conductedwith a BigDye Terminator CycleSequencing Ready Reaction Kit in an ABI 3730xl automatic DNAsequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide anddeduced amino acid sequences were analyzed with the MEGA 3.1program.

RNA isolation and semiquantitative reverse transcription–PCR (RT-PCR)

Total RNA was isolated from blood using TRI reagent (Ambion,Austin, TX, USA). After DNA digestion (DNase I; Fermentas, Burling-ton, ON, Canada), cDNA equivalent to 1 μg of total RNA was preparedby one-step RT-PCR (AccessQuick RT-PCR system; Promega) usinggene-specific primers according to the manufacturer's protocol.Semiquantitative RT-PCR was performed to amplify GSTP1 mRNAlevels and to compare its expression in healthy subjects with P. vivaxand P. falciparum infection. The primers used in reactionmixture werefor GSTP1, as described by Ghobodloo et al. [25]; this generates aproduct of 433 bp. The housekeeping gene β-actin primers, forward(5′-GCGGGAAATCGTGCGTGACATT-3′) and reverse (5′-GATGGAGTT-GAAGGTAGTTTCGTG-3′), were used to normalize the RNA as aninternal control, which generates a product of 250 bp. Densitometerevaluation of the ethidium bromide-stained bandwas performedwiththe ImageQuant TL software (GE Healthcare, Chalfont St Giles, UK). AGSTP1 gene expression study was carried out on isolates with the Val/Val genotype from subjects infected with P. vivax and also thoseinfected with P. falciparum using RT-PCR and assessed by densitom-etry and labeled as mRNA expression relative to that of healthysubjects. The human β-actin gene was used as a normalizing factorand data represent the means±SE of two independent experiments,and the statistical significance was calculated using Student's t test(N=5 for each bar represented).

Preparation of GST from malaria patients and Western blot analysis

For preparation of total GST, 1 ml of blood from P. vivax- and P.falciparum-infected patients was thoroughly washed with PBS andthen homogenized with a Potter–Elvehjem homogenizer in aminimum volume of PBS. The homogenate was further processed aspreviously described by Ahmad et al. [26] to obtain mitochondrial,postmitochondrial, cytosolic, and microsomal fractions. All thefractions were analyzed for the activity of GST, and purification of

GSTwas carried out by ammonium sulfate precipitationmethods [26].Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis was performed on a 12% gel according to the methodof Laemmli [27]. For Western blot analysis, the fraction having thehighest GST activity was used as homogenate. Thirty micrograms oftotal protein was resolved by gradient polyacrylamide gel electro-phoresis and electrotransferred to a polyvinylidene difluoridemembrane (Millipore, Billerica, MA, USA). Immunodetection ofGSTP1 proteins was accomplished with anti-GSTP1 antiserum(Biogenes, NH, USA). Visualization was achieved with an alkalinephosphatase-conjugated goat anti-rabbit IgG. Immunoreactive bandswere digitally scanned and analyzed with NIH ImageJ for quantifica-tion. GSTP1 protein expression study was carried out on isolates withthe Val/Val genotype from subjects infected with P. vivax and alsothose infected with P. falciparum using Western blot and assessed bydensitometry and labeled as protein expression relative to that ofhealthy subjects. The human β-actin gene was used as a normalizingfactor and data represent the means±SE of two independentexperiments, and the statistical significance was calculated usingStudent's t test (N=5 for each bar represented).

Quantitative RT-PCR

Real-time PCR was used to determine the induction of GSTP1expression after total RNA isolation from Plasmodium-infected bloodand uninfected blood from healthy subjects with the RNeasy Mini Kit(Qiagen). cDNA was synthesized using an iScript cDNA kit (Bio-Rad)following the manufacturer's protocol. qRT-PCR was performed in theIQ5 thermal cycler (Bio-Rad) using the iQ SYBR Green I Supermix(Bio-Rad). The following PCR program was used: 95 °C for 10 min,followed by 45 cycles of 95 °C for 15 s for denaturation, 60 °C for 1 minfor annealing, and 72 °C for 30 s for extension. The gene-specificprimers used were GSTP1-F, 5′-ACCTTCATTGTGGGAGACCA-3′, andGSTP1-R, 5′-GGACCTCATGGATCAGCAG-3′, for GST and β-actin for-ward primer, 5′-GCGGGAAATCGTGCGTGACATT-3′, and reverse prim-er, 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′, for β-actin. Each reactioncontained 10 μl IQ SYBR Green Supermix reagent (Bio-Rad), 10 ngcDNA, and 300 nM each gene-specific primer in a final volume of20 μl. All qRT-PCRs were performed in duplicate. Data were analyzedusing the ΔΔCT method with β-actin used as normalizer.

Calculation of association strength

The odds ratio (OR) and 95% confidence interval (95% CI) provide ameasure of the strength of association, e.g., for a given vivax infectiondemonstrating a particular genotype compared with healthy subjects,a higher odds ratio is reflective of a disease association. The modelused for risk assessment was the logistic regression adjusted forgender and age. All tests were conducted at the Pb0.05 level ofsignificance.

Statistical analysis

SPSS 10 and EPIINFO 6 software were used for statistical analysis.EPIINFO was used for the power calculation. With an α value of 0.05the study provides a power (two-tailed) of N60% for the differencesobserved between the genotype and the allele frequency comparedwith healthy subjects. A goodness-of-fit test was used for testing theHardy–Weinberg equilibrium and a χ2 test compared the genotypeand allele frequencies of the GSTP1 Ile105Val polymorphism betweenthe two groups. OR and 95% CI for malarial isolates of each genotypewere calculated with logistic regression to quantitatively assess thedegree of association and were used to compare categorical variables.All biochemical data were expressed as means±SE. The means of theparameters for malaria patients and healthy subjects were compared

Table 1Physical characteristics of malaria patients and healthy subjects

Parameter P. vivax (mean±SE) P a Healthy subjects (mean±SE) P. falciparum (mean±SE) P a

No. of subjects (gender, F/M) 116 (11/105) 26 (8/18) 54 (8/46)Age (years) 30.3±0.86 NS 28.4±1.73 28.6±1.98 NSTemperature (°F) 99.7±0.16 0.0001 98.3±0.01 99.7±0.31 0.0001Weight (kg) 53.7±0.87 0.001 59.46±1.43 56.8±1.65 NSLipid peroxidation (μmol/L) 6.16±0.11 NS 6.43±0.17 1.91±0.08 0.0001Reduced glutathione (μmol/mg) 5.21±0.08 0.0001 3.21±0.03 4.38±0.54 0.0001Glutathione peroxidase (nmol/mg) 5.85±0.12 0.0001 3.15±0.01 6.05±0.13 0.0001Glutathione S-transferase(U/L) 4.64±0.07 0.0001 1.41±0.01 2.1±0.03 0.0001

a Compared with healthy subjects. NS, not significant.

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using Student's t test. Data analysis was performed nonparametrically.Correlation coefficients (r2) were calculated using the Spearman rankcorrelation test. Coefficients of variation were generally well under10%. The differences were considered significant when Pb0.05.

Results

Biochemical parameters and GST levels in malarial and healthy subjects’sera

A total of 196 subjects (116 (11 F/105M) vivax patients, 54 (8 F/46M)falciparum patients, and 26 (8 F/18 M) healthy subjects) were matchedand included for analysis as shown inTable1.Weobservedameanoverallsignificant elevation of serum reduced glutathione, glutathione peroxi-dase, and GST (P=0.0001, P=0.0001, P=0.0001, respectively) andmarginal butnonsignificantdepletionof LPO inP. vivaxpatients comparedto healthy subjects. A similar trendwas observed (P=0.0001, P=0.0001,P=0.0001, respectively), except for a significant depletion of LPO(P=0.0001), in P. falciparum infection as shown in Fig. 1. In addition tothis,we observed that LPO, GSH, andGST levels (P=0.001, P=0.001, and

LPO-PV LPO-H LPO-PF0

2

4

6

8NS P=0.0001

HEALTHY SUBJECTP.FALCIPARUM

P.VIVAXA

LP

O A

CT

IVIT

Y (

µmo

le/m

l.)

GPX-PV GPX-H GPX-PF0

2

4

6

8P=0.0001 P=0.0001

HEALTHY SUBJECTP.VIVAX

P.FALCIPARUM

C

GP

X A

CT

IVIT

Y (

µmo

le/m

l.)

Fig. 1. (A) Level of LPO in P. vivax and P. falciparum compared with healthy subjects. (B) LeveP. vivax and P. falciparum compared with healthy subjects. (D) Level of GST in P. vivax and

P=0.001, respectively) are significantly higher in vivax infectioncompared to falciparum infection, depicted in Fig. 1.

Polymorphism at the Ile105Val position of the GSTP1 gene

Amplification of the 433-bp fragment for variants of codon 105 ofthe GSTP1 gene in exon 5 was carried out on the blood samplescollected from vivax and falciparum patients and healthy subjects andcloned sequences were submitted to GenBank (Accession Nos.GQ141555 and GQ141556). Digestion of the PCR products withBsmAI restriction enzyme resulted in complete digestion of theproduct. The gel showed two fragments of 329 and 104 bp for thehomozygous wild (TT) genotype, two fragments of 222 and 104 bp forthe homozygous mutant (CC) genotype, and three fragments of 329,222, and 104 bp for the heterozygous mutant (TC) genotype.

Distribution of GSTP 1Ile105Val genotype in malarial patients

The genotype distributions for GSTP1 at position Ile105Val ofexon 5 in vivax- and falciparum-infected patients were compared

GSH-PV GSH-H GSH-PF0

2

4

6 P=0.0001

P=0.0001

HEALTHY SUBJECTP.VIVAX

P.FALCIPARUM

B

GS

H A

CT

IVIT

Y (

µmo

le/m

l.)

GST-PV GST-H GST-PF0

1

2

3

4

5

P=0.0001

P.VIVAX

P.FALCIPARUMP=0.0001HEALTHY SUBJECT

D

GS

T A

CT

IVIT

Y (

µmo

le/m

l.)

l of GSH in P. vivax and P. falciparum compared with healthy subjects. (C) Level of GPX inP. falciparum compared with healthy subjects.

Table 2Genotypic distribution of the GSTP1 Ile105Val polymorphism in P. vivax and P. falciparum patients compared to healthy subjects

Genotypesa GSTP1 –105 Ile/Ile Ile/Val Val/Val χ2 P OR (95% CI)

P. vivax (N=26) 8 (30.76%) 12 (46.15%) 6 (23.07%) 6.24 0.044 4.5 (1.33–13.56)Healthy subjects (N=26) 17 (65.38%) 6 (23.07%) 3 (11.53%) 1.09 0.578P. falciparum (N=26) 17 65.38%) 6 (23.07%) 3 (11.53%) 0.107 0.948 1.0 (0.319–3.135)

OR, odds ratio; CI, confidence interval.a Ile/Ile-Ile/Val vs Val/Val.

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with healthy subjects and are shown in Table 2. A total of 52patients, 26 each in the P. vivax and P. falciparum groups and 26healthy subjects, were genotyped for the GSTP1 Ile105Val mutation.We observed that the GSTP1 genotype was associated with anincreased risk of vivax infection (OR 4.5, 95% CI 1.33–13.56)compared to healthy subjects and decreased risk of falciparuminfection (OR 1.0, 95% CI 0.319–3.135) compared to healthy subjects(Table 2). However, genotype frequency data of vivax and falciparumpatients were analyzed and interestingly the difference was foundstatistically significant in the case of vivax infection (P=0.04),whereas it was found nonsignificant in falciparum infectioncompared to healthy subjects (Table 2).

Association of GSTP1 mutation with GST levels and clinical parameters inmalarial patients

The level of GST in the Ile105Val and Val105Val genotypes atposition 105 was significantly higher in vivax infection compared tothe ancestral genotype (Ile105Ile), as shown in Fig. 2A, whereas asignificant but marginally higher GST level was observed in falciparum

Fig. 2. (A) Comparison of GST level among P. vivax, P. falciparum, and healthy subjectsbased on the Ile105Val polymorphism in the GSTP1 gene. (B) Effect of the Ile105ValGSTP1 polymorphism on GST level in P. vivax compared with P. falciparum patients.

infection compared to the ancestral genotype, as shown in Fig. 2A. Inaddition, these genotypes were also correlated with clinical para-meters such as temperature, weight, and age in healthy subjects(Table 3). Interestingly, we observed significantly elevated GST levelduring vivax infection in all the GSTP1 genotypes compared tofalciparum infection (P=0.0001, P=0.0002, P=0.024) as shown inFig. 2B.

GSTP1 expression in the P. vivax- and P. falciparum-infectedblood isolates

Oxidative stress plays an important role in cell regulation, witheffects on gene regulation. GSTP1 activity is known to defend againstoxidative stress caused by parasitic and parasite-induced host factors[28]. To explore the physiological and pathological roles of GSTP1 inPlasmodium infection in humans, we investigated whether Plasmodi-um infection has any effect on the expression of GSTP1. Theexpression of GSTP1 RNA in blood isolated from patients was analyzedby RT-PCR, and the cDNA sequences were deposited with GenBank(Accession Nos. GQ141553 and GQ141554). Relatively high expres-sion of GSTP1 was observed in vivax infection compared to healthysubjects and P. falciparum infection. However, in the case of falciparuminfection an induction of GSTP1 was observed, compared to healthysubjects, but its expression was lower than that in vivax infection, asshown in Supplementary Figs. 1A and B. Furthermore, Western blotanalysis of the parasite-infected cell lysate revealed a 1.2-fold increasein GSTP1 protein during vivax infection compared to the levels in thehealthy subjects, whereas this increase was 0.7-fold compared tofalciparum infection, as shown in Figs. 3A and B.

Based on these results of semiquantitative RT-PCR and Westernblotting, we hypothesized that Plasmodium-specific infection mod-ulates the induction of GSTP1 and that the different genotypes ofGSTP1 are differentially expressed during malaria pathology. To testthese hypotheses, we analyzed expression changes in both Plasmo-dium-infected and healthy subjects. Real-time PCR assay showed theexpression levels of GSTP1 mRNA in P. vivax infection to be more than2.5- and 1.5-fold higher than those of healthy and P. falciparum-infected subjects, respectively (Fig. 4A). Indeed, the transcriptionalresponse analysis by qRT-PCR revealed the different quantitativeexpression of the different genotypes of the GSTP1 gene comparinghealthy subjects with Plasmodium-infected subjects (Fig. 4B). Inaddition, Plasmodium species-specific differences in GSTP1 expressionwere evident between P. vivax and P. falciparum, as significantlyhigher expression of GSTP1 was modulated during P. vivax infection(Fig. 4C). The above data validate the results of qualitative estimationand immunoblot, that polymorphism promotes the expression ofGSTP1 in response to Plasmodium infection. These results collectivelysuggest that the presence of a mutant genotype (Val/Val) enhancesGSTP1 expression in response to Plasmodium infection, resulting inincreased production of GST and other enzymes associated withmalarial pathology.

Discussion

In this study, we set out to systematically identify a common GSTP1Ile→Val genetic variation in two groups of patients infected with

Table 3Association of GSTP1 gene polymorphism with GST level in malarial patients compared to healthy subjects

Genotype Ile/Ile P a Ile/Val P a Val/Val P a

P. vivax N=8 N=12 N=6GST (U/L) 4.74±0.11 NS 5.76±0.32 0.007 6.25±0.45 0.031Age (years) 24.25±1.8 NS 28.33±3.01 NS 31.17±3.85 NSTemperature (°F) 101.3±0.63 0.0001 99.84±0.39 0.01 99.1±0.25 0.03Weight (kg) 55±1.45 NS 61.17±3.71 NS 53.67±3.39 NSHealthy subjects N=17 N=6 N=3GST (U/L) 4.32±0.16 4.44±0.3 4.53±0.43Age (years) 27.06±1.79 27±4.32 22.67±1.76Temperature (°F) 98.34±0.02 98.32±0.07 98.37±0.06Weight (kg) 58.41±2 55.33±1.82 58.33±1.2P. falciparum N=17 N=6 N=3GST (U/L) 2.22±0.04 0.0001 2.29±0.08 0.0001 2.57±0.27 0.025Age (years) 30.12±2.61 NS 24.33±2.36 NS 29±8.02 NSTemperature (°F) 99.75±0.35 0.0004 99.48±0.83 NS 99.83±1.1 NSWeight (kg) 57.82±2.43 NS 55.33±1.66 NS 54.67±2.6 NS

a Compared with healthy subjects. NS, not significant.

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vivax or falciparum and compared with a group of healthy subjects,followed by functional genomics studies designed to define thebiological significance of that variation. Specifically, we genotypedand sequenced GSTP1 exon 5, which contains the SNP at Ile105Val. Wealso performed functional genomics studies, including enzymeactivity assays, Western blots, and mRNA expression analysis.

There has been no published study examining the associationbetween vivax malaria and GSTP1 polymorphism. However, to thebest of our knowledge, our investigation is the first report an attemptto evaluate the association between GSTP1 polymorphism at position105 and GST levels in clinical vivax malaria and their association withdisease in an adult population residing in a high P. vivax prevalentregion (Delhi, North India).

In this study, we found evidence for an association of the GSTP1codon 105 polymorphism with vivax infection (P=0.044). Similarly,when we analyzed the falciparum patients no association withpolymorphism and the disease was observed (Table 2). Interestingly,we observed a significant presence of GSTP1 Ile105Val and Val105Val,

Fig. 3. (A) Immunoblot of partially purified GSTP1 from Plasmodium-infected isolates ofthe Val105Val genotype compared with healthy subjects. Lane 1, homogenate fromhealthy subjects; lane 2, homogenate from P. falciparum-infected sample; lane 3,homogenate from P. vivax-infected sample. (B) The induced GST activity in P.falciparum- and P. vivax-infected isolates was expressed as the mean fold increaserelative to the quantity of GSTP1 induced in the healthy subjects and normalized to thelevel of cellular actin, as assessed by densitometry. The data represent the means±SEof two independent experiments and the statistical significance was calculated usingStudent's t test (N=5 for each column).

with high frequency (46.5 and 23.07%, respectively), in vivax-infectedpatients compared to healthy subjects and falciparum-infectedpatients (23.07 and 11.53%, respectively). Thus, our frequencydistribution data suggest the existence of a mutation(s) within theGSTP1 gene that is more likely to influence the risk of vivax malaria inthe population, whereas in the case of falciparum infection a lowerfrequency distribution of mutated amino acid implies that it couldprovide protection from infection and disease progression. The reasonfor genetic disparities and varied degrees of association with diseasesusceptibly may be a highly polymorphic chromosomal location,biological effect, and extensive linkage disequilibrium to the HLAlocus; such differences are likely to exist [29]. In addition, the differentages, ethnicities, and geographical locations in the studied populationcan also contribute to genetic association and disease outcome [30].Apart from this, the mutation covering the amino acid segment frombp 99 to 121 influences the binding of GSTP1 to JNK, suggesting thatthe Ile105Val and Ala114Val substitutions may be crucial for JNKbinding and potentially other protein–protein interactions [31]. It isalso likely that the impact of GSTs is not direct on erythrocytes but onother cells that are involved in the immune responsemechanisms andthat severe malarial anemia as an outcome can partly be attributed tosuch responses. Holley et al. recently reported differential effects ofGSTP1 haplotypes on cell proliferation and apoptosis [32]. Thusaccumulating data suggest that GSTP1may be crucial in regulating theactivation of multiple stress kinase cascades, including the ASK1,MEKK1, mitogen-activated protein kinase, ERK, and IKK–NF-κBsignaling pathways. Among the substrates for these signaling cascadesare p53, NF-κB, c-Jun, ATF2, and c-Fos, which dictate protection from,or promotion of, cell death [33,34]. Our findings showed that amutation at position 105 of GSTP1 significantly correlates with theelevated GST level in vivax infection compared to healthy subjects anddoes not mediate clinical severity and symptoms but is stronglyassociated with susceptibility to disease (Table 2), which is wellreflected by the high odds ratio. However, in falciparum infectionmarginal elevation was observed compared to ancestral genotype,and significant depletion was not associated with disease suscepti-bility compared to vivax infection. Our observations regardingelevated GST level due to transition polymorphisms are consistentwith observations of high serum concentration of GST in patients withvarious other infectious diseases, including rodent malaria, falciparuminfection, and cancers [19,28,35–37], and suggest that GST poly-morphisms can change the enzyme activity, which can lead toreduced detoxification of the host cell or increased availability of hostGSH that might be used by the parasite. In both cases the malariapathology could be accelerated. However, our observations arepertinent and their interpretation can be attributed to several reasonsbecause GSTP1 is an enzyme for which genetic variation in the

Fig. 4. Fresh PBMCswere collected from healthy subjects and P. vivax- and P. falciparum-infected patients for RNA isolation and cDNA. (A) Differential expression of the GSTP1polymorphic genotype during Plasmodium infection; (B) mutation-specific expressionduring Plasmodium infection; and (C) species-specific modulation of GSTP1 expression.Fold induction was calculated using the ΔΔCT method of qRT-PCR, in which uninfectedsamples were compared to infected samples relative to β-actin levels. Data arepresented as means and error bars represent±SE from four isolates. Results arerepresentative of at least three independent experiments. *P≤0.05; **P≤0.0005compared with healthy subjects by one-way analysis using Tukey's multiplecomparison test through GraphPad Prism version 5.0.

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encoded amino acid sequence is responsible for alterations in its levelof enzyme activity as a result of at least two mechanisms—changes inthe active site, e.g., Val105Ile, and differences in levels of protein. Inaddition, noncytokine factors, e.g., nitric oxide intermediates, reactiveoxygen intermediates, antioxidants, and leukotrienes, have beenshown to influence GST level and subsequent inhibition of parasitegrowth [38]. An effect of physiological factors and perturbation in themetabolic cascade could also contribute to elevated GST, althoughthere is no strong biological rationale to provide support for such an

effect. Lipid peroxides have been implicated in a number of diseases,includingmalaria, and have been shown to be cytotoxic to various celltypes. They could kill cells in several ways. Because the parasites donot have a de novo triglyceride synthesis pathway [39], they mustobtain all their lipid requirements from the host serum. If oxidizedlipids are taken up by the parasite, they may have a direct cytotoxiceffect on the parasite, for example, in cross-linking proteins. However,they aremore likely to destabilize the cell membrane, in particular theerythrocyte membrane, which could then lead to parasite deathwithout necessarily destroying the erythrocyte. Our observation of asteep drop in lipid peroxide levels during falciparum infection duringoxidation of LDL is most likely due to a breakdown of lipid peroxidesinto a variety of secondary products of lipid peroxidation, such asaldehydes, some of which may react in turn with the apoproteinmoiety of LDL [40], as well as epoxides and other compounds [41].

SNPs did not seem to explain the full extent of variation inexpression, and their frequencies varied widely among ethnic groups,raising the possibility of variations in disease risk and differentresponses to drug therapy and subsequent influence on transcription[19], which was observed in our experimental data, with an increasein GSTP1 mRNA level as well as protein during vivax infectioncompared to healthy subjects (Supplementary Fig. 1A, Figs. 2A and B,respectively), indicating that signal cascade activation might occur toupregulate the expression of those genes particularly against vivaxinfection (Figs. 4A–C). Most importantly, our findings suggest thatoverall elevation of oxidative stress enzymes such as GSH, GPX, andGST during vivax infection (Figs. 1B, C, and D) in association withreactive oxygen intermediates enhances GSTP1 expression by themechanism mediated by an enhancer (GSTP1 enhancer I), located2.5 kb upstream of the transcriptional initiation site of the GSTP1 gene[42,43]. We show here . We show here an induction of GSTP1 mRNAand proteins, which suggests that the induction of these GST isoformsoccurred at the transcriptional and posttranslational levels. Thepresence of an antioxidant responsive element (ARE) in theirpromoters may at least partly explain the induction, as the ARE iscapable of mediating transcriptional induction after administration ofa variety of chemicals and parasitic products producing oxidativestress [44]. Despite the presence of this element, the fact that GSTP1expressionwas not distinctly correlatedwith GST activity in this studysuggests that other mechanisms are probably involved in itsregulation. It is worth noting that a discrepancy was found in otherisoforms, with an increase at the protein level suggesting aposttranscriptional rather than a transcriptional mechanism couldbe involved [45]. The differential regulation of GSTP activity might berelated to the expression of tissue-specific transacting factorscontrolling the expression of these enzymes. However, the contribu-tion of GST polymorphisms to malarial pathology may differ betweenpopulations or geographic areas. These findings do not undermine theimportance of oxidative stress in malaria clearance, but rather providea broader perspective on the impact of oxidative stress on both thehost and the parasite cells. Interestingly, we observe relatively lowermRNA level as well as protein during falciparum infection compared tovivax infection (Supplementary Fig. 1B), suggesting a differentregulatory mechanism in falciparum infection and host response.Based on RT-PCR and immunoblotting results it seems that GSTP1 ispredominantly expressed in vivax infection compared to falciparum.The significant correlations between GST enzyme and GSTP1 proteinactivities andmRNA levels in polymorphic isolates further support theidea that high antioxidant capacity in vivax infection probablypromotes enhanced GSTP1 expression, thus favoring its monomericconformation, and variation can be considered a developmentalmechanism of GST gene regulation, and involvement of other GSTclasses cannot be ruled out [46]. Furthermore, cellular redox statusmay also influence the transcription of GSTP1 [47]. Thus, we believethat high GST levels in mutant isolates modulate cysteine-rich redox-sensitive domains of GSTP1 and alter the transcription of GSTP1 [48].

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Apart from enzymatic functions, GSTs have been found to act asregulatory proteins serving a structural role (S-crystallins). GSTs areinvolved in sequestering and transport of exogenous hydrophobiccompounds, regulation of stress response, detoxification of lipidperoxidation products, and drug resistance. In human and rat, GSTP isnot expressed constitutively [49]. GSTP is a single gene, highlyconserved in evolution, the promoter region of which contains aCpG island, which is invariably expressed in cell lines, in culture, andin most somatic cells in vivo, indicating that this gene has importantphysiological functions.

GSTP1 expression and activity are redox dependent; we examinedthe relationship between GSTP1 expression at the protein and mRNAlevels, as well as the activities of antioxidant enzymes in malarialinfection. Differential expression and activity reflect differences in thebinding of electrophilic substrates to the hydrophobic binding site ofthe GTSP1, and the polymorphism at the GSTP1 locus has shown anassociation with malaria anemia [15].

Regardless of the exact functional relevance, based on existingevidence and our findings, we hypothesize that the relevant role ofthe transition polymorphism Val105Val in the GSTP1 gene isassociated with circulating levels of GST and increased risk ofdisease susceptibility or progression in vivax infection, whereas thismutation has no significant association with disease susceptibilityduring falciparum infection. Our results expand the previousobservations and are consistent with the an association, in otherstudies, between the GSTP1 polymorphism and other malarias[19,28,35–37].

In view of the present observation of GSTP1 polymorphism withGST level and other clinical parameters of vivax infection, we suggestthat evaluation of the GST level and its polymorphism in the exon 5region may be a reliable molecular and biochemical marker,possessing a promising rationale for potential diagnostic andchemotherapeutic interventions in clinical vivax malaria. Geneticvariation in exon 5 is of biological significance and may play a pivotalrole in host defense mechanisms against vivax pathology byenhancing antioxidant enzymes and stimulating the protectivebiochemical cascade against falciparum pathology.

Irrespective of whether therapies directed against GST will beuseful in the management of clinical malaria, the study of the effectsof selective activation and evaluation of GST during natural infectionwill provide unique and valuable insights into the understanding ofprotective immunity and pathobiology of clinical malaria, becauseour current understanding of these mechanisms is limited. As thesefindings are novel and from a small number of subjects, they deserveto be investigated in a large study group. In conclusion, these dataprovide a link between clinically acquired immunity, infection, andpolymorphism through a complex interplay of the oxidant–antioxidant cascade in a population exposed to vivax and falciparummalaria and could be beneficial in the development of recombinantGSTs as antimalarial drug targets. However, the reliability andclinical usefulness of GSTs and their polymorphisms as sensitivebiochemical, genetic, and diagnostic markers await further well-conducted clinical investigations so as to permit interventions tocontrol vivax infection.

In summary, in this study we have applied a comprehensive andsystematic genotype-to-phenotype research strategy to characterizecommon genetic variations in GSTP1, a gene that encodes a proteinthat plays diverse roles, from phase II drug metabolism to theregulation of apoptosis. Knowledge of common GSTP1 SNPs andhaplotypes, as well as the understanding of their functionalimplications, should contribute to both mechanistic and epidemi-ologic studies of the involvement of GSTP1 in malarial pathogenesisas well as individual variation in response to antimalarial drugtherapy.

Supplementary data to this article can be found online at doi:10.1016/j.freeradbiomed.2010.09.004.

Acknowledgments

We thank Professor M. Raziuddin, Vinoba Bhave University,Jharkhand, India, for critical comments on parts of the manuscriptand analytical help in experimental planning. M.S. acknowledges theIndian Council of Medical Research (ICMR), India, for a SeniorResearch Fellowship. We also acknowledge the ICMR for financialassistance for the work.

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