Glutathione S-transferase M1, T1, and P1 genotypes and rheumatoid arthritis

Glutathione S-transferase M1, T1, and P1 genotypes and rheumatoid arthritis
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  Glutathione  S -transferase M1, T1, and P1 genotypes and breastcancer risk: a study in a Portuguese population Ana Cristina Ramalhinho  • Jose´ Alberto Fonseca-Moutinho  • Luiza Breitenfeld Received: 13 February 2011/Accepted: 28 April 2011/Published online: 11 May 2011   Springer Science+Business Media, LLC. 2011 Abstract  Glutathione  S  -transferases are a superfamily of multifunctional enzymes that play a key role in Phase IImetabolism, detoxifying therapeutic drugs, and variouscarcinogens by conjugation with glutathione. We under-took a case–control study in Central-Eastern Portuguesepopulation to evaluate the association of null genotype inGSTM1 and GSTT1 along with the polymorphism inGSTP1 (A/G) and susceptibility to breast cancer. Thepopulation sample consisted of 85 patients with histologi-cal diagnosis of breast cancer and 102 healthy women.Genomic DNA was extracted from blood samples, andgenotyping analyses were performed by PCR-basedmethods. Odds ratios (ORs) and 95% confidence intervals(95% CIs) were calculated by unconditional logisticregression. We found a increased breast cancer risk asso-ciated with GSTM1 null genotype (OR  =  3.597; 95%CI  =  1.849–6.999;  P  =  0.0001) and GSTT1 (OR  =  2.592;95% CI  =  1.432–4.690;  P  =  0.002), but the presence of valine alleles compared to isoleucine alleles in codon 105in GSTP1 did not increase the risk of breast cancerdevelopment. The two-way combination of GSTM1 andGTTT1 null genotypes resulted in 8-fold increase for breastcancer risk (OR  =  8.287; 95% CI  =  3.124–21.980; P  =  0.0001) and the three-way combination of GSTP1105AA/AG and null genotypes for both GSTM1 andGSTT1 resulted in 5-fold increase for breast cancer risk (OR  =  5.040; 95% CI  =  1.392–18.248;  P  =  0.016). Ourresults suggest that GSTM1 and GSTT1 null genotypealone, both combined or combined with GSTP1 valinealleles, are associated with higher susceptibility to breastcancer development. Keywords  GSTM1    GSTT1    GSTP1   Polymorphisms    Breast cancer Introduction Breast cancer is the most common cause of cancer-relateddeaths among women in Western world [1]. Mutations inhigh penetrance genes account for only a small percentageof cases of familial and sporadic breast cancer, because thelow frequencies of risk alleles in the general population. Soit seems interesting to analyze the role of other geneticfactors such as low penetrance candidate genes that mayalter breast cancer predisposition. Low penetrance genescan participate on detoxification of environmental carcin-ogens, steroid hormone metabolism, and DNA damagerepair pathways [2]. Glutathione  S  -transferases (GSTs) area superfamily of ubiquitous, dimeric, and multifunctionalenzymes that play a key role in Phase II metabolism, whichgenerally detoxifies carcinogens, by conjugation withglutathione (GSH) [3]. A multiplicity of exogenous andendogenous compounds with electrophilic functionalgroups can be degraded by glutathione (GSH) conjugationby neutralizing their electrophilic sites, increasing theirwater solubility and excretability, and also protecting thecells from DNA damage and adducts formation [4]. Inestrogen metabolism, GSTs play a role in the catalysisof GSH conjugation of catechol estrogen quinones, the A. C. Ramalhinho ( & )    J. A. Fonseca-Moutinho   L. BreitenfeldCICS-UBI—Centro de Investigac¸a˜o em Cieˆncias da Sau´de,Universidade da Beira Interior, Av. Infante D. Henrique,6200-506 Covilha˜, Portugale-mail: cramalhinho@fcsaude.ubi.ptA. C. Ramalhinho    J. A. Fonseca-MoutinhoCentro Hospitalar Cova da Beira, E.P.E., Quinta do Alvito,6200-251 Covilha˜, Portugal  1 3 Mol Cell Biochem (2011) 355:265–271DOI 10.1007/s11010-011-0863-9  reactive intermediates of estrogen metabolism capable tobind to DNA [2]. In humans, GSTs can be differentiatedinto at least three major families of proteins: cytosolic orsoluble, mitochondrial and peroxisomal, and microsomal(currently named membrane-associated proteins involvedin eicosanoid and glutathione metabolism, MAPEG) GSTs[5]. Seven classes of cytosolic GSTs were created based onamino acid sequence similarities, physical structure of thegenes, and immunological crossreactivity, and are termedalpha ( a ), mu ( l ), kappa ( j ), pi ( p ), theta ( h ), omega ( X ),and zeta ( f ) [5–7]. The active site of a GST protein is formed by the glutathione-binding site, which is preservedin the different classes, and the hydrophobic substrate-binding site, that has variations in the forming residues,leading to wide substrate specificity [2]. GSTM1, GSTT1,and GSTP1 isoenzymes are present in both normal andbreast tumor tissues and belong to the best-characterizedclasses, along with class alpha and omega. GSTM1 iscodified by a gene located on chromosome 1p13.3 [8, 9]. The homozygous deletion (null genotype) of the GSTM1gene results in the total absence of a functional geneproduct, and then a total absence of the respective enzymeactivity [8]. Like GSTM1, GSTT1 gene, located at22q11.2, has a genetic variant that consists of a completedeletion of the whole gene, resulting in the lack of thecodified enzyme [10]. GSTP1 is a polymorphic genelocated on chromosome 11q13. One of the functionalgenetic variants identified is the point mutation at nucleo-tide 313 that results in a single amino acid change fromisoleucine (Ile) to valine (Val), at codon 105 [11]. As thisresidue lies in close proximity to the hydrophobic-bindingsite for electrophilic substrates, the variant allele seems tohave altered specific activity and affinity depending on thesubstrate [12, 13]. GSTM1 and GSTP1 isoenzymes cata- lyze the nucleophilic conjugation of glutathione to severalexogenous and endogenous chemicals molecules withelectrophilic functional groups, including polyaromaticcompounds, responsible for the formation of lipophilicpolyaromatic adducts that can be stored in breast tissue[14]. GSTT1 is better known by its role in the detoxifica-tion of smaller reactive hydrocarbons like activatedmetabolites of the heterocyclic amines formed by cookingmeat at high temperatures [15, 16]. Carcinogen metabo- lizing enzymes like GSTs are involved in the activation anddeactivation of diverse chemical carcinogens, includingxenobiotics and sex hormones. Individuals carrying a var-iant in one of these low penetrating genes are estimated tohave low risk to develop breast cancer when compared tocarriers of mutations in high penetrating genes like BRCA1and BRCA2, but the high frequency in the population of some of the variants makes the population to be interpretedas being of high risk. This study was designed to investi-gate the oncologic risk of genotypic variations of GSTM1,GSTT1, and GSTP1 low penetrance genes involved inxenobiotic metabolic pathways in breast cancer. Materials and methods Study populationCase subjects, 85 women with histologically confirmedbreast cancer, diagnosed at Child and Women HealthDepartment, Gynecologic Oncology Division, HospitalCentre of Cova da Beira, Covilha˜—Portugal, were enrolledbetween May 2008 and March 2009. 102 female controlswith no previous history of any type of cancer and withoutfamily histories of cancer, from the same geographicregion, were also studied. All subjects were Caucasian.Both cases and controls completed a questionnaire thatassessed parameters such as age, family history of breastcancer, parity, ethnicity, reproductive and menstrual his-tory, and history of breast diseases and procedures. Aninformed consent was obtained before entering in thestudy. The study was approved by the Institutional ReviewBoard of Hospital Centre Cova da Beira, Covilha˜—Portugal.DNA extraction and genotypingBlood was collected by venous puncture in EDTA-con-taining tubes, and genomic DNA was extracted usingWizard Genomic DNA Purification Kit (Promega, USA)according to the manufacturer’s protocol, and stored at4  C. Genotyping for GSMT1, GSTT1, and GSTP1 wasperformed using PCR-based methods slightly modifiedfrom procedures earlier described [17, 18]. For GSTM1 and GSTT1, the presence of the wild-type and/or the nullalleles was analyzed by multiplex PCR together with co-amplification of a fragment of beta-globulin gene as apositive control. Regarding to GSTP1, the variant allelescontaining a base substitution at the nucleotide 313,resulting in Ile105Val amino acid change, were differen-tiated from the wild-type allele (Ile/Ile) by BsmA1restriction enzyme digestion (New England Biolabs) sub-sequent to PCR amplification. In brief, each PCR reactionmixture was carried out in a total volume of 25  l l andcontained 10 pmol of each primer, 1.5 mM of MgCl 2 ,100 nM of each deoxynucleotide triphosphate, 1 unit of Taq DNA polymerase (Promega, USA), and 100 ng of genomic DNA, using MyCycler Thermal Cycler (Bio-Rad,Munich, Germany). Reaction mixtures were pre-incubatedfor 10 min at 95  C, and the amplified or digested DNA waselectrophoresed through agarose gels stained with ethidiumbromide. The genotyping assays used for GSTM1 andGSTT1 do not identify homozygous wild-type and 266 Mol Cell Biochem (2011) 355:265–271  1 3  heterozygous individuals but classifies as ‘‘present’’ indi-viduals with one or two copies of the relevant gene andindividuals with homozygous deletions as ‘‘null.’’ Theassay for GSTP1 classifies individuals as homozygousIle/Ile, homozygous Val/Val, or heterozygous Ile/Valdepending on the band pattern created by the digestion.Results were confirmed by re-genotyping 10% of randomlyselected samples, and all the results were in agreement withthe ones obtained previously.Statistical analysisLogistic regression method was used to access odds ratio(ORs) and 95% confidence intervals (95% CI) as estimatesof relative risk. The frequencies of the GSTP1 Val/Valgenotype were too low, thus the genotype data were divi-ded into two groups, GSTP1 Ile/Ile and GSTP1 Ile/ Val  ?  Val/Val to increase the statistical power. Jointeffects were estimated using GST putative low risk geno-types combination (GSTM1 present, GSTT1 present, andGSTP1 Ile/Ile) as referent group.  P  values less than 0.05were considered statistically significant. Calculations weredone using computer software SPSS for Windows (version16.0). Results The mean age was 55.5 years (SD, 20.3 years; range,17–84 years) for the controls and 62.8 years (SD,12.9 years; range, 34–85 years) for the cases. The associa-tion between presumed risk factors and the development of breast cancer is shown in Table 1. The majority of studyparticipants were postmenopausal at the time of blooddonation (64.5% controls and 72.9% cases). The risk of breast cancer was lower for women who had at least one fullterm pregnancy (OR  =  0.248; 95% CI  =  0.089–0.694) andin general, other factors like menopausal status, history of first degree family history ofbreastcancer, history of benignbreastdisease,orBMIhavenotincreasedordecreasedbreastcancer risk significantly. Genotypes distribution of GSTM1,GSTT1, and GSTP1 in cases and controls is summarized inTable 2.GSTM1deletion occurred in59.8%ofthecontrols.GSTM1 null genotype was significantly more commonamong breast cancer cases compared to controls (OR 2.592;95%CI1.432–4.690; P  =  0.002).GSTT1nullgenotypewasalsosignificantlymorefrequentincasesthanincontrols(OR3.597; 95% CI 1.849–6.999;  P  =  0.0001). We did not findany significant increase of breast cancer risk associated withGSTP1 genotypes (OR 1.103; 95% CI 0.611–1.989; P  =  0.765) when compared Val allele carriers in homozy-gosity or heterozygosity with Ile/Ile carriers. To furtherinvestigate if GSTs genotypes are related with breast cancerrisk, we analyzed their association in two and three-waycombinations to evaluate the impact of gene–gene interac-tion. Presence of GSTM1, GSTT1 alleles along with Ile/Ilegenotype in GSTP1 were considered as low risk genotypes,and null genotype in both GSTM1 and GSTT1 along withValalleleinhomozygosityorheterozygosityforGSTP1wasreferred as the presumed high-risk genotype combination.Table 3 exhibits the risk of breast cancer with the two-waycombinationofGSTM1andGSTT1.Morethanobservinganincreased risk for breast cancer associated with GSTM1 andGSTT1 null genotypes, we found a 8-fold increased breastcancer risk in women who carry null genotype both inGSTM1 and GSTT1 (OR 8.287; 95% CI 3.124–21.980; P  =  0.0001). It was not observed any association of breastcancerriskwithGSTP1polymorphismsincombinationwithGSTM1 (Table 4). In relation to GSTT1 combined withGSTP1, it was found a significant elevation of breast cancerrisk associated with GSTP1 Ile/Ile genotypes when associ-ated to GSTT1 null genotype (OR 3.627; 95% CI Table 1  Selected characteristics of the population studiedControls, n  (%)Cases, n  (%)OR (95% CI)Total 102 (100) 85 (100)Age at menarche B 12 years 51 (50.0) 58 (68.2) 1.013–14 years 35 (34.3) 19 (22.3) 2.095 (1.068–4.108) C 15 years 16 (15.7) 8 (9.4) 2.275 (0.899–5.755)Age at first full term pregnancy B 25 years 56 (54.9) 39 (45.9) 1.026–30 years 12 (11.8) 18 (21.2) 0.464 (0.201–1.072) C 31 years 13 (12.7) 16 (18.8) 0.566 (0.245–1.308)Number of full-term pregnanciesNulliparous 21 (20.6) 12 (14.1) 1.01 10 (9.8) 23 (27.1) 0.248 (0.089–0.694)2 36 (35.3) 27 (31.8) 0.762 (0.320–1.814)3 ?  35 (34.3) 23 (27.1) 0.870 (0.360–2.103)Menopausal statusPremenopausal 36 (35.3) 23 (27.01) 1.0Postmenopausal 66 (64.7) 62 (72.9) 0.680 (0.363–1.274)First degree family history of breast cancerNo 91 (89.2) 70 (82.3) 1.0Yes 11 (10.8) 15 (17.6) 0.564 (0.244–1.304)History of benign breast diseaseNo 55 (53.9) 37 (43.5) 1.0Yes 47 (46.1) 48 (56.5) 0.659 (0.369–1.176)BMI B 25 56 (54.9) 36 (42.3) 1.0 [ 25 46 (45.1) 49 (57.6) 0.603 (0.338–1.079)Mol Cell Biochem (2011) 355:265–271 267  1 3  1.448–9.084;  P  =  0.008) and less with Ile/Val  ?  Val/Valgenotypes (OR 3.455; 95% CI 1.273–9.372;  P  =  0.016), asshown in Table 5. After analyzing the three-way combina-tion of GSTM1, GSTT1, and GSTP1 polymorphisms(Table 6), we found that Ile/Ile genotype seems to bestrongly associated with risk of breast cancer when com-bined with both GSTM1 and GSTT1 null genotypes (OR12.600; 95% CI 2.358–67.315;  P  =  0.001). Also, thecombination of GSTM1 and GSTT1 null genotypes withpresence of the Val allele in GSTP1 was associated with a5-foldincreaseinbreastcancerriskwhencomparedwiththecombinationofGSTM1present,GSTT1present,GSTP1Ile/ Ile (OR 5.040; 95% CI 1.392–18.248;  P  =  0.016), so itappears that the increase of breast risk is mainly given byGSTM1 together with GSTT1 deletion, rather than by anygenotype of GSTP1. Table 2  Association betweenGSTs genotypes and breastcancer a ORs  odds ratio;  CI   confidenceintervalControls,  n  (%) Cases,  n  (%) OR (95% CI) a P  value GSTM1 Present 61 (59.8) 31 (36.5) 1.0Null 41 (40.2) 54 (63.5) 2.592 (1.432–4.690) 0.002 GSTT1 Present 84 (82.4) 48 (56.5) 1.0Null 18 (17.6) 37 (43.5) 3.597 (1.849–6.999) 0.0001 GSTP1 Ile/Ile 48 (47.1) 39 (45.9) 1.0Ile/Val  ?  Val/Val 54 (52.9) 46 (54.1) 1.103 (0.611–1.989) 0.765 Table 3  Association between GSTM1 and GSTT1 genotype combinations and breast cancer GSTM1 GSTT1  Controls,  n  (%) Cases,  n  (%) OR (95% CI) a P  value ? ?  49 (48.0) 17 (20.0) 1.0 ? -  12 (11.8) 14 (16.5) 3.363 (1.303–8.679) 0.014 - ?  33 (32.3) 31 (36.5) 2.708 (1.294–5.664) 0.011 - -  8 (7.8) 23 (27.0) 8.287 (3.124–21.980) 0.0001 a ORs  odds ratio;  CI   confidence interval Table 4  Association between GSTM1 and GSTP1 genotype combinations and breast cancer GSTM1 GSTP1  Controls,  n  (%) Cases,  n  (%) OR (95% CI) a P  value ?  Ile/Ile 29 (28.4) 19 (22.3) 1.0 ?  Ile/Val  ?  Val/Val 32 (31.4) 12 (14.1) 0.572 (0.237–1.380) 0.271 -  Ile/Ile 19 (18.6) 24 (28.2) 1.928 (0.837–4.443) 0.144 -  Ile/Val  ?  Val/Val 22 (21.6) 30 (35.3) 2.081 (0.937–4.624) 0.076 a ORs  odds ratio;  CI   confidence interval Table 5  Association between GSTT1 and GSTP1 genotype combinations and breast cancer GSTT1 GSTP1  Controls,  n  (%) Cases,  n  (%) OR (95% CI) a P  value ?  Ile/Ile 38 (37.2) 22 (25.9) 1.0 ?  Ile/Val  ?  Val/Val 46 (45.1) 26 (30.6) 0.976 (0.479–1.990) 1.000 -  Ile/Ile 10 (9.8) 21 (24.7) 3.627 (1.448–9.084) 0.008 -  Ile/Val  ?  Val/Val 8 (7.8) 16 (18.8) 3.455 (1.273–9.372) 0.016 a ORs  odds ratio;  CI   confidence interval268 Mol Cell Biochem (2011) 355:265–271  1 3  Discussion We examined the association of three common GSTM1,GSTT1, and GSTP1 polymorphisms, both alone and incombination, with breast cancer risk in a population-based, case–control study of Caucasian women in aregion of Portugal. Genotypes distribution in controlsubjects was in agreement with previous reports forCaucasian populations. GSTM1 deletion in 59.8% of thecontrols agrees with a meta-analysis of 30 studies [19]involving over 10,000 individuals, which identified theGSTM1 null genotype in 53% Caucasians (with a rangeof 42–62%). About 20% of Caucasians are homozygousfor a GSTT null allele [20], and the occurrence of thedeletion in our control group was somewhat lower,17.6%. Concerning GSTP1, the frequency of GSTP1 Valallele containing genotypes in our population study(52.9%) was similar to that reported for Caucasians(45–60%) [21]. It was not found any significant breastcancer risk associated with GSTs genotypes and meno-pausal status, parity, or BMI, however, we observed anincreased risk for breast cancer related with GSTM1 andGSTT1 null genotype, both alone or in a two-way com-bination, confirming previous reported data [22–25]. In the used assay, the absence of a PCR product indicatesthe GSTM1 or GSTT1 null/null genotype, and studyparticipants were categorized as either ‘‘present’’ (wild-type) or ‘‘null’’ genotypes. This analytical approach doesnot positively identify the null allele and, therefore, can-not distinguish homozygous wild type from heterozygouspresent/null individuals, but it conclusively identifies thenull/null genotypes. GSTM1 and GSTT1 null genotypeshave been the focus of several studies because of theirspecial condition: the majority of polymorphisms affect-ing genes involved in carcinogen metabolism are singlenucleotide polymorphisms, deletions are less common,and the complete absence of a gene in form of a nullallele is rare. GSTs are phase II detoxifying enzymes, andthe polymorphisms in the codifying genes may beassociated with the increased susceptibility to breastcancer, as normal or increased GSTs activity may facili-tate detoxification of electrophilic carcinogens, protectingsusceptible tissues from somatic DNA mutations [20, 22]. Thus, population with reduced GST enzyme activity, asresult of homozygous deletions of GSTM1 or GSTT1,may be at greater risk for developing cancers due to theirimpaired ability to metabolically eliminate carcinogeniccompounds and reduced detoxification efficiency. Wefound no significant elevation of breast cancer risk asso-ciated with GSTP1 genotypes alone but significantlyhigher breast cancer risk when all genotypes were ana-lyzed together. Women who carry null genotype both inGSTM1 and GSTT1 and wild-type genotype in GSTP1have shown 12-fold increased risk for breast cancer. Thismeans that Ile/Ile genotype seems to increase risk forbreast cancer only when associated to GSTT1 nullgenotype or both GSTM1 and GSTT1 null genotypes, andthat Val allele tends to act as a protective factor ratherthan as a risk factor, in agreement with the findings of other research groups [22, 24]. It has also been reported that GSTP1 Ile allele acted in the opposite way, pro-tecting against the risk of breast cancer, and Val allelewas associated with the risk of developing the disease[23, 26]. Another study that comprised 500 breast cancer cases and 395 controls did not found association of breastcancer risk with any combination of GSTM1, GSTT1, andGSTP1 genotypes [21]. The inconsistent results betweenstudies might be due to the fact that the enzyme encodedby GSTP1 Val allele exhibits different activity, affinity,and thermostability according to substrates [26]. GSTP1variants encode the change at position 105 from Ile toVal. It was hypothesised that this amino acid substitutionmay limit free access of the substrate to the H-siterequired for conjugating with glutathione as well as itsthermostability [27, 28]; however, the exact biological mechanism and effect of the polymorphism is not com-pletely understood [26, 29]. Furthermore, GSTs are known to have overlapping substrate specificities, and the Table 6  Association between GSTM1, GSTT1, and GSTP1 genotype combinations and breast cancer GSTM1 GSTT1 GSTP1  Controls,  n  (%) Cases,  n  (%) OR (95% CI) a P  value ? ?  Ile/Ile 21 (20.5) 10 (11.8) 1.0 - ?  Ile/Ile 17 (16.6) 12 (14.1) 1.482 (0.516–4.258) 0.593 ? -  Ile/Ile 8 (7.8) 9 (10.6) 2.362 (0.702–7.955) 0.221 ? ?  Ile/Val and Val/Val 29 (28.4) 8 (9.4) 0.579 (0.196–1.716) 0.411 - -  Ile/Ile 2 (2.0) 12 (14.1) 12.600 (2.358–67.315) 0.001 - ?  Ile/Val and Val/Val 17 (16.7) 18 (21.1) 2.224 (0.815–6.064) 0.140 ? -  Ile/Val and Val/Val 3 (2.9) 4 (4.7) 2.800 (0.524–14.959) 0.387 - -  Ile/Val and Val/Val 5 (4.9) 12 (14.1) 5.040 (1.392–18.248) 0.016 a ORs  odds ratio;  CI   confidence intervalMol Cell Biochem (2011) 355:265–271 269  1 3
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