Toxicology Letters 156 (2005) 331–339

CYP1A2 phenotype and genotype in a population from theCarboniferous Region of Coahuila, Mexico

Fabiola Castorena-Torresa, Ania Mendoza-Cantua, Mario Bermudez de Leonb,Bulmaro Cisnerosb, Omar Zapata-Perezc, Lizbeth Lopez-Carrillod,

Juan E. Salinase, Arnulfo Alboresa,∗

a Seccion Externa de Toxicolog´ıa, Centro de Investigaci´on y de Estudios Avanzados del IPN, Av. Instituto Polit´ecnico Nacional 2508,Col. San Pedro Zacatenco, Mexico City C.P. 07360, M´exico

b Departamento de Gen´etica y Biolog´ıa Molecular, Centro de Investigaci´on y de Estudios Avanzados del IPN, Av. Instituto Polit´ecnicoNacional 2508, Col. San Pedro Zacatenco, Mexico City C.P. 07360, M´exico

c Departamento de Ciencias del Mar, Centro de Investigaci´on y de Estudios Avanzados del IPN, Antigua Carretera aProgreso Km 6, Santiago, M´erida, Yuc. C.P. 97310, M´exico

d Instituto Nacional de Salud P´ublica, Av. Universidad, Col. Santa Ma. Ahuacatitl´an,Cuernavaca, Mor. C.P. 62508, M´exico

e Jurisdiccion Sanitaria No. 3, Secretar´ıa de Salud, Zaragoza 346,Co. Centro. Sabinas, Coah. C.P 26700, M´exico

Received 4 August 2004; received in revised form 7 December 2004; accepted 8 December 2004Available online 13 January 2005

46 malexcretionenotype

nces for,nzyme

; DU,leotide

Abstract

CYP1A2 regulation by polycyclic aromatic hydrocarbons (PAHs) exposure and polymorphism was investigated involunteers from the Carboniferous Region in northern Coahuila, Mexico. PAH exposure was estimated by the urinary eof 1-hydroxypyrene (1-OHP), whereas the regulatory effects were assessed by the caffeine metabolic ratio (CMR). Gwas evaluated by determining 5′-flanking region (−2964) and intron I (734) polymorphisms. A statistically significant differein the urinary 1-OHP geometric means of Barroteran, Cloete and Juarez (2.30, 0.45 and 0.04, respectively) was observed. Athe genotype, the intron I distribution was 0% C/C, 46% C/A and 54% A/A, whereas that of the 5′-flanking region was 26% G/G42% G/A and 32% A/A. Both distributions were in agreement with the Hardy-Weinberg equilibrium model. A greater eactivity was observed in the A/A compared to C/A individuals according to the CMR (P< 0.001), whereas the 5′-flanking region

Abbreviations:ANOVA, analysis of variance; BEL, biological exposure limit; CMR, caffeine metabolic ratio; MX, 1-methylxanthine1,7-dimethyluric; MU, 1-methyluric; AFMU, 5-acetylamino-6-formamylamine-3-methyluracil; CYP, cytochrome P450; SNP, single nucpolymorphism; PCR, polymerase chain reaction; PAHs, polycyclic aromatic hydrocarbons; 1-OHP, 1-hydroxypyrene

∗

Corresponding author. Tel.: +11 52 55 5061 3308; fax: +11 52 55 5747 7111.

0

E-mail address:aalbores@cinvestav.mx (A. Albores).

378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.toxlet.2004.12.005

332 F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339

polymorphism showed no effect on CYP1A2 enzymatic activity. These results suggest that intron I polymorphism and PAHexposure are relevant factors that modulate CYP1A2 enzymatic activity.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords:CYP1A2 polymorphism; PAH; Genotype; Phenotype; 1-Hydroxypyrene

1. Introduction

Industrial processes frequently contaminate the en-vironment. For example, coke factories constantly re-lease polycyclic aromatic hydrocarbons (PAHs). Thesearomatic compounds are considered as aethilogical fac-tors in the development of lung cancer that may af-fect exposed workers (Jongeneelen, 1992) and, possi-bly, populations living in the neighboring areas. PAHsare complex mixtures, and their constituents are sub-strates of the cytochrome P450 (CYP) enzymes. PAHmetabolic products can be eliminated or bioactivatedinto highly reactive and toxic electrophilic intermedi-aries (Guengerich, 1991). PAHs are also inducers of theenzymes that catalyze their own biotransformation, forexample, CYP1A1, 1A2 and 1B1 (Anzenbacher andAnzenbacherova, 2001). In addition, the extent of PAHmetabolism, regulation and toxicity depends on thecontaminants nature and concentration, but also on thepolymorphism of xenobiotic metabolism enzymes ofexposed subjects (Georgiadis et al., 2004).

lPto(tCccte(ttp

ieo

with PAHs due to coal mining and coke production, andto evaluate in this population the relationship betweenthe genetic polymorphism and CYP1A2 enzymatic ac-tivity.

2. Materials and methods

2.1. Study populations

The present study was performed in Barroteran,Cloete and Juarez, three localities of the CarboniferousRegion located in northern Coahuila, Mexico (Fig. 1)from which soil samples were collected and PAH an-alyzed. Soil samples were extracted according to theEPA 3540C method and assayed by gas chromatog-raphy, then PAHs were identified and quantified us-ing standards (Ultra Scientific, North Kingstown, RI).Quality assurance procedures included addition of in-ternal standards and blank analysis for each set ofsamples.

CYP1A2, a constitutive enzyme expressed in theiver, is among the phase I enzymes responsible forAH metabolism, but its participation has received lit-

le attention. In humans, the CYP1A2 gene is locatedn chromosome 15 and presents 13 polymorphic sites

Corchero et al., 2001; Oscarson, 2003), of which onlywo polymorphisms have been related to change on

′ -de-

sedlismu-

iva-m-

e toA2er-ted

F n inC

YP1A2 enzymatic activity: the 5-flanking region loated at the−2964 position and associated with arease of CYP1A2 activity (Nakajima et al., 1999) andhe intron I at position 734 associated with an increanzymatic activity, as evaluated by caffeine metaboSachse et al., 1999). Thus, the presence of such mations may alter the relative rate of metabolic action of PAHs and the susceptibility toward these coounds.

Therefore, the aims of the present study wernvestigate the effects of PAH exposure on CYP1nzymatic activity in a population from the Carbonifus Region of northern Mexico, an area highly pollu

ig. 1. Map of Mexico and location of the Carboniferous Regiooahuila.

F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339 333

Table 1Characteristics of the study population from Carboniferous Region, Coahuila, Mexico

Variables Barroteran Cloete Juarez Total

n (%) 14 (100) 13 (100) 19 (100) 46 (100)Occupational status Coke plant workers Coke plant ex-workers FarmersAge (years)

Mean± S.D. 44.50± 13.77 47.07± 9.48 50.05± 19.42 49.14± 15.07Range 23–60 31–60 20–84 20–84

Quetelet index (Kg/m2)Mean± S.D. 26.94± 5.33 26.90± 3.55 30.41± 4.38 27.64± 5.2Range 20.04–38.23 20.76–31.37 21.28–38.62 20.04–38.62

Smoking statusNon-smoker (%) 7 (50) 10 (76.69) 13 (68.43) 30 (64.44)Smoker (%) 7 (50) 3 (23.31) 6 (31.57) 16 (35.56)

Drinking statusa (%)Less than 1 3 (21.40) 1 (7.70) 1 (5.26) 5 (11.11)1–3 6 (42.80) 6 (46.15) 14 (73.68) 26 (57.77)3 or more 5 (35.70) 6 (46.15) 3 (15.79) 14 (31.11)

Grilled meatb (%)Less than 1 2 (14.28) 2 (15.38) 2 (10.52) 6 (14.63)1–2 7 (50) 7 (53.85) 2 (10.52) 16 (39.02)3 or more 5 (35.71) 4 (30.77) 10 (55.26) 19 (46.34)

Beveragesc

Yes 1 (7.15) 0 (0) 1 (5.26) 2 (4.44)No 13 (92.85) 13 (100) 17 (89.47) 43 (93.48)

a Number of beer or spirit cups per week.b Meat servings per week.c Beverages with a low caffeine content.

The population of this cross-sectional study com-prised 46 adult male healthy volunteers distributed asfollows: 14 workers from the Barroteran coke plant, 13ex-workers from the Cloete coke plant and 19 farm-ers from Juarez.Table 1summarizes the demographiccharacteristics of the study populations. Each partici-pating subject filled in a questionnaire containing infor-mation related to PAH exposure and factors affectingCYP1A2 phenotype, such as age, smoking habits, al-cohol consumption (defined as beers or spirit cups perweek), occupation, and dietary habits, such as meatconsumption (number of servings per week). Biologi-cal samples were obtained following informed writtenconsent; the protocol of this study was approved bythe Ethics Committee of Centro de Investigation y deEstudios Avanzados del IPN.

2.2. Biological sample collection

A blood sample was collected from each subjectby venous puncture and placed in an EDTA tube to

evaluate CYP1A2 genotype, and a urine sample wascollected to quantify PAH exposure, as estimated bythe urinary 1-hydroxypyrene (1-OHP) concentration.In addition, CYP1A2 enzymatic activity was assessedby the caffeine metabolism ratio in urine. All sampleswere kept at 4◦C for transportation, then after DNAisolation from lymphocytes or urine storage, at−80◦Cuntil analyzed.

2.3. Analysis of urinary 1-OHP

Urinary 1-OHP was quantified by HPLC accordingto Jongeneelen et al. (1987). Urine samples were di-gested with�-glucuronidase/arylsulfatase (Sigma, St.Louis, MO) at 37◦C overnight. They were then purifiedusing Bakerbound C18 cartridges (T.J. Baker, Phillips-burg, NJ), washed with 10 mL of deionized water andeluted from the cartridge with 10 mL of methanol. Thesolvent was evaporated to 1 mL at 30◦C under a softnitrogen jet. The volume of the residue was adjusted to2 mL with methanol and ultrasonicated for 4 min.

334 F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339

2.4. Evaluation of caffeine metabolism

The enzymatic activity of CYP1A2 was eval-uated by studying caffeine metabolism. Caffeineand the following metabolites: 1-methylxanthine(MX), 1,7-dimethyluric (DU), 1-methyluric (MU)and 5-acetylamino-6-formamylamine-3-methyluracil(AFMU), were analyzed. They were extracted fromurine according toGrant et al. (1983)and separatedby HPLC according toBerthou et al. (1989). Briefly,an oral dose of 140 mg caffeine, prepared as instantcoffee (Nescafe special filter, Nestle, Mexico) wasgiven to each volunteer. Urine was collected for 8 hand acidified with 1 mL of 6N HCl. Urine samples(1 mL) were adjusted to pH 3.5–4.0 with 1N HCl,and saturated with (NH4)2SO4. Then, 8 mL of a chlo-roform:methanol solution (85:15, v/v) were added toeach sample, following the addition of 200�L of 8-chlorotheophyline (50 mg/L) as internal standard. Themixture was shaken for 1 h. Samples were subsequentlycentrifuged at 2500 rpm for 5 min and the organic layerwas removed and filtered through a column contain-ing glass fiber and anhydrous Na2SO4. The filtratewas evaporated to dryness at 40◦C under a nitrogenstream and dissolved in 500�L of mobile phase A(see below).

Caffeine and all its metabolites were separatedby HPLC using a gradient elution program. Theywere analyzed in a Perkin Elmer liquid chromato-graph, equipped with a 50�L injection loop andd gth.A mn( ith5 weree o-b aseB an:a 8-c 2%.F bolicr ws:C ,1

2

y-m .

(1999)andSachse et al. (1999), respectively, as mod-ified by us. Briefly, the intron I sequence (Accessionno. NM00761) was amplified by PCR using 500 ngof genomic DNA and the following primers: forwardprimer P1f, 5′-CAACCCTGCCAATCTCAAGCAC-3′ (located on exon 1) and reverse primer R, 5′-AGAAGCTCTGTGGCCGAGAAGG-3′ (located onexon 2). PCR was performed with an initial denatura-tion for 4 min at 94◦C followed by 35 cycles of 1 min at94◦C, 30 s at 60◦C, 1 min at 72◦C, and a terminal ex-tension for 4 min at 72◦C. PCR products were furtherdigested with Apa I (Fermentas Inc., Hanover, MD)and visualized on agarose gels stained with ethidiumbromide. The bands corresponding to each genotypeof 5′-flanking region and intron I polymorphism areshown inFig. 1.

2.6. Statistical analysis

Statistical analyses were performed using the Stataversion 8.0 software (Stata Corporation, TX). Geomet-ric means and standard deviations were calculated be-cause urinary 1-OHP and CMR showed no normal dis-tribution. In addition, CMR values for selected geno-types and 1-OHP concentrations were compared byANOVA test and Pearson correlation analysis. Sim-ple and multiple linear regression models were usedto evaluate the relationship between PAH exposureand CYP1A2 enzymatic activity including stratifica-tion by selected genotypes. Confounding factors werea owerf t PSv ille,T

3

3

tet riedo ofc ew ast i-fi ne,a thene,

iode array detector, set at 280 nm wavelen250 mm× 4.6 mm Spherisorb Ps phaseb colu

Phase Separation Inc., Norwalk, CT), packed w�m C18 reverse phase was used. Compoundsluted with a 1 mL/min flow, using a mixture of mile phase A (2.75:3.30:10:83.95) and mobile ph

(2.75:7.5:75:14.75) (acetic acid:tetrahydrofurcetonitrile:water, v/v). The mean recovery ofhloroteophyline, used as internal standard, was 8inal results are expressed as the caffeine metaatio (CMR) calculated for each subject as folloMR = (AFMU + MU + MX)/DMU ( Campbell et al.987).

.5. CYP1A2 genotype determination

The 5′-flanking region and intron I CYP1A2 polorphisms were analyzed according toNakajima et al

nalyzed by regression and correlation test. The por this study was calculated using the program Geersion 2.1.31 (Vanderbilt Medical Center, NashvN) (Fig. 2).

. Results

.1. PAH levels in soil samples

A preliminary environmental monitoring to evaluahe magnitude of the PAH contamination was carut with soil samples obtained from the localitiesoncern. PAHs concentration in Barroteran and Cloetere 1708.74 and 1876�g/g soil, respectively, where

hose in Juarez was 2.1�g/g soil. The PAHs idented in Barroteran and Cloete soils were naphthalecenaphthene, phenanthrene, anthracene, fluoran

F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339 335

Fig. 2. PCR-restriction fragment length polymorphism-based analysis of (A) CYP1A2 5′-flanking region polymorphism: (C/C) wild type,(C/A) heterozygous, (A/A) mutated and (B) intron I polymorphism: (G/G) wild type, (G/A) heterozygous, (A/A) mutated, (M) 1 kb DNA ladder(Invitrogen). Intron I polymorphism standardization was carried out with samples not included in the study population.

pyrene, and some chemicals classified as carcinogens,like benzo[a]pyrene and dibenz[a,h]anthracene.

3.2. Demographical characteristics of the generalpopulation

The demographical characteristics of the study pop-ulations are given inTable 1. There were no significantdifferences in any of the parameters considered in thisstudy. Eighty percent of the subjects were obese ac-cording to the Quetelet index.

3.3. Urinary 1-OHP concentrations

Table 2shows the participants urinary 1-OHP con-centration. A urinary 1-OHP gradient showed the fol-lowing trend 57.5 > 11.2 > 1 for Barroteran, Cloete andJuarez, the latter with a geometric mean of 0.04; the dif-ference of the urinary 1-OHP geometric means amongthe subjects of the three localities was statistically sig-

nificant (P< 0.015). The 1-hydroxypyrene values of thewhole population were also correlated with the smok-ing habit (P< 0.048). Finally, the values observed wereconsistent with the subjects environmental exposure toPAH.

3.4. CYP1A2 genotype

The study population was allocated according to theCYP1A2 single nucleotide polymorphisms (SNP) lo-cated in the 5′-flanking region and intron I. Genotypefrequencies for the 5′-flanking region polymorphismfound in the entire population were published else-where (Mendoza-Cantu et al., 2004). With reference tothe intron I polymorphism, the genotype frequenciesobtained were 0, 46 and 54% for wild (C/C), heterozy-gous (C/A) and mutated (A/A), respectively. These val-ues are in accordance with the Hardy-Weinberg equi-librium model. A representative gel of these polymor-phisms is shown inFig. 2.

Table 2Urinary 1-hydroxypyrene concentrations of subjects from the Carboniferous Region, Coahuila, Mexico

Variables Barroteran Cloete Juarez Total

1-OHPa,* 2.30 0.45 0.04 1.32Range ND−6.38 ND−2.80 ND−0.45 ND−6.38Caffeine metabolic ratio 2.36± 1.81 2.80± 3.16 1.98± 0.86 2.52Range 0.2–6.93 0.2–12.70 0.11–3.63 0.11–12.7

ND: not detected (detection limit = 1 nmol/L).

a �mol/mol creatinine, expressed as geometric mean.∗ Statistically significant differences among groups atP< 0.015.

336 F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339

Fig. 3. Caffeine metabolic ratio for the different CYP1A2 genotypesof (A) intron I polymorphisms (*P< 0.01) and (B) 5′-flanking re-gion, assayed in the population from the Carboniferous Region fromCoahuila, Mexico.

3.5. Effect of genotype on caffeine metabolic ratio

This preliminary study, with a reported power of76.88%, shows a statistically significant increase ofthe CMR mean value in individuals showing the intronI mutant homozygous subjects (A/A) (n= 26) (0.68,

range 0.27–2.54) compared to heterozygous subjects(C/A) (n= 20) (0.3, range 0.10–1.56) when regres-sion analysis results were applied (R2 = 0.188,β = 0.70,P= 0.001) (Fig. 3A). No significant effects were ob-served when values were separated according to the 5′-flanking region SNP (R2 = 0.096,β = 0.073,P= 0.548)(Fig. 3B). These results suggest a close relationshipbetween intron I mutant genotype and CYP1A2 cat-alytic activity; this association was unaffected by othervariables such as smoking, obesity, consumption offood containing low amounts of PAHs, sweets or bever-ages with low caffeine content, and alcoholic beverages(Table 3).

3.6. Effect of PAH exposure and intron Ipolymorphism on enzymatic activity

The effect of PAH exposure and intron I polymor-phism on CYP1A2 catalytic activity was assessed bystratifying the relationship between urinary 1-OHPconcentrations and CMR for each genotype. No sta-tistical significance was found between CMR and uri-nary 1-OHP concentration in the genotypes analyzed(R2 = 0.096,β = 0.073,P= 0.475).

4. Discussion

Polymorphism affects the activation and eliminationo tivee od-u bee thea ffecto ityi

Table 3Correlation coefficients and statistical significance between character d CMR

Variables 1-OHPa

Smoking status 0.290Grilled meat −0.340 8Drinking status −0.128 9Beverages with low caffeine content – 174

a Pearson correlation coefficients.∗ Statistically significant difference atP< 0.05.

f xenobiotics. CYP1A2, a phase I hepatic constitunzyme, is polymorphic and its activity can be mlated also by PAHs; this cytochrome, then, canxpressed differently in each individual. Therefore,im of this preliminary study was to assess the ef CYP1A2 polymorphism and its metabolic activ

n populations exposed to PAH.

istics of the population and concentration of urinary 1-OHP an

P CMRa P

0.048* 0.202 0.1770.207 0.082 0.570.426 0.049 0.75

– 0.227 0.

F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339 337

4.1. PAHs in the environment and in the studypopulation

Environmental concentrations were assessed by afew PAH measurements in the soil of the study loca-tions. Barroteran and Cloete showed to be highly con-taminated with coke-oven emissions and wastes im-properly disposed. Hydrocarbons concentrations in thesoil of Barroteran and Cloete were similar despite theCloete coke plants closure a year before this study be-gan. As expected, hydrocarbon levels were the lowestand aliphatic compounds were undetectable in the soilof Juarez, the agricultural location.

Individual exposure to PAHs was assessed as uri-nary 1-OHP. A gradient of urinary 1-OHP concentra-tions was observed and Barroteran subjects showed thegreatest concentrations followed by those of Cloete andJuarez, the latter with the smallest values. The gradientobserved was consistent with the PAH environmentalexposure, since Barroteran volunteers were coke-ovenworkers, whereas Cloete subjects were coke-oven ex-workers and those from Juarez were farmers. In thisstudy, six Barroteran coke-oven workers and 1 Cloetesubject showed urinary 1-OHP concentrations higherthan 2.3�mol/mol creatinine, which is the thresholdvalue suggested for this industry (Jongeneelen, 1992).The urinary 1-OHP values reported here are similar tothose reported by other authors for coke-oven work-ers (Jongeneelen, 1992; Pan et al., 1998; VanRooij etal., 1994). Urinary 1-OHP results from Cloete subjectss duesa ex-w lace.A tra-t op-u eo tieso ok-i ary1 rtedb ndC

4t

eend as

two polymorphic sites, which have been associatedwith altered enzymatic activity. One SNP is located inintron I at position 734 and the other in the 5′-flankingregion at position−2963.

The frequency of CYP1A2 intron I SNP in the studypopulation was 20 heterozygous and 26 mutated ho-mozygous individuals, but no native homozygous in-dividuals were detected, as the frequency of the latterappears to be very low. These results thus gave 23 and77% frequencies for the native and mutated alleles, re-spectively. Those values are similar to those describedin Asian and Caucasian populations (Ozdemir et al.,2001; Sachse et al., 1999; Shimoda et al., 2002). As forthe frequencies of the 5′-flanking region SNP in a Mex-ican population, we previously reported that 43% werenative allele and 57% mutated allele (Mendoza-Cantuet al., 2004). These values are different from those re-ported in Asian populations, where a 76–78% nativeallele frequency occurs (Chida et al., 1999; Nakajimaet al., 1999; Shimoda et al., 2002).

Considering both CYP1A2 polymorphisms in thestudied population, the differences between observedfrequencies and those calculated by the Hardy-Weinberg equations were not statistically significant,indicating that it was representative of the populationstudied (Klug and Cummings, 2000). Since the Mexi-can population originated from a complex ethnic mix-ture, it is of relevance to know its genetic background,especially the polymorphisms responsible for the ef-fects on xenobiotic metabolism enzymes.

4a

A2g sti-g sta-t eent andC R;t igherC ta re-s er,H i-n MRi cto A2

uggest a poor disposition of the coke plants resind the accumulation of PAH in the coke plantorkers even a year after the plants closure took ps for the Juarez farmers, urinary 1-OHP concen

ions were similar to those reported for the general plation (Dor et al., 1999). However, there was still somverlapping in the 1-OHP values of the three localif study. In spite of the 48 h withdrawal period, sm

ng was still significantly associated with the urin-OHP concentrations; similar results were repoy other authors (Alexandrie et al., 2000; Granella alonfero, 1993).

.2. CYP1A2 polymorphism in the population ofhe Carboniferous Region

The detection of SNP in CYP1A2 has recently bescribed (Oscarson, 2003). This autonomic gene h

.3. Influence of CYP1A2 genotype on its enzymectivity in exposed subjects

When the influence of PAH exposure and CYP1enotype on CYP1A2 enzymatic activity was inveated in the Carboniferous Region population, a

istically significant association was observed betwhe presence of CYP1A2 intron I mutant genotypeYP1A2 enzymatic activity as estimated by the CM

he mutated homozygous subjects showed a hMR than the heterozygous ones (Fig. 3A). Sachse el. (1999)observed a greater activity in smokers penting the intron I mutation in both alleles. Furthan et al. (2001)found similar association in a Chese population of non-smokers, measuring the C

n plasma.Sachse et al. (1999)suggest that the effef a mutation in a non-coding region, as in CYP1

338 F. Castorena-Torres et al. / Toxicology Letters 156 (2005) 331–339

intron I, may result from differential interactions withregulatory proteins or from an imbalance with othermutations that may affect the inducibility of this cy-tochrome. The regulatory mechanisms of intron muta-tions on transcriptional activity have been described forseveral genes (Le Hir et al., 2003). However,Shimodaet al. (2002)observed no positive relationship betweenthe presence of the intron I mutation of CYP1A2 andits catalytic activity in a schizophrenic population, us-ing haloperidol as substrate, but this chemical is pri-marily metabolized by CYP3A4. The data all-togethersuggest that intron I mutated homozygous genotypecontribute to metabolize PAHs and other CYP1A2 sub-strates more efficiently, which ultimately may affect thesusceptibility to chemicals with carcinogenic potential.

The CYP1A2 5′-flanking region polymorphism re-sults in no apparent effect on CYP1A2 enzymatic ac-tivity in our population. These results differ from thosereported byNakajima et al. (1999), who observed an as-sociation between the 5′-flanking region homozygousmutated genotype and decreased CYP1A2 enzymaticactivity in smokers. The low PAH exposure found inthe study population reported here may account for thislack of association, sinceNakajima et al. (1999)ob-served such an effect only in heavy smokers exposedto high PAH levels, but not in non-smokers.

When we stratified the relationship between uri-nary 1-OHP concentrations and CMR according to theCYP1A2 intron I genotype, however, a positive trendwas found. It is possible that A/A individuals displaya ayb ci-a velsb ntp cen-t ma o-s l.( in-d pt),a , ont seds ed inu pa-t ol-lT ntra-t the

pyrene 1-hydroxylation and CMR can be found, andthis effect should be more evident in subjects with aCYP1A2 intron I A/A polymorphism.

In summary, the present study suggests that intronI polymorphism is a relevant factor that modulatesCYP1A2 enzymatic activity in PAH-exposed subjects.Here we show that such regulatory effect can be ob-served even at low PAH exposure levels. The genotyp-ing and phenotyping of the these populations may leadto identifying the individuals most susceptible to PAHexposure.

Acknowledgements

This study was supported by: EC contract: ERBIC18-CT980341. FCT was supported by a CONACyTgrant. We gratefully acknowledge R. Reyes and V.Ruız-Perez for technical assistance in 1-OHP quantita-tion, N. Cirnes for artistic work, and Dr. J.R. Bend andL. Sarkissian for their critical review of this manuscript.

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