e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 3 ( 2 0 1 2 ) 226–232

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jo ur nal homep age: www.elsev ier .com/ locate /e tap

The relationship among IL-13, GSTP1, and CYP1A1polymorphisms and environmental tobacco smoke in apopulation of children with asthma in Northern Mexico

Balam Munoza,∗, Jonathan J. Maganab, Israel Romero-Toledoa, Evelyn Juárez-Péreza,Andrea López-Moyaa, Norberto Leyva-Garcíab, Celsa López-Camposc, Víctor M.Dávila-Borjad, Arnulfo Alboresa

a Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV del IPN),México, DF 07360, Mexicob Departamento de Genética, Instituto Nacional de Rehabilitación, México, DF 14389, Mexicoc Departamento de Pediatría UMAE 71, IMSS, Torreón, Coahuila, Mexicod Departamento de Toxicología Genética, Instituto Nacional de Pediatría, México, DF 04530, Mexico

a r t i c l e i n f o

Article history:

Received 19 April 2011

Received in revised form

30 November 2011

Accepted 4 December 2011

Available online 10 December 2011

Keywords:

Asthma

Environmental Tobacco smoke

a b s t r a c t

Exposure to environmental tobacco smoke (ETS) during early childhood increases the risk

of developing asthma. The intention of this study was to genotype a population of children

from Coahuila state in Northern Mexico and to determine whether polymorphisms of the

CYP1A1, GSTP1, and IL13 genes are associated with exposure to ETS and subsequently a

higher risk for asthma. IL13 plays an important role in the development of allergic response,

particularly those related with airway inflammation. CYP1A1 and GSTP1 are xenobiotic-

metabolizing enzymes induced by repeated exposure to toxicants. Polymorphisms of these

genes have been related with ETS exposure and increased risk for asthma. To assess the

effect of IL13 (−1112 C > T and Arg110Gln), GSTP1 (Ile105Val), and CYP1A1 (Ile462Val) on

asthma risk and ETS exposure, we recruited 201 unrelated children and classified them into

Polymorphism

GSTP1

IL-13

CYP1A1

four groups: (1) control without ETS exposure; (2) control with ETS exposure; (3) with asthma

and with ETS exposure and (4) with asthma and without ETS exposure. No association

among ETS exposure, asthma, and the studied polymorphisms was denoted by multivariate

analysis of this population.

The contribution of ETS exposure to wide range of respira-

1. Introduction

Asthma is a multifactorial and polygenic disease charac-terized by Bronchial hyper-responsiveness (BHR) and atopy,

affecting more than 300 million people worldwide (Oh et al.,2010). Bronchial asthma is a common disorder that affects12% of the Mexican population and it is likely to increase

∗ Corresponding author at: Departamento de Toxicología, Centro de InveNacional No. 2508, Col. San Pedro Zacatenco, 07360 México, D.F., Mexic

E-mail address: rbmunoz@cinvestav.mx (B. Munoz).1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.etap.2011.12.007

© 2011 Elsevier B.V. All rights reserved.

over the next 10 years (Vargas Becerra, 2009). Air pollution,common allergens, and exposure to environmental tobaccosmoke (ETS) are a few of the major environmental factorsthat influence asthma development (Jindal and Gupta, 2004).

stigación y de Estudios Avanzados del IPN, Av. Instituto Politécnicoo. Tel.: +52 55 5061 5476; fax: +52 55 5747 7111.

tory diseases in children have been extensively reported, themost prevalent being impaired lung function and aggravationof asthma in childhood (Gergen, 2001). However, asthma and

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sthma-related traits possess an evident hereditary com-onent. Some studies have related bronchial asthma witholymorphisms localized in or adjacent to different geneequences (Vercelli, 2008). The relationship between genesnd the environment is the key to understanding how eachactor contributes to disease susceptibility.

IL-13 plays a crucial role in the development of aller-ic asthma by several mechanisms, including inductionf IgE antibodies, airway eosinophilia, and hyper-eactivity (Hunninghake et al., 2007). Furthermore, someenetic polymorphisms and expression levels of IL13re highly associated with certain respiratory illnessesuch as pulmonary fibrosis and Chronic obstructive pul-onary disease (COPD) (Beghe et al., 2009). The role

f IL13 in the pathogenesis of asthma has been exten-ively documented and several studies demonstrate anssociation among polymorphisms of this gene and preva-ence of the disease (Arima et al., 2002; Black et al., 2009;adeghnejad et al., 2008). Recently, the association betweenwo Single nucleotide polymorphisms (SNP) at the IL13 locusrs1800925 [−1112 C/T] and rs20541 [R130Q]), has been linked

ith the risk for asthma or atopy in both children and adultsBeghe et al., 2009; Sadeghnejad et al., 2008).

In addition, the metabolism of some toxic ETS com-ounds are produced via a complex network of enzymes andydrolysis, reduction, or oxidation reactions (phase I) and con-

ugation reactions (phase II). Among these, cytochrome P450A1 (CYP1A1) metabolizes polycyclic aromatic hydrocarbons,ommon components of ETS, while Glutathione-S-transferaseGST) conjugates molecules with glutathione and facilitatesxcretion (Nerurkar et al., 2000). Both genes are highly poly-orphic. Some reports have demonstrated that the variant,

YP1A1, increases its expression in a murine model of inducedsthma (Haag et al., 2002). Meanwhile, the GST pi 1 geneGSTP1) is a polymorphic gene that encodes active, func-ionally different, GSTP1 variant proteins, which are thoughto function in xenobiotic metabolism and to play a role inusceptibility to cancer, asthma, and other diseases (Mappt al., 2002). The GSTP1 variant (rs1695 [Ile105Val]) may protectgainst the increased risk of asthma associated with ozonexposure (Islam et al., 2009) and has also been associated withsthma in an Asian population (Hanene et al., 2007). Poly-orphisms in CYP1A1 and GSTP1 are therefore likely to be

ssociated with ETS exposure and asthma.In the present study, we analyzed the association between

wo polymorphic markers located in the IL13 gene and two SNPn the CYP1A1 and GSTP1 genes with risk of asthma develop-

ent through ETS exposure in a sample of Mexican children.e address possible interactions between passive ETS expo-

ure and IL13 and xenobiotic-metabolizing enzymes.

. Materials and methods

.1. Study subjects

he study population was composed of 201 unrelated nativeexican children (101 males and 100 females aged between

and 9 years) from Torreón City, Coahuila, who providednformed consent to participate in this study (all subjects

m a c o l o g y 3 3 ( 2 0 1 2 ) 226–232 227

and their parents) and whose families have lived in Mexicofor at least three generations. The children were dividedinto two groups; the first group was composed of 90 chil-dren with asthma, and the second group, of 111 childrenwithout asthma, all recruited by the Instituto Mexicano delSeguro Social (IMSS) of Torreón City, Coahuila. Parents of allchildren were interviewed by trained personnel to record infor-mation concerning the International Study of Asthma andAllergies in Children (ISAAC) questionnaire on respiratory andallergic symptoms (ISAAC, 1993),and provided information onexposure to tobacco smoke, asthma and allergy antecedents,interactions with pets, and household conditions. Each groupwas analyzed for ETS exposure (see Section 2.2). This studywas approved by the Faculty of Medicine Ethics Committeesat the University of Coahuila and at CINVESTAV-IPN.

2.2. Exposure to tobacco smoke

All children were analyzed for ETS exposure through detec-tion of cotinine in urine. Information on tobacco smoking bymothers (during pregnancy and after), by fathers, or by anyother family member inside the home was recorded at eachfollow-up interview. ETS in the household and maternal smok-ing during pregnancy were combined and classified into threegroups: ETS-0 (mothers did not smoke during pregnancy andthere was no exposure to household ETS); ETS-1 (householdmembers smoked within the home at some point up to thechild’s age of 10 years), and ETS-2 (mothers smoked duringpregnancy and the children were also exposed to householdETS at some point up to the age of 10 years) (Sadeghnejadet al., 2008). Studies dealing with decomposition products ofnicotine (such as cotinine and nicotine in urine) as indicatorsfor ETS exposure are readily available (Benowitz, 1996) andtheir validity has been confirmed (Matt et al., 2007). To deter-mine cotinine levels in urine, a DRG Cotinine Urine ELISA kit(Catalog # EIA-1377) (DRG International, Inc., Mountainside, NJ,USA) was used according to the manufacturer’s instructions.Briefly, 10 �L of sample, calibrator, and control was added ineach of the plate’s wells. Then, 100 �L of cotinine enzyme wasadded and incubated for 30 min at room temperature. Theplate was washed four times and 100 �L of substrate solutionwas added to each well. After 30 min at room temperature,100 �L of stop solution was added. Absorbance was measuredat 450 nm employing a microplate reader (Infinite M200, Tecan,Grödig, Austria). Cotinine concentrations were corrected withcreatine concentrations according to the method reported byFried et al. (Fried et al., 1995).

2.3. Genotyping

Genomic DNA was extracted from peripheral blood leukocytesutilizing the FlexiGene DNA extraction kit (QIAGEN, Hilden,Germany), according to the manufacturer’s instructions. ForSNP genotyping of IL13 (−1112 C > T [rs1800925], Arg110Gln(110G > A) [rs20541]), and GSTP1 (Ile105Val (562A > G) [rs1695]),the 5′ nuclease assay was performed using minor groove

binder probes fluorescently labeled with FAM or VIC (Taq-man SNP genotyping assays and SNP Genotyping Master Mix)and the protocol recommended by the supplier was followed(Applied Biosystems, Foster City, CA, USA). Reactions were run

228 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 3 ( 2 0 1 2 ) 226–232

Table 1 – General characteristics of the study population.

Exposure to tobacco smoke Without exposure to tobacco smoke

Children with asthma Control children Children with asthma Control children

Subjects 43 51 47 60Gender

Male 29 23 25 24Female 14 28 22 36

Age (years)a 7.18 ± 0.87 7.03 ± 0.86 7.31 ± 1.05 7.50 ± 0.78Range (years) (6–9) (6–9) (5–9) (6–9)

Exposure to maternal smoking in uteroETS-0 23 28 41 51ETS-1 18 22 6 8ETS-2 2 1 0 1

Familial asthmaNo 18 29 20 38Yes 25 22 27 22

Familial allergyNo 16 22 17 29Yes 27 29 30 31

Household typeNo traffic on street 15 22 10 20Continuous traffic 17 18 25 25Industrial area 11 11 12 15

Household petNo 16 18 22 22Yes 27 33 25 38

a Expressed as mean ± standard deviation (SD). ETS = Environmental tobacco smoke.[ETS-0 (mothers did not smoke during pregnancy and there was no exposure to household ETS); ETS-1 (household members smoked within

2 (mo

the home at some point up to the child’s age of 10 years), and ETS-to household ETS at some point up to the child’s age of 10) years].

in 96-well plates and the 10-�L PCR reaction mixture included20 ng of genomic DNA, 100 nM of each probe, 900 nM of eachprimer, and 1X TaqMan PCR Master Mix (Applied Biosystems).PCR cycling conditions consisted of a 2-min preincubationperiod at 50 ◦C, an initial denaturation period of 10 min at95 ◦C, a 15-s annealing step at 62 ◦C, and a 30-s denaturationstep at 95 ◦C for 47 cycles. The ABI Prism® 7700 Real Time PCRSystem (Applied Biosystems) was employed for data acquisi-tion.

For the CYP1A1 (Ile462Val [rs1048943]) polymorphism, weused the method reported by Hayashi et al. (Hayashi et al.,1991) with some modifications. The PCR reaction was per-formed in a total volume of 25 �L containing 2 �g/mL ofeach primer (1A1A; 5′-GAAGTGTATCGGTGAGACCA-3′, 1A1Gor 5′-GAAGTGTATCGGTGAGACCG-3′), together with (C53; 5′-GTAGACAGAGTCTAGGCCTCA-3′), 50 ng of human genomicDNA and 15 �L of RED-Taq Ready Mix (Sigma–Aldrich, St. Louis,MO, USA). The thermocycling procedure consisted of 30 cyclesand each cycle was divided into the following three steps:denaturation for 60 s at 95 ◦C; annealing for 60 s at 65 ◦C, andextension for 60 s at 72 ◦C. The 2720 thermal cycler (AppliedBiosystems) was employed for this amplification. The PCRproduct was electrophoresed on 2% agarose gel to analyze thereaction.

2.4. Statistical analysis

All results are expressed as the mean ± Standard error(SE). Differences in allelic and genotype distributions wereassessed by chi-square tests (�2). Simple and multiple logistic

thers smoked during pregnancy and the children were also exposed

regression analyses were carried out to determine an associ-ation among asthma, ETS, and the analyzed polymorphisms.The �2 test and the multiple regression models were adjustedby confounders employing the STATA 8.0 software package(Stata Corporation, College Station, TX, USA). The level ofstatistical significance was set at p < 0.05. Analysis of the devi-ation of frequencies from the Hardy-Weinberg expectation(HWE) was performed with GENÈTIX software (University ofMontpellier, Montpellier, France).

3. Results

3.1. Characteristics of the studied population

To genotype the Mexican child population under study, weclassified, into the group with asthma, 90 children withasthma defined to by ISAAC questionnaire parameters andinto the control group, 111 healthy children. In summary, ofthe participants, 50.3% were males and 49.7% were femaleswith a mean age of 7.2 years.

Children with and without asthma were subdivided intotwo groups according to their exposure to ETS (Table 1). Ofthe total population, 53.3% was classified as having receivedno exposure to tobacco smoke and 46.7%, as having receivedexposure. Table 1 depicts detailed data of the study population.

We confirmed ETS exposure by measuring the level of coti-nine in the urine of all children in the four groups (Fig. 1).Fig. 1A denotes that in ETS-exposed children, cotinine levelsshowed a significant increase when compared with those of

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 3 ( 2 0 1 2 ) 226–232 229

Table 2 – Allelic and genotypic distribution of all investigated polymorphisms.

Exposure to tobacco smoke Without exposure to tobacco smoke p

Children withasthma (n = 43)

Control children (n= 51) Children withasthma (n = 47)

Control children (n = 60)

IL13 (−1112 C/T)C allele 65.6 72.82 70.58 72.03 0.84T allele 34.4 27.18 29.42 27.97 0.73CC genotype 44.44 52.17 52.94 50.85 0.55CT genotype 42.22 41.3 35.29 42.37 0.62TT genotype 13.33 6.52 11.76 6.78 0.73

IL13 (R130Q)A allele 42.22 52.17 65.22 47.46 0.07G allele 57.78 47.83 34.75 52.54 0.06AA genotype 11.11 26.09 31.37 15.25 0.001AG genotype 62.22 52.17 54.9 64.41 0.26GG genotype 26.67 21.74 13.73 20.34 0.3

GSTP1 (Ile105Val)A allele 48.88 47.82 51 40.67 0.59G allele 51.12 52.18 49 59.33 0.64AA genotype 20 26.09 21.57 16.95 0.39AG genotype 57.78 43.48 56.86 47.46 0.81GG genotype 22.22 30.43 19.61 35.59 0.66

CYP1A1 (Ile462Val)A allele 21.13 26.80 23.71 28.35 0.94G allele 24.39 25.61 21.34 28.66 0.68AA genotype 15.91 25.00 27.27 31.82 0.59AG genotype 22.67 27.33 22.67 27.33 0.84GG genotype 42.86 7.14 7.14 42.86 0.77

chi-sq

nsShEc

3

Tf

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Differences in allele and genotype distribution were assessed by the

on-exposed groups. Fig. 1B presents the cotinine levels mea-ured from the parents of the children in the four groups.imilarly, parents who smoked or who were exposed to ETSad higher cotinine levels than non-smoking parents. Clearly,TS exposure causes increased cotinine levels in ETS-exposedhildren and their parents.

.2. Genotyping analyses

able 2 illustrates the allelic and genotypic frequencies of theour SNP studied. A chi-square analysis was performed to

Children

Cot

inin

e (µ

g/m

L)

0

10

20

30

40

50

60

70

No ETSNo asthma

ETSNo asthma

No ETSasthma

ETSasthma

*

Cot

inin

e (µ

g/m

L)

A *

ig. 1 – Urine cotinine levels in children and their parents. UrineB) adults. Values were corrected with creatine concentrations. Mxperiments performed in triplicate are shown. *P < 0.05 Student

uared test (�2). Significant values (p = 0.05) are indicated in bold.

determine differences among groups. From IL13 (−1112 C/T),the C allele and CC genotype were the most common amongall groups, with no statistically significant difference amonggroups. From IL13 (R130Q), frequencies of the A allele andgenotype AA were higher in children without asthma withETS exposure than in children with asthma with ETS exposure(Table 2); however, multivariate analysis displayed no statisti-

cally significant differences. Finally, for GSTP1 (Ile105Val) andCYP1A (Ile462Val) polymorphisms, allelic and genotypic fre-quencies were similar among all groups and no significantstatistical differences were observed.

0

2,000

2,500

3,000

No ETSNo asthma

ETSNo asthma

No ETSasthma

ETSasthma

ParentsB

cotinine analysis performed by ELISA in (A) children andean values and standard error (±SE) from two independent

t test. ETS, Environmental tobacco smoke.

230 e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Table 3 – Polymorphisms and their relationship withbronchial asthma.

Locus OR 95% CI P value

IL13 (−1112 C/T)C allele 0.59 0.13–2.60 0.49T allele 1.34 0.57–3.12 0.5CC genotype 0.83 0.36–1.91 0.67CT genotype 1.02 0.44–2.35 0.97TT genotype 1.69 0.38–7.47 0.49

IL13 (R130Q)A allele 1.37 0.52–3.55 0.52G allele 0.47 0.13–1.69 0.25AA genotype 2.11 0.59–7.60 0.25AG genotype 0.91 0.39–2.09 0.83GG genotype 0.73 0.28–1.90 0.52

GSTP1 (Ile105Val)A allele 1.63 0.65–4.07 0.31G allele 0.92 0.33–2.56 0.87AA genotype 1.09 0.39–3.04 0.87AG genotype 1.46 0.61–3.50 0.39GG genotype 0.61 0.25–1.53 0.29

CYP1A1 (Ile462Val)A allele 0.81 0.15–4.35 0.79G allele 1.71 0.57–5.17 0.34AA genotype 0.58 0.19–1.75 0.34AG genotype 1.4 0.54–3.67 0.49GG genotype 1.25 0.23–0.79 0.79

For the Odds ratio (OR), the models were adjusted by age, gender,Environmental tobacco smoke (ETS), familial asthma antecedents,familial allergy antecedents, type of household, and presence of

study suggests the potential role of CYP1A1 and other xenobi-

household pets.95% CI = 95% confidence interval; significant values (p <0.05).

There were no significant deviations from theHardy–Weinberg equilibrium (HWE) for any of the poly-morphisms.

3.3. Polymorphic association with asthma

Table 3 depicts the determination of risk for bronchial asthmain the study population of Mexican children, taking intoaccount some asthma confounders, including gender, age,level of maternal exposure to smoke in utero, household con-dition, pet contact, and familial history for allergy or asthma.Under this analysis, we found no overall association betweenasthma and the polymorphisms analyzed.

4. Discussion

In this study, we analyzed a population of Mexican childrenwith Environmental tobacco smoke (ETS) exposure for thepresence of an association between bronchial asthma andfour single nucleotide polymorphic markers located on theIL13, GSTP1, and CYP1A1 genes. ETS is a mixture of gases andparticles from the lighted cigarette and exhaled mainstreamsmoke and contains >1014 oxidative molecules per puff ofsmoke, including both nitric oxide and superoxide. The impor-tance of ETS in the etiology of asthma in children has been

established. Experimental studies on the acute effect of ETSin adult asthma supports its contribution to exacerbation ofasthma (Comhair et al., 2011). Because children are commonly

a r m a c o l o g y 3 3 ( 2 0 1 2 ) 226–232

exposed to cigarette smoke at home and in public places, thestudy of an interaction between ETS exposure and polymor-phisms is important. Furthermore, the polymorphisms of keygenes such as cytokines, adhesion molecules, and receptorsappear to be related with disease severity. Several cytokinesare highly related with asthma development; in particular,IL13 plays an important role in the allergic response and inexpression level increases in persons with asthma (Arimaet al., 2002). IL13 polymorphisms (particularly R130Q) havebeen associated with increased IgE levels, possibly influenc-ing atopy (Beghe et al., 2009). In Costa Rican children, IL13polymorphisms have also been associated with increased lev-els of IgE and eosinophils (Hunninghake et al., 2007). Recently,two IL13 polymorphisms, namely −1112 C > T and R130Q, weresignificantly associated with rhinitis, IgE serum levels, andasthma in a Dutch population (Bottema et al., 2011). Occu-pational asthma induced by di-isocyanates was associatedwith the R130Q polymorphism in Canadian workers (Bernsteinet al., 2011). Moreover, according to several studies, IL13 vari-ants are highly associated with asthma and tobacco smokeexposure (Colilla et al., 2003; Meyers et al., 2005). The A alleleof IL13 R130Q was significant associated with increased risk ofasthma in a Chinese child population (Wu et al., 2010). How-ever, in our study, neither the R130Q polymorphism nor the−1112 C/T variant was associated with asthma or ETS expo-sure. The reason for these discrepancies is not clear; however,it is likely that different ethnic or environmental conditionsplay a role, as well as on the design of each study.

The strengths of this study include genotyping of anasthma-related gene and the use of an internal biologicalmarker, urinary cotinine, to assess individual ETS exposurelevels. However, our study was smaller in scale comparedwith previous reports; thus, to obtain conclusive results on theassociation of different polymorphic markers with asthma, itis necessary to increase the sample size. The role of polymor-phisms of xenobiotic-metabolizing enzymes remains unclear.Cytochrome P450 (CYP) is an important enzyme in detox-ification processes. Variants of this enzyme determine therate at which pollutants can be metabolized. ETS contains>4000 substances, at least 50 of which are highly carcinogenic(Borgerding and Klus, 2005). ETS components are primarilyconverted into oxidized forms by CYP and are then conjugatedwith other molecules. Polymorphisms of CYP genes, due totheir important role in metabolizing xenobiotics, have beenassociated with lung cancer risk and ETS exposure. The roleof CYP in asthma has recently been explored. In rats, expo-sure to toluene di-isocyanate (an asthma inducer) increasesCYP1A1 expression (Haag et al., 2002). In a Russian popula-tion, polymorphisms of CYP1A1, CYP2D6, and CYP1B1 werefound to be associated with bronchial asthma. Vavilin et al.(2002) compared the CYP1A1 valine allele between childrenwithout asthma and children with bronchial asthma. In bothgroups, the authors found that the parameters of trait associa-tion with the disease depended on passive smoking. However,the Odds ratio (OR) of CYP1A1 for passive smoking remainedunchanged, suggesting that its role is discrete. Another recent

otic enzymes in the development of atopic diseases (Fedorovaet al., 2009). Meanwhile, Glutathione-S-transferases (GST)are phase II xenobiotic-metabolism enzymes that catalyze

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he conjugation of molecules with glutathione. In asthma,hese molecules induce airway inflammation and remodeling.ecause GST are highly polymorphic, their role in asthma haseen of recent interest. Downregulation of GSTPi in a murineodel of asthma increases oxidative stress and disrupts the

edox homeostasis (Schroer et al., 2011). GSTP1 is considered biomarker of asthma in children (Cortina et al., 2011). GSTP1le105Val and a promoter polymorphism of this gene haveeen shown to be associated with asthma risk in children ofmoking mothers (Li et al., 2008). In Taiwanese ETS-exposedhildren, polymorphisms of GSTM1 and GSTP1 are highlyelated with asthma severity (Lee et al., 2007). A recent studyn an occupational model of di-isocyanate-induced asthma,

polymorphism in GSTP1 could be protective (Broberg et al.,010). A study involving a Russian population confirms theelationship between bronchial asthma and GST polymor-hisms (Vavilin et al., 2000). However, another recent findingrgues that genotypes coding for low antioxidative-enzymehase II activity, including GSTP1, may not be associated withsthma (Malling et al., 2011). Our study examined the role ofYP1A1 and GSTP1 polymorphisms and failed to indicate anssociation with asthma or ETS exposure. Both frequenciesor CYP1A1 and GSTP1 polymorphisms are highly variable inatin-American subjects. In a study of a Brazilian population,olymorphisms of several GST differed from those found inopulations with a lower degree of ethnic mixture (Rossinit al., 2002). Some authors have established that associationtudies are frequently difficult to reproduce between differentthnic populations, particularly when different phenotypicarkers are analyzed. The lack of a significant relationship

n this study regarding these polymorphisms does not fullyxclude the possibility of a relationship with other polymor-hisms in these genes or in other xenobiotic-metabolismenes (Polonikov et al., 2009). Future studies should includehe protein expression levels of each gene and determinationf enzyme activity.

In conclusion, the polymorphisms analyzed in this studyere not useful markers for predicting bronchial asthma in aopulation of Mexican children, suggesting that they may notossess a functional role in the development of this disease.inally, further studies with larger samples, clinical assess-ents, and individuals from different regions would be helpful

n confirming that these polymorphisms are not associatedith susceptibility to asthma.

onflict of interest statement

one.

cknowledgment

his work was supported by grants 60463 and 104316 fromONACyT-México to AA and to BM, respectively.

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