Environmental polycyclic aromatic hydrocarbon (PAH) exposure and DNA damage in Mexican children

Mutation Research 742 (2012) 66–71

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Environmental polycyclic aromatic hydrocarbon (PAH) exposure and DNA damage in Mexican children Marco Sánchez-Guerra a , Nadia Pelallo-Martínez b , Fernando Díaz-Barriga b , Stephen J. Rothenberg a,c , Leticia Hernández-Cadena c , Sylvain Faugeron d , Luis F. Oropeza-Hernández e , Margarita Guaderrama-Díaz a , Betzabet Quintanilla-Vega a,∗ a

Departamento de Toxicología, CINVESTAV-IPN, Mexico, D.F. 07360, Mexico Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, San Luis Potosí 78290, Mexico c Instituto Nacional de Salud Pública, Cuernavaca 62100, Mexico d Facultad de Ciencias Biológicas, Pontifica Universidad Católica de Chile, Santiago, Chile e Laboratorio de Toxicología Industrial, Hospital General-PEMEX, Nanchital, Veracruz 96360, Mexico b

a r t i c l e

i n f o

Article history: Received 22 September 2011 Received in revised form 2 December 2011 Accepted 8 December 2011 Available online 17 December 2011 Keywords: PAH metabolism 1-Hydroxypyrene DNA damage Environmental exposures Genetic polymorphisms

a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants presenting a public health risk, particularly to children, a vulnerable population. PAHs have genotoxic and carcinogenic properties, which depend on their metabolism. Many enzymes involved in PAH metabolism, including CYP1A1, CYP1B1, GSTM and GSTT are polymorphic, which may modulate the activation/deactivation of these compounds. We evaluated PAH exposure and DNA damage in children living in the vicinity of the main petrochemical complex located in the Gulf of Mexico, and explored the modulation by genetic polymorphisms of PAH excretion and related DNA damage. The participants (n = 82) were children aged 6–10 y attending schools near the industrial area. Urinary 1-hydroxypyrene (1-OHP; a biomarker of PAH exposure) was determined by reverse-phase-HPLC; DNA damage by the comet assay (Olive Tail Moment (OTM) parameter); CYP1A1*2C and CYP1B1*3 polymorphisms by real time-PCR; and GSTM1*0 and GSTT1*0 by multiplex PCR. The median value of 1-OHP was 0.37 ␮mol/mol creatinine; 59% of children had higher 1OHP concentrations than those reported in environmentally exposed adults (0.24 ␮mol/mol creatinine). A stratified analysis showed increased DNA damage in children with 1-OHP concentrations greater than the median value. We observed higher 1-OHP concentrations in children with CYP1A1*2C or GSTM1*0 polymorphisms, and a positive influence of CYP1A1*2C on OTM values in children with the highest PAH exposure. The data indicate that children living in the surroundings of petrochemical industrial areas are exposed to high PAH levels, contributing to DNA damage and suggesting an increased health risk; furthermore, data suggest that polymorphisms affecting activation enzymes may modulate PAH metabolism and toxicity. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) include over 100 chemical compounds formed during the incomplete combustion

Abbreviations: CYP, cytochrome P450; GST, glutathione-S-transferase; 1-OHP, 1-hydroxypyrene; PAH, polycyclic aromatic hydrocarbon; UGT, UDP-glucuronosyl transferase. ∗ Corresponding author at: Ave. IPN 2508, Col. Zacatenco, Mexico City 07360, Mexico. Tel.: +52 55 57473800x5446; fax: +52 55 57473395. E-mail addresses: [email protected] (M. Sánchez-Guerra), na [email protected] (N. Pelallo-Martínez), [email protected] (F. Díaz-Barriga), [email protected] (S.J. Rothenberg), [email protected] (L. Hernández-Cadena), [email protected] (S. Faugeron), [email protected] (L.F. Oropeza-Hernández), [email protected] (M. Guaderrama-Díaz), [email protected] (B. Quintanilla-Vega). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.12.006

of organic matter [1]. Exposure to these ubiquitous environmental contaminants is a public health problem in cities with high industrial activity and heavy vehicular traffic, and also in some rural communities, due to indoor coal burning. PAHs are genotoxicants and human carcinogens [2]. Some studies of PAH exposure in occupational settings and in environmentally exposed children reported increased DNA damage, by means of the comet assay [3–5], a suitable measure of genotoxicity used in molecular epidemiology [6]. Children constitute a susceptible population, in part because they tend to have higher PAH exposure, due to behavioral patterns such as hand-to-mouth behavior, time spent outside in contact with soil and dust, and higher inhalation rate per bodyweight unit [7]; therefore, risk evaluation of this group has high priority. Some of the largest industrial complexes of Mexico (including petrochemicals) are located in Coatzacoalcos County in the state

M. Sánchez-Guerra et al. / Mutation Research 742 (2012) 66–71

of Veracruz bordering the Gulf of Mexico. The Coatzacoalcos River and the areas surrounding these industrial complexes are among the most contaminated sites in the country [8]. PAHs such as benzo[a]pyrene undergo metabolic activation to form bay-region dihydrodiol epoxides, with cytochromes P450 (CYP) 1A1 and 1B1 playing important roles [9]. Detoxication of PAH reactive metabolites can be catalyzed by glutathione-S-transferases (GST: Mu and Theta classes) and UDP-glucuronosyltransferases (UGT) [10]. The pyrene metabolite 1-hydroxypyrene (1-OHP) has been considered the most suitable biomarker of PAH internal dose recent exposure, since pyrene is emitted in large amounts and is always found as a component of PAH mixtures [11]. Many genes encoding enzymes involved in PAH metabolism are polymorphic, a factor which may explain much of the inter-individual variability in their rates of activation/deactivation [9,12]. Since children are one of the populations most vulnerable to environmental pollutants, we have conducted a pilot study on a sample of children living near a petrochemical complex, an important source of environmental PAHs, to evaluate PAH exposure and its potential genotoxic effects. In addition, we explored the influence of genetic polymorphisms of some PAH-metabolizing enzymes on urinary 1-OHP concentrations (PAH internal dose) and DNA damage. 2. Materials and methods 2.1. Study population The study area, Coatzacoalcos County in the State of Veracruz, bordering the Gulf of Mexico, has many petrochemical industries, representing 80% of the total petrochemical activity of Mexico. This community has about 280,000 inhabitants, of whom 30% are children aged < 14 y, representing a high percentage of children at risk to environmental pollutants. This cross-sectional study was conducted from April to May, 2008 in children 6–10 y, selected from three public elementary schools located less than 5 km from the main petrochemical complexes of the region. The schools were located in the cities of Allende (school 1; north of the main petrochemical complex), Mundo Nuevo (school 2; south), and Coatzacoalcos (school 3; west). Parents of 1st to 5th grade students from the selected schools were invited to take part in the study, by invitation through the school authorities. A total of 82 children were included; informed consent was obtained from each participant’s parent or guardian. A structured questionnaire was administered to parents by trained personnel to obtain information about demographic variables, socioeconomic status, lifestyle, health and nutrition status, and potential exposures to PAHs, such as wood smoke, burning garbage, and grilled meat intake. Children were scheduled the following morning for urine and blood sampling. The study was approved by the Institutional Ethical Committee for Conducting Research in Humans (COBISH) from CINVESTAV-IPN, Mexico. 2.2. Sample collection and analysis On the first morning, a urine sample was collected from each participant, in a clean sterile container, and whole blood was collected in EDTA or heparinized tubes; samples were frozen at −20 ◦ C until analysis. Urine samples were thawed at room temperature and homogenized before the analyses. Urinary 1-OHP concentrations were determined following the methods of Jongeneelen et al. [13] and Kuusimaki et al. [14]. Analyses were performed using HPLC1100 with a fluorescence detector (G1321A) (Agilent Technologies). Urinary 1-OHP concentrations were normalized to urinary creatinine, determined by the Jaffe colorimetric method [15]. A urinary metabolite of benzene, trans, trans muconic acid (t,t-MA) was determined by HPLC [12] and included in the multivariate regression analyses. PAHs and benzene have been proposed as the major carcinogenic compounds found in urban air pollution [4]. Urinary cotinine levels were used as a biomarker of passive smoking and determined using Accutest, NicAlert Strips (Jant Pharmaceutical, Encino, CA), as recommended by the manufacturer. A NicAlert result of Level 3 (100–200 ng/mL) or higher indicates exposure to tobacco products. 2.3. DNA extraction and genotyping DNA extraction from whole blood samples was done by the phenol:chloroform technique. Genotyping for CYP1A1*2C (rs1048943) and CYP1B1*3 (rs1056836) polymorphisms was done by real-time PCR using an ABI Prism 7000 Sequence Detection System with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA).

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Primers and probes sequences used for CYP1A1*2C genotyping were: 5 -GGCAAGCGGAAGTGTATCG-3 (forward), 5 -CAGGATAGCCAGGAAGAGAAAGAC-3 (reverse); *1A probe (VIC) 5 -TGAGACCATTGCCC-3 ; *2C probe (FAM) 5 -TGAGACCGTTGCCC-3 . For the CYP1B1*3 polymorphism, TaqMan® Drug Metabolism Genotyping Assays were used following the manufacturer’s instructions (ID. C 3099976 30; Applied Biosystems, Foster, CA). Subjects were categorized as wild-type homozygous, mutant homozygous, or heterozygous. The null polymorphisms of GSTM1 and GSTT1 were analyzed by multiplex PCR according to Arand et al. [16] and subjects were scored as null or non-null. Not all samples were successfully genotyped; the exact number for each polymorphism is shown in Table 3. 2.4. Comet assay DNA damage (strand breaks) was evaluated in heparinized whole blood by the comet assay [17]. Duplicate slides were prepared and 50 random cells were analyzed for each sample. Damage level was evaluated as Olive Tail Moment (OTM) using Komet software v. 4 (Kinetic Imaging Ltd., Liverpool, UK). The assay was successfully performed in 77 samples; for five subjects, there was insufficient blood sample to perform the analysis. 2.5. Statistical analyses Arlequin software v. 3.11 was used to evaluate the concordance of all polymorphisms with the Hardy–Weinberg Law. Urinary 1-OHP concentrations and OTM were natural log-transformed to normalize residuals for regression analyses. The influences of school location, age, gender, body-mass index (BMI), and exposures to wood smoke, passive cigarette smoke, grilled meat intake, and garbage-burning activities, as potential factors that may modify urinary 1-OHP concentrations, were evaluated by multiple linear regression analysis. Only two children showed high cotinine levels corresponding to cigarette smoke exposure (Level 3), and they were excluded from further analyses. We assessed the odds ratio (OR) between each genetic polymorphism and urinary 1-OHP concentrations by multiple logistic regression, using the median value (0.37 ␮mol/mol creatinine) as a cut-off. Since there were few CC-genotyped individuals for the CYP1B1*3 polymorphism they were combined with CT subjects, grouped as heterozygous (CC + CG). Regarding the CYP1A1*2C polymorphism, we combined subjects with at least one risk allele into the risk group (AG + GG), to increase the statistical power. Furthermore, the additive effects of the polymorphisms on 1-OHP concentrations were evaluated by a multiple regression analysis, taking the sum of risk alleles for each participant according to the following coding: 0 = wild-type homozygous, 1 = heterozygous and 2 = mutant homozygous for CYP1A1 and CYP1B1; and 0 = wild-type homozygous or heterozygous and 2 = homozygous for the GST deletions. We also used multiple regression analysis to test the association between PAH exposure and DNA damage, considering t,t-MA levels as a covariate. Since there is no established regulatory limit of environmental PAH exposure to children, we decided to stratify 1-OHP concentrations using two cuts-offs: the NOAEL for genetic damage (1.4 ␮mol/mol creatinine) [18] and the median value observed in the sample. The regression coefficients are presented as percent of the difference from the referent group, according to the formula %effect = 100(exp(ˇ) − 1), for categorical comparisons of natural log-transformed data [19,20]. To explore the genetic susceptibility of the sample of children to PAH genotoxicity, we evaluated the joint effect of PAH exposure and each polymorphism on the DNA damage through a multiple regression model, stratifying PAH exposure by the median 1-OHP value. The significance level was set as p < 0.05 in all statistical analyses, performed using the STATA software v. 10.1 (Stata Corp, College Station, TX).

3. Results 3.1. Children’s characteristics and their influence on urinary 1-OHP concentrations The general characteristics of participants and their influences on urinary 1-OHP concentrations are summarized in Table 1. The mean age of the participants was 7.9 ± 1.3 y, gender was approximately balanced (45% boys and 55% girls), and the BMI geometric mean was 17.5 ± 3.5; over 30% of children were overweight or obese and nine children (11%) were underweight according to the BMI-for-age growth chart from CDC. The median value of urinary 1OHP was 0.37 ␮mol/mol creatinine (Table 1). A high percentage of children (59%) had 1- to 17-fold higher 1-OHP concentrations than those previously reported in non-occupationally exposed adults (0.24 ␮mol/mol creatinine) [11], representing high PAH exposure.

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Table 1 Effects of subject characteristics on urinary 1-OHP concentrations. n (%)a Age (years) 82 All subjects Gender 37 (45) Males 45 (55) Females BMIc Healthy weight 47 (57) Underweight 9 (11) Overweight 16 (20) Obesity 10 (12) School locationd 1 36 (44) 2 31 (38) 3 15 (18) Exposure to wood smoke 56 (69) No Yes 25 (31) Burning garbage No 48 (61) Yes 31 (39) Grilled meat intake 53 (66) No 27 (34) Yes

1-OHP concentrationb (␮mol/mol creatinine)

p-Value

Genotype (n)

0.38 (0.30–0.48)

0.48e

0.34 (0.23–0.49) 0.42 (0.30–0.58)

0.38

0.38 (0.27, 0.53) 0.33 (0.14, 0.76) 0.52 (0.29, 0.94) 0.27 (0.14, 0.51)

0.73 0.31 0.39

0.21 (0.15, 0.28) 0.43 (0.29, 0.64) 1.26 (1.03, 1.52)

0.001* 0.001**

0.37 (0.28, 0.51) 0.38 (0.24, 0.58)

0.97

0.35 (0.25, 0.49) 0.43 (0.29, 0.62)

0.42

0.37 (0.27, 0.52) 0.36 (0.25, 0.52)

0.89

a

Percent of sample in parentheses. b Geometric mean and 95% confidence interval (95% CI). c Body mass index was calculated as the weight divided by height squared (kg/m2 ) and the corresponding BMI-for-age percentile was based on the CDC growth chart. d School location 1 = Allende, 2 = Mundo Nuevo and 3 = Coatzacoalcos. e Included as a continuous variable * Compared to school location 1 (p < 0.001). ** Compared to school locations 1 or 2 (p < 0.001).

The concentration of urinary 1-OHP was significantly influenced only by school location; children attending the school located in Coatzacoalcos city (West of petrochemical complexes) presented the highest 1-OHP concentrations. The geometric mean value of urinary t,t-MA was 370 ␮g/g creatinine (95% CI, 309, 442); no difference was observed among the school locations. 3.2. DNA damage We found positive associations between 1-OHP concentrations and genetic damage (OTM parameter) in the stratified analyses (Table 2). The median split of 1-OHP (0.37 ␮mol/mol creatinine) was associated with OTM values (Table 2). DNA damage was different according to school location, in a pattern similar to PAH exposure (1-OHP concentrations); children attending the school located in the city of Coatzacoalcos showed the greatest Table 2 Associations between genetic damage and 1-OHP urinary concentrations. Variable (n)

DNA damage (OTM)a

All subjects (77) school 1. Schools were located at similar distance to one of the main petrochemical complexes (5 km). Prevailing winds usually go from North to South of the County, explaining the high concentrations observed in school 2 (South of the industrial area), and we believe that school 3, located in Coatzacoalcos city (West of the industrial area), had the highest PAH level because it has a high vehicular traffic as well, compared to the other sites. It is well known that traffic is a dominant source to emission of PAHs in street air [24]. Consistent with 1-OHP concentrations, we observed higher DNA damage according to school location in children from Coatzacoalcos city. However, to enhance statistical power, we evaluated DNA damage in all children (n = 77) in a stratified analysis (at the median 1-OHP value), and found an increase in OTM at 1-OHP concentrations higher than 0.37 ␮mol/mol creatinine. Ruchirawat et al. [4] observed higher DNA damage in urban children with 0.18 ␮mol 1-OHP/mol creatinine compared to rural children with 0.10 ␮mol/mol creatinine. Introducing the parameter “relative index of damage”, as suggested by Valverde and Rojas [25], the index observed in our study in children with 1-OHP concentrations higher than the median value was 1.2, which is similar to the index observed in workers from a steel company with 10times higher 1-OHP levels [5]. These data strikingly indicate that children environmentally exposed to PAHs by living near petrochemical industrial areas may have similar genetic effects and probably health risks as adult populations exposed to higher PAH concentrations. This emphasizes the need for establishing a maximum limit for PAH exposure to general population to protect most vulnerable groups, such as children. Genetic polymorphisms affecting enzymes involved in PAH metabolism may modulate 1-OHP concentrations and DNA damage. We found a positive influence of some polymorphisms on PAH metabolism by means of urinary 1-OHP concentrations, after controlling for potential confounders. Urinary 1-OHP concentrations in heterozygous and homozygous individuals to CYP1A1*2C polymorphism were higher than in wild-type children. This finding observed in environmentally exposed children is in agreement with studies conducted in PAH occupationally exposed workers [26,27] and in smokers [28]. There was only one study conducted in children [4] that did not find a significant association between 1-OHP concentrations and CYP1A1*2C polymorphism. On the other hand, the higher 1-OHP concentrations observed in polymorphic GSTM1*0 homozygous individuals suggest that this variant may also positively modulate PAH excretion, as reported by others [27,29]. Interestingly, Alexandrie et al. [27] found that the highest 1-OHP concentrations were observed in individuals carrying both CYP1A1*2C and GSTM1*0 polymorphisms, in a study of 98 aluminum workers and 55 controls; this combined effect could not be evaluated in the present study, due to the small sample size.

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The comet assay is a reliable technique for assessing DNA damage in molecular epidemiology studies [6,25] and has been widely used in studies of PAH exposure [3–5]. Since we observed that the presence of CYP1A1*2C or GSTM1*0 polymorphisms may alter PAH metabolism by modifying urinary 1-OHP concentrations, we explored the joint effect of the risk alleles and PAH exposure on the DNA damage. The presence of the CYP1A1*2C variant in children with the highest 1-OHP concentrations increased the risk of higher DNA damage (OTM values). This result must be regarded with caution because of the small sample size. Other authors reported a positive association between the number of risk alleles of CYP1A1, CYP1B1, GSTM1, and GSTT1 and PAH-DNA adducts in healthy inhabitants from Mexico City Metropolitan Area (n = 92) [30], while Topinka et al. [31] observed that the combination of CYP1A1 and GSTM1 polymorphisms was associated with an increased formation of PAH-DNA adducts in non-smoking policemen (n = 109). CYP1A1*2C polymorphism has also been associated with increased risk for lung cancer in GSTM1-deficient subjects [32]. Although the sample size of this study is a limitation, CYP1A1*2C polymorphism was consistently associated with higher urinary 1OHP concentrations as well as with higher DNA damage, suggesting that this polymorphism affecting a PAH activation enzyme may indeed increase susceptibility to genotoxic effects. Another limitation was the lack of a direct biomarkers of PAH exposure other than urinary 1-OHP concentration; there are no previously reported PAH measurements to define air pollution in the studied area.

5. Conclusions Our study indicate that children from Coaltzacoalcos County are exposed to high PAH levels that induce genetic damage and may represent a significant health risk factor for children. This emphasizes the need for establishing a maximum limit for PAH exposure of the general population, especially to protect the most vulnerable groups. In addition, 1-OHP excretion was associated with CYP1A1*2C and GSTM1*0 polymorphisms, suggesting that PAH metabolism may be altered by the presence of variant alleles. Moreover, children carrying at least one risk allele for CYP1A1*2C polymorphism were more likely to have higher genetic damage. The presence of risk alleles may have implications for PAH toxicity, and children with certain polymorphisms may have higher levels of toxic intermediate metabolites, increasing their risk for PAHrelated adverse effects. More studies are needed to further test the possible role of the polymorphisms evaluated in this pilot study on PAH metabolism and toxicity.

Conflict of interest statement All authors declare that there are no conflicts of interest.

Financial source This study was financially supported by CONACYT-Mexico Grant #87234, given to BQV.

Acknowledgments The authors thank Gerardo Martinez Aguilar for his technical assistance and Evelyn Jimenez Mendoza and Susana Lara Montoya for helping on genotyping. MSG received a scholarship from CONACYT-Mexico.

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