Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage

Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage

STOTEN-21906; No of Pages 11 Science of the Total Environment xxx (2017) xxx–xxx Contents lists available at ScienceDirect Science of the Total Envi...

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STOTEN-21906; No of Pages 11 Science of the Total Environment xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage Bruno A. Rocha a,b, Alexandros G. Asimakopoulos b,c, Fernando Barbosa Jr. a, Kurunthachalam Kannan b,d,⁎ a

Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo 14040-903, Brazil Wadsworth Center, New York State Department of Health, Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12201, United States c Department of Chemistry, The Norwegian University of Science and Technology (NTNU), 7491, Trondheim, Norway d Biochemistry Department, Faculty of Science, Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• 25 phthalate metabolites were measured in children’s urine from 5 regions of Brazil. • Concentrations of monoethyl and di(2-ethylhexyl) phthalate metabolites were higher than in other children populations. • Exposure patterns varied among the geographic regions of Brazil. • 14 phthalate metabolites were positively associated with 8-hydroxy-2’deoxyguanosine.

a r t i c l e

i n f o

Article history: Received 30 November 2016 Received in revised form 26 January 2017 Accepted 27 January 2017 Available online xxxx Editor: Jay Gan Keywords: Human exposure Phthalates Children Daily intake Risk assessment Oxidative stress

a b s t r a c t Exposure of humans to phthalates has received considerable attention due to the ubiquitous occurrence and potential adverse health effects of these chemicals. Nevertheless, little is known about the exposure of the Brazilian population to phthalates. In this study, concentrations of 25 phthalate metabolites were determined in urine samples collected from 300 Brazilian children (6–14 years old). Further, the association between urinary phthalate concentrations and a biomarker of oxidative stress, 8-hydroxy-2′-deoxyguanosine (8OHDG), was examined. Overall, eleven phthalate metabolites were found in at least 95% of the samples analyzed. The highest median concentrations were found for monoethyl phthalate (mEP; 57.3 ng mL−1), mono-(2-ethyl-5-carboxypentyl) phthalate (mECPP; 52.8 ng mL−1), mono-isobutyl phthalate (mIBP; 43.8 ng mL−1), and mono-n-butyl phthalate (mBP; 42.4 ng mL−1). The secondary metabolites of di(2-ethylhexyl) phthalate (DEHP), and mEP, mIBP, and mBP were the most abundant compounds, accounting for N90% of the total concentrations. On the basis of the measured concentrations of urinary phthalate metabolites, we estimated daily intakes of the parent phthalates, which were 0.3, 1.7, 1.8, 2.1, and 7.2 μg/kg-bw/day for dimethyl phthalate, di-n-butyl phthalate, diisobutyl phthalate, diethyl phthalate, and DEHP, respectively. Approximately one-quarter of the Brazilian children had a hazard index of N 1 for phthalate exposures. Statistically significant positive associations were found between 8OHDG and the concentration of the sum of phthalate metabolites, sum of DEHP metabolites, mEP, mIBP, mBP, monomethyl phthalate, mono(3-carboxypropyl) phthalate, monobenzyl phthalate, monocarboxyoctyl

⁎ Corresponding author at: Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States. E-mail addresses: [email protected] (B.A. Rocha), [email protected] (A.G. Asimakopoulos), [email protected] (F. Barbosa), [email protected] (K. Kannan).

http://dx.doi.org/10.1016/j.scitotenv.2017.01.193 0048-9697/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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B.A. Rocha et al. / Science of the Total Environment xxx (2017) xxx–xxx

phthalate, monocarboxynonyl phthalate, monoisopentyl phthalate, and mono-n-propyl phthalate. To the best of our knowledge, this is the first study to report the exposure of a Brazilian population to phthalates. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Phthalates are synthetic organic chemicals used in numerous consumer products, including personal care products, medical devices, and foodstuffs, as well as building materials. Low molecular weight phthalates, including diethyl phthalate (DEP), dimethyl phthalate (DMP), and diisobutyl and di-n-butyl phthalates (DIBP and DBP) are used as additives in personal care products and in packaging materials for foodstuffs and pharmaceuticals. High molecular weight phthalates, including di-(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), benzyl butyl phthalate (BzBP), and di-n-octyl phthalate (DOP), are used mainly in the manufacturing of polyvinyl chloride (PVC), which is found in building materials and medical devices (Ejaredar et al., 2015; Katsikantami et al., 2016; Kumar and Sivaperumal, 2016; Mariana et al., 2016; Marie et al., 2015; Net et al., 2015). Phthalates are not chemically bound to the polymeric matrix and, therefore, can be easily released into the environment. The general population is exposed to phthalates through ingestion, inhalation, and dermal absorption (Colacino et al., 2010; Gao et al., 2016; Gomez Ramos et al., 2016; Guo and Kannan, 2013; Guo et al., 2011a, 2011b, 2014a; Hauser and Calafat, 2005; Hernandez-Diaz et al., 2009; Katsikantami et al., 2016; Koniecki et al., 2011; Net et al., 2015; Wang et al., 2015; Wittassek et al., 2011). Several studies have shown a link between phthalate exposure and adverse health outcomes on human reproductive, endocrine, and cardiovascular systems (Bamai et al., 2016; Ejaredar et al., 2015; Hauser and Calafat, 2005; Johns et al., 2015; Katsikantami et al., 2016; Kumar and Sivaperumal, 2016; Mariana et al., 2016). The mechanisms by which phthalates induce these adverse effects are not well established, but there is a growing evidence that oxidative stress may be involved (Wu et al., 2017). Recent human studies have suggested a positive association between urinary concentrations of phthalate metabolites and oxidative stress (Asimakopoulos et al., 2016; Ferguson et al., 2011, 2012, 2015, 2016; Guo et al., 2014b; Holland et al., 2016; Kim et al., 2014; Wang et al., 2011; Wu et al., 2017), which arises from an imbalance in the redox state that can result in an overload of reactive oxygen species in cells and tissues. Additionally, several experimental studies have shown that exposure to phthalates may lead to activation of peroxisome proliferator-activated receptors, increase in fatty acid oxidation, and reduction in the ability of cells to cope with the augmented oxidative stress which lead to reproductive organ malformations, reproductive defects, and decreased fertility (Mathieu-Denoncourt et al., 2015a, 2015b). Phthalates are metabolized in humans and eventually excreted in urine (Johns et al., 2015; Katsikantami et al., 2016; Kumar and Sivaperumal, 2016). Phthalate metabolites are considered suitable biomarkers for assessing exposure to parent compounds, and their occurrence has been established in human urine from countries around the world, including the United States, China, Germany, Canada, Denmark, Saudi Arabia and Australia (Asimakopoulos et al., 2016; Barr et al., 2003; Calafat et al., 2016; CDC, 2015; Gao et al., 2016; Gomez Ramos et al., 2016; Guo et al., 2011a; Hartmann et al., 2015; Hauser and Calafat, 2005; Johns et al., 2015; Kasper-Sonnenberg et al., 2012; Kumar and Sivaperumal, 2016; Saravanabhavan et al., 2013; Wang et al., 2015; Wittassek et al., 2011). 8OHDG is a biomarker of oxidative stress. Oxidation of deoxyribonucleic acid (DNA) occurs normally, but is augmented by elevated exposure to oxidizing agents. The oxidized DNA derivatives, including 8OHDG, are excreted in urine and reflect an equilibrium between the rates of DNA damage and repair. Therefore, the levels of oxidative stress

can be assessed non-invasively by measuring the urinary concentrations of 8OHDG (Angerer et al., 2007; Asimakopoulos et al., 2016; Ferguson et al., 2012; Ferguson et al., 2015; Kim et al., 2014; Guo et al., 2014b; Wang et al., 2011; Zhang et al., 2013). With this as background, the present study aimed to establish urinary levels (total concentrations) of 25 phthalate metabolites in a population of children from different geographic regions of Brazil to assess exposures and to delineate the association with oxidative stress in that population. Inter-correlations between phthalate metabolites were examined, and the association between phthalates and 8OHDG was assessed. 2. Materials and methods 2.1. Chemicals and materials Urine samples were analyzed for 25 phthalate metabolites (Table S1) viz., monoethyl phthalate (mEP), mono(2-ethyl-5-carboxypentyl) phthalate (mECPP), mono[(2-carboxymethyl) hexyl] phthalate (mCMHP), mono(2-ethyl-5-oxohexyl) phthalate (mEOHP), mono(2ethyl-5-hydroxyhexyl) phthalate (mEHHP), mono(2-ethylhexyl) phthalate (mEHP), monoisobutyl phthalate (mIBP), mono-n-butyl phthalate (mBP), monomethyl phthalate (mMP), monocyclohexyl phthalate (mCHP), monobenzyl phthalate (mBzP), monooctyl phthalate (mOP), mono(7-carboxyheptyl) phthalate (mCHpP), mono(3carboxypropyl) phthalate (mCPP), monoisononyl phthalate (mINP), monocarboxyoctyl phthalate (mCOP), mono(8-methyl-1-nonyl) phthalate (mIDP), monocarboxynonyl phthalate (mCNP), mono-npentyl phthalate (mPeP), monoisopentyl phthalate (mIPeP), mono-npropyl phthalate (mPrP), monoisopropyl phthalate (mIPrP), monohexyl phthalate (mHxP), monoheptyl phthalate (mHpP), and phthalic acid (PA). Specific information with regard to phthalate metabolites analyzed and their respective parent compound are presented in Table S2. Urine samples also were analyzed for 8OHDG, creatinine, and specific gravity. Detailed information with regard to phthalate metabolite standards is presented elsewhere (Asimakopoulos et al., 2016). However, we added new phthalate metabolites to this study that were not included previously, and the details for those are given below. mCOP (purity N 95%) and mCNP (purity N 95%) were purchased from CanSyn Chem Corp (Toronto, Ontario, Canada). mPeP and mIPrP were purchased from Accustandard Inc. (New Haven, CT, U.S.), and mPrP was purchased from Toronto Research Chemical Inc. (North York, Ontario, Canada). The isotopically labeled standards for these analytes, 2D4mPeP, 2D4-mIPeP, and 2D4-mIPrP, were purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). The individual stock solutions of each compound and internal standards were prepared by dissolution in acetonitrile and stored in amber glass vials, at −20 °C. The calibration and working standard solutions were prepared daily from the stock solutions through serial dilution with acetonitrile:milli-Q water (1:9), and stored in amber glass vials at 6 °C until analysis. 2.2. Study population and sample collection Urine samples were collected from 300 urban resident Brazilian school children aged 6 to 14 years from five geographic regions in Brazil (Southeast, South, Central-west, Northeast, and North) (Fig. S1) in 2012–2013. The demographic characteristics (distributions by gender, age, and region) of the population studied are given in Table S1. Spot urine samples were collected in polypropylene conical tubes from

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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healthy donors and stored at −80 °C until analysis. Informed consent was obtained from the legal guardian(s) of every child. The study was approved by the Institutional Ethical Review Board of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil. 2.3. Sample preparation and instrumental analysis The method for the analysis of phthalate metabolites in urine samples is described in Asimakopoulos et al. (2016). Briefly, phthalate metabolites were extracted after enzymatic deconjugation of urine samples, followed by solid phase extraction. Phthalate metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-ESI(−)MS/MS). Urinary concentrations of 8OHDG and creatinine were determined by LC-ESI(+)MS/MS after dilution of the urine samples with milli-Q water. Specific gravity of urine samples was determined by a refractometer (Asimakopoulos et al., 2014a, 2014b, 2016). Quantification was performed by the isotope dilution method. For the newly added compounds, corresponding labeled standards listed above were used for quantification; 2D4-mIPeP was used to measure mIPeP (the native standard for this compound was not available). Detailed information on tandem MS transitions for each phthalate metabolite and internal standards are provided in Table S3. 2.4. Quality assurance/quality control Contamination that arises from laboratory materials and solvents was monitored by the analysis of procedural blanks. A 15 to 20-point instrumental calibration curve was prepared in acetonitrile:water (1:9) at concentrations that ranged from 1 to 100 ng mL−1. The regression coefficients of the calibration curves were N 98%. For each batch of 25 samples, one procedural blank was analyzed. Throughout the analysis, 12 pre-extraction matrix spikes in solvent acetonitrile:water (1:9) were prepared (one in every 25 samples) by spiking known concentrations (40 ng mL−1) of target analytes and passing them through the entire analytical procedure. In addition, the Standard Reference Materials 3672 (Organic Contaminants in Smokers' Urine) and 3673 (Organic Contaminants in Non-Smokers' Urine) provided by the National Institute of Standards & Technology (NIST), which contain certified values for 11 phthalate metabolites (Schantz et al., 2015), were analyzed with every 50 samples. Our results for NIST SRMs were within ±15% of the certified values. A calibration check standard was performed, and methanol was injected after every 25 samples as a check for drift in instrumental sensitivity and carry-over between samples, respectively. The limits of detection (LODs) of phthalate metabolites varied from 0.01 to 0.84 ng mL−1 (Table S3). The recoveries of target chemicals spiked in 12 pre-extraction matrix spikes ranged from 72.5 to 148% (Table S3).

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was applied on log-transformed concentrations to detect similarities in exposure patterns among different groups (i.e., gender, age, and region). Compounds that were detected in b50% of the samples were excluded from statistical assessment. All statistical tests were considered significant if the two-tailed P-value was b0.05. The concentrations of the sum of DEHP metabolites (mECPP, mCMHP, mEOHP, and mEHHP) and the sum of all 25 phthalate metabolites are referred to as ∑4DEHP and ∑25Pht, respectively. All statistical analyses reported herein were calculated from volume-based concentrations (ng mL−1). 2.6. Estimation of daily intake (EDI) Urinary concentrations of the commonly detected phthalate metabolites in Brazilian children (volume-based) were used to estimate the daily intake of phthalates (DEP, DEHP, DIBP, DBP, and DMP) (Eq. (1)). Nevertheless, a creatinine-based calculation was also performed. The EDI was calculated using the following equation, previously described (Koch et al., 2003, 2007; Gao et al., 2016; Wang et al., 2015): EDI ¼

C pht  V u  MW pht f  bw  MW met

ð1Þ

where EDI is the estimated daily intake of the parent phthalate (μg/kgbw/day), Cpht is the respective urinary concentration of the phthalate metabolite (μg L− 1), Vu is the daily excretion volume of urine (L day−1), f is the molar fraction of the urinary metabolite excreted in relation to the oral intake of the parent phthalate, bw is the body weight (kg), and MWpht and MWmet are the respective molecular weights of the parent phthalate and its metabolite (g mol−1). Vu was calculated from the normal daily urine excretion rate of 0.0224 L kg−1 body weight (Szabo and Fegyverneki, 1995; Wang et al., 2015) and the average of body weights for the gender and age of Brazilian children (Table S4; IBGE, 2010). The f values used were derived from previous studies on the urinary concentrations of phthalate metabolites after oral doses of respective parent phthalates, and the f values were 0.69, 0.13, 0.11, 0.15, 0.70, 0.84, and 0.69 for mEP, mECPP, mEOHP, mEHHP, mIBP, mBP, and mMP, respectively (Anderson et al., 2001; Anderson et al., 2011; Itoh et al., 2007; Koch et al., 2012; Wang et al., 2015). The EDI of DEHP was calculated as the average of EDI for the metabolites mECPP, mEOHP, and mECPP. The EDI and risks were calculated only for major phthalates found in this study. 2.7. Risk assessment Hazard quotients (HQ) for exposure to DEP, DEHP, DIBP, and DBP were calculated as follows (Eq. (2)): HQ ¼

EDI Reference limit value ðTDI; RfD or RfD AAÞ

ð2Þ

2.5. Data analysis Data analysis was performed using SPSS software, Version 20, and Microsoft Excel 2013®. Median, mean, geometric mean (GM) and percentiles were calculated on volume-based, creatinine-adjusted, and specific-gravity adjusted concentrations. Concentrations below the LOQ were substituted with a value equal to the LOQ divided by 2 for the calculation of GM, arithmetic mean, and median. Only concentrations ≥ LOD were used to calculate the descriptive statistics. Because the urinary concentrations were not normally distributed (as determined by Kolmogorov-Smirnov and Shapiro-Wilk tests), data were log-transformed for statistical and correlation analysis. The nonparametric Mann-Whitney U test was used to study the differences between two groups of data, whereas the nonparametric Kruskal-Wallis test was used to examine the differences among three or more groups. To examine the relationship between phthalate metabolites, we applied Spearman's correlation and principal component analysis (PCA). PCA

where reference values are the tolerable daily intakes (TDI), reference doses (RfD) or a reference dose for anti-androgenicity (RfD AA) reported for individual phthalates. The TDI values for DEHP, DIBP, and DBP reported by the European Food Safety Authority (EFSA) were 50, 10, and 10 μg/kg-bw/day, respectively (EFSA, 2005a, 2005b, 2005c). The RfD values for DEP, DEHP, DIBP, and DBP, as reported by the United States Environmental Protection Agency (USEPA), were 800, 20, 100, and 100 μg/kg-bw/day, respectively (USEPA, 1990, 1993a, 1993b, 1993c). The RfD AA values for DEHP, DiBP, and DBP, were reported by Kortenkamp and Faust (2010), and they were 30, 200, and 100 μg/kgbw/day, respectively. To determine the cumulative risk from exposure to several phthalates, we calculated a hazard index (HI) for each Brazilian child by the summation of HQ values for individual phthalates. The RfDs established by the U.S. EPA are not suitable, because they are based on endpoints other than anti-androgenicity (Gao et al., 2016; Hartmann

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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et al., 2015; Søeborg et al., 2012; Wang et al., 2015). Therefore, HITDI and HIRfD AA were calculated by the summation of the HQTDI and HQRfD AA values of DEHP, DBP, and DIBP, as shown below (Eqs. (3–4)): HITDI ¼ HQ DEHP TDI þ HQ DBP TDI þ HQ DiBP TDI

ð3Þ

HIRfD AA ¼ HQ DEHP RfD AA þ HQ DiBP RfD AA þ HQ DBP RfD AA

ð4Þ

3. Results and discussion 3.1. Urinary concentrations of phthalate metabolites The urinary concentrations of phthalate metabolites found in Brazilian children are summarized in Table 1 and Fig. 1. Descriptive statistics and statistical analyses among different demographic groups (i.e., gender, age, and region) for creatinine-adjusted and specific-gravityadjusted concentrations are summarized in Tables S5–S7 and Tables S8–10, respectively. Eight metabolites, namely, mEP, mECPP, mCMHP, mEHHP, mCPP, mIBP, mBP, and mIPeP, were detected in all samples (DR = 100%). The rank order of DR for other predominant derivatives (DR N 50%) was: mMP (99.7%) N mEOHP (98.7%) = mPrP (98.7%) N mCNP (94.7%) N mCOP (93.3%) N mCHP (67.0%) N mBzP (59.2%) N mIPrP (55.7%). mCHpP, mHpP, mINP, and mPeP were not found in any of the samples. The highest median concentrations were

found for mEP (57.3 ng mL− 1), mECPP (52.8 ng mL−1), mIBP (43.8 ng mL−1), and mBP (42.4 ng mL−1). The median urinary concentration of mEP (57.3 ng mL−1) in Brazilian children was two to three times higher than that reported for children from Germany (33.6 ng mL−1), the United States (33.0 ng mL− 1), Canada (23.6 ng mL− 1), Austria (20.0 ng mL− 1), Denmark (20.0 ng mL−1), and China (18.7 ng mL−1) (Table 2). High concentrations of mEP were associated with the use of personal care products (PCPs) (Hartmann et al., 2015; Heudorf et al., 2007; Koniecki et al., 2011). In 2012, Brazil ranked third worldwide, behind the United States and Japan, in PCP consumption. It is noteworthy that Brazil is the global leader in the youth market for PCPs (Euromonitor, 2016a, 2016b; Oetterer, 2015; Oetterer and Nunes, 2015). The rank order of median concentrations of secondary DEHP metabolites in urine was: mECPP (52.8 ng mL−1) N mEHHP (23.8 ng mL−1)mEOHP (16.7 ng mL− 1) N mCMHP (13.1 ng mL− 1). The median urinary concentrations of ∑4DEHP (110 ng mL−1) in Brazilian children were higher than those reported for children from Denmark (69.0 ng mL−1), China (64.7 ng mL−1), and Germany (57.2 ng mL−1) (Table 2). Diet is a major pathway for DEHP exposure (Colacino et al., 2010; Gao et al., 2016; Koch et al., 2013; Schecter et al., 2013; Serrano et al., 2014), and in particular, high concentrations of DEHP were reported in meat and grains, such as rice (Cheng et al., 2016; Fierens et al., 2014). In Brazil, meat and rice are major dietary choices (IBGE, 2011; Pereira et al., 2014), and high concentrations of DEHP are thought to be through dietary sources.

Table 1 Urinary concentrations of phthalate metabolites and 8OHDG (ng mL−1) in Brazilian children (n = 300). Phthalate metabolite

DR%a

GMb

Mean

25th

Median (50th)

75th

Minc

Maxd

Spearman correlations with 8OHDG

mEP mECPP mCMHP mEOHP mEHHP ∑4DEHPe mEHP mIBP mBP mMP mCHP mBzP mOP mCHpP mCPP mINP mCOP mIDP mCNP mPeP mIPeP mPrP mIPrP mHpP mHxP PA ∑25Phtg 8OHDG

100 100 100 98.7 100 100 15.3 100 100 99.7 67.0 59.2 2.0 0 100 0 93.3 1.0 94.7 0 100 98.7 55.7 0 3.3 42.0 100

70.0 54.0 13.5 12.7 18.5 110 17.2 31.4 31.4 7.61 0.98 2.21 1.36 ndf 1.37 nd 2.10 3.31 1.04 nd 4.14 0.62 0.12 nd 0.66 46.7 437 94.6

278 106 26.3 36.6 53.8 223 36.1 73.8 113 10.4 2.43 3.83 1.66 nd 2.58 nd 6.25 4.95 2.88 nd 14.7 1.47 0.28 nd 1.20 91.1 774 4.40

22.6 27.7 7.47 3.67 6.14 54.4 7.63 10.6 10.7 4.94 0.46 1.13 1.05 nd 0.65 nd 0.82 1.80 0.47 nd 1.05 0.27 0.05 nd 0.32 21.4 217 5.58

57.3 52.8 13.1 16.7 23.8 110 19.2 43.8 42.4 8.30 0.88 1.91 1.15 nd 1.22 nd 2.21 1.90 0.93 nd 6.58 0.64 0.12 nd 0.53 60.3 489 2.80

243 106 24.8 38.6 56.7 226 38.5 89.1 96.7 12.6 1.86 3.93 1.21 nd 2.58 nd 5.18 6.58 2.13 nd 14.9 1.34 0.26 nd 1.35 119 957 4.40

0.95 1.52 0.72 0.36 0.09 3.81 1.03 1.23 0.07 0.42 0.05 0.15 0.85 nd 0.07 nd 0.06 1.70 0.07 nd 0.02 0.02 0.01 nd 0.15 0.61 16.2 7.31

9530 2070 1080 1220 1610 5090 396 869 3350 91.6 137 52.8 4.59 nd 50.4 nd 473 11.3 127 nd 261 23.1 4.07 nd 4.40 746 10,300 0.40

0.161⁎⁎ 0.261⁎⁎ 0.293⁎⁎ nsh nci 0.226⁎⁎ nc 0.143⁎ 0.172⁎⁎ 0.363⁎⁎ nc 0.166⁎ nc nc 0.312⁎⁎ nc 0.159⁎⁎ nc 0.187⁎⁎ nc 0.151⁎ 0.168⁎⁎ ns nc nc nc 0.211⁎⁎ 29.5



Abbreviations: Correlation is significant at the 0.05⁎ and 0.01⁎⁎ level (2-tailed). a DR%, detection rate %. b GM, geometric mean. c Min, minimum. d Max, maximum. e ∑4DEHP, sum of the 4 secondary metabolites of DEHP. f nd, not detectable. g ∑25Pht, sum of 25 phthalate metabolites. h ns: no significant. i nc: no calculated.

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

B.A. Rocha et al. / Science of the Total Environment xxx (2017) xxx–xxx

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Fig. 1. Urinary concentrations of phthalate metabolites (ng mL−1) in Brazilian children. The horizontal lines represent 10th, 50th and 90th percentiles and the boxes represent 25th and 75th percentiles.

The median urinary concentrations of mIBP and mBP were 43.8 and 42.4 ng mL−1, respectively, and these were higher than those found in children from the U.S., comparable to those found in Chinese children, and lower than those found in German children (Table 2). The median concentration of mMP in Brazilian children was higher than that found for other countries, except for China (Table 2). mCHP was not detected in children's urine from other countries (except for China) and was found in urine from 67% of Brazilian children. Metabolism of DMP and DCHP can yield mMP and mCHP, respectively (CDC, 2015). DMP is widely used in PCPs, as a solvent and as a plasticizer, and in insect repellents, lacquers, paints, plastics, and rubbers (Koniecki et al., 2011; Serrano et al., 2014). DCHP is used in the stabilization of rubbers, resins, and polymers, including nitrocellulose, polyvinyl acetate, and PVC (Li et al., 2016). Regulations exist on the use of DCHP and DMP in Europe and the United States, whereas no regulations on their use have been imposed in Brazil yet. The median urinary concentration (1.91 ng mL−1) and detection rate of mBzP (59.2%) were similar between Brazilian and Chinese

children, but lower than those reported for children in other countries (Table 2). mBzP was frequently detected in urine samples from the United States, Canada, and Germany, but was less frequently detected in Brazil (Table 2), which can be explained by the regulations on the use of benzyl butyl phthalate (BBzP) in Brazil (ANVISA, 2008, 2016; INMETRO, 2007). mOP and mCHpP are minor metabolites of DOP (Silva et al., 2005). mOP was detected in only 2.0% of the samples, and the highest concentration found was 4.59 ng mL−1. mCHpP was not detected in any samples. mCPP is the main metabolite of DOP (Calafat et al., 2006; Silva et al., 2005) and a minor metabolite of DBP (Silva et al., 2007b). The median concentration of mCPP found in children in this study (1.22 ng mL−1) was lower than that reported in the United States (4.9 ng mL− 1), Canada (3.1 ng mL− 1) and Germany (2.6 ng mL− 1) (Table 2). DOP was restricted for use in food packaging and toys in 2007 in Brazil (INMETRO, 2007). To the best of our knowledge, mIPeP, mPrP, and mIPrP were detected for the first time in human urine samples at high detection rates.

Table 2 Reported median urinary concentrations (ng mL−1) of phthalate metabolites in children worldwide. Country

Year

Age

N

mEP

mECPP

mCMHP

mEOHP

mEHHP

mEHP

∑4DEHP

mIBP

mBP

mMP

mCHP

mBzP

mCPP

mCOP

mCNP

Brazila Chinab USAc Germanyd Denmarke Canadaf Austriag

2012–2013 2012 2009–2010 2007–2009 2011 2007–2009 2010–2012

6–14 8–11 6–11 6–7 6–11 6–11 7–15

300 782 415 234 143 1037 220

57.3 18.7 33.0 33.6 20.0 23.6 20.0

52.8 20.7 29.4 42.1 15.0 – 16.0

13.1 11.8 – – – – –

16.7 17.6 11.1 26.4 12.0 20.3 3.0

23.8 11.8 17.0 31.0 23.0 31.6 4.0

19.2 5.9 1.7 4.0 2.0 3.3 nd

109.5 64.7 – 57.2 69.0 – –

43.8 38.5 10.9 68.7 54.0 – 35.0

42.4 47.1 23.3 54.2 32.0 32.6 12.0

8.30 8.4 2.4 3.8 – nd –

0.88 0.8 nd nd – nd nd

1.91 0.3 12.6 11.7 7.0 21.4 2.6

1.22 – 4.9 2.6 – 3.1 –

2.21 – 14.2 – – – –

0.93 – 3.6 – – – –

a b c d e f g

This study. Wang et al., 2015. CDC, 2015. Kasper-Sonnenberg et al., 2012. Frederiksen et al., 2013. Saravanabhavan et al., 2013. Hartmann et al., 2015.

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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B.A. Rocha et al. / Science of the Total Environment xxx (2017) xxx–xxx

mIPeP was found in all urine samples at a median concentration of 6.58 ng mL− 1 and a maximum concentration of 261 ng mL− 1. mPeP was not detected in any samples. The widespread occurrence of mIPeP indicates the occurrence of specific sources to Brazilian children. Diisopentyl phthalate (DIPeP) has been registered for use in the manufacture of nitrocellulose propellants and explosives, and, as with other low molecular weight phthalates, this compound also is used as a plasticizer in PVC products and other polymers (ECHA, 2016). DIPeP has been reported to occur in landfill leachates from municipal solid wastes in Brazil (do Nascimento Filho et al., 2003). DIPeP and di-n-pentyl phthalate (DPeP) were banned in 2016 in Brazil in cosmetics due to their toxicity (ANVISA, 2016). The median concentrations of mIPrP and mPrP found in our study were 0.12 and 0.64 ng mL− 1 respectively. Di-n-propyl phthalate (DPrP) has been reported to occur in cleaning products, such as bleach, baking soda, and borax (Dodson et al., 2012). PA is a non-specific biomarker of phthalate exposures and is formed through the hydrolysis of phthalate monoester metabolites (Meeker et al., 2009). The median concentration of PA found in Brazilian children was 60.3 ng mL−1. The median concentrations of mCOP and mCNP found in Brazilian children were 2.21 and 0.93 ng mL−1, respectively, which were lower than those reported for children from the United States (14.2 and 3.6 ng mL−1, respectively) and Norway (6.0 and 2.1 ng mL−1, respectively) (Bertelsen et al., 2013; CDC, 2015). In Brazil, regulations have been imposed on the use of phthalates in toys, food packaging, and cosmetics (ANVISA, 2008, 2016; INMETRO, 2007). DEHP, DBP, and BBzP are allowed for use at levels of ≤0.1% by mass of toys. In addition, DINP, DIDP, and DOP are allowed at levels of ≤0.1% by mass of toys that are intended for use by children under the age of 3 years (INMETRO, 2007). Further, restrictions are in place with regard to the use of DEHP, DBP, BBzP, DCHP, DEP, DIDP, and DOP in food-packaging materials. DEHP, DBP, DIBP, BBzP, DPeP, and DIPeP have been banned from PCPs in Brazil (ANVISA, 2016). Despite the regulations on the usage of phthalates, no systematic monitoring is in place to establish the baseline levels and to evaluate the effectiveness of regulations. A study investigated phthalate levels in Brazilian food-packaging materials and found levels 5 to 10-fold higher than the acceptable limits proposed by the Brazilian government (Freire et al., 2006). Our study established baseline values for monitoring future trends of phthalate exposure in Brazilian children. 3.2. Correlations and composition profiles of phthalate metabolites A significant correlation was found between the concentrations (log-transformed) of phthalate metabolites, except for mEP, mMP, and mBzP (Table S11). Significant correlations between phthalate metabolites suggest concomitant exposure to several phthalates by children. Significant correlations were found between the secondary metabolites of DEHP (r N 0.70), which reiterates that they originate from the same parent compound (Hartmann et al., 2015; Saravanabhavan et al., 2013). The correlation between mBP and mIBP was strong, which suggests similar sources and routes of exposure (Hartmann et al., 2015; Lorber and Koch, 2013). Moderate to strong correlations were observed among the isomers mIBP and mBP and the metabolites of DEHP (mECPP, mCMHP, mEOHP, and mEHHP). These results may indicate that the source of DEHP, DIBP, and DBP in Brazilian children was diet. A significant correlation was found between mCPP and mBP concentrations (the main metabolite of DBP) (r = 0.621, p b 0.01). This result may suggest that the main source of mCPP is DBP (instead of DOP). Concentrations of mCOP and mCNP, the main metabolites of DINP and DIDP, respectively, were strongly correlated (Kato et al., 2007; Silva et al., 2006; Silva et al., 2007a), and these two phthalates have been used as substitutes for DEHP (Bertelsen et al., 2013; Serrano et al., 2014; Wittassek et al., 2011). Urinary concentrations of phthalate metabolites are affected by several factors, including age, gender, and lifestyle (Bamai et al., 2015;

Becker et al., 2009; Cutanda et al., 2015; Gao et al., 2016; Gomez Ramos et al., 2016; Guo et al., 2011a, 2011b; Hartmann et al., 2015; Kasper-Sonnenberg et al., 2012; Saravanabhavan et al., 2013; Serrano et al., 2014; Wang et al., 2015). Concentrations of phthalate metabolites stratified according to gender and age are summarized in Table 3. Further details of phthalate concentrations divided into four additional groups, 6–10 and 11–14 years for males and females, are shown in Table S12. There was no statistical difference in the concentrations of phthalate metabolites between male and female individuals (p N 0.05) (Table 3), except for mEP, for which females had significantly higher concentrations (67.7 ng mL−1) than did males (47.5 ng mL−1). mECPP was higher in males (56.8 ng mL−1) than in females (45.4 ng mL−1). This result is consistent with the data published for other populations (Bertelsen et al., 2013; CDC, 2015; Cutanda et al., 2015; Hartmann et al., 2015). Significant differences were found for mBP, mIBP, and mIPeP concentrations between the two age groups [6–10 vs. 11–14 year old]. Higher concentrations of these phthalate metabolites were found in older children. For other phthalate metabolites, no significant differences were observed between the two age groups. The composition profiles of urinary phthalate metabolites in Brazilian children from various geographic regions of Brazil are presented in Fig. 2, Fig. S2, and Table 4. ∑4DEHP, mEP, mIBP, and mBP accounted for 40, 21, 16, and 15%, respectively, of the total concentrations (Fig. 2). This result is in accordance with the previous studies reported for Chinese, U.S., Canadian, Japanese, Australian, and Danish children, for whom the metabolites of DEHP were the most abundant of all phthalates (Bamai et al., 2015; Becker et al., 2009; CDC, 2015; Frederiksen et al., 2011; Gomez Ramos et al., 2016; Saravanabhavan et al., 2013; Wang et al., 2015). Among the four DEHP metabolites analyzed, mECPP and mEHHP accounted for 49.6 and 22.3%, respectively, of the total DEHP metabolite concentrations, followed by mEOHP (15.7%) and mCMHP (12.3%). The composition profiles of the secondary DEHP metabolites in children's urine from five geographic regions of Brazil are shown in Fig. S3. It is noteworthy that lifestyle, culture, and consumer habits vary among the several Brazilian regions. The urinary concentrations of phthalate metabolites, except for mMP and mBzP, showed significant differences (Table 4) among the five regions of Brazil. Similarly, the distribution of phthalate metabolites was different among the five geographic regions of Brazil. In all of the regions, the secondary metabolites of DEHP were the predominant derivatives, accounting for 32–55% of the total concentrations. In the Central-west, the secondary metabolites of DEHP accounted for 55% of the total, whereas, in the Northeast and North, they accounted for approximately 30% of the total concentrations. The proportion of mIBP and mBP found in urine from the South and Central-west was higher than that for mEP, whereas in the Southeast, North, and Northeast, mEP was more abundant than mIBP and mBP (Fig. 2). Overall, the median concentrations of phthalate metabolites were higher in the Central-west than in the other regions of Brazil. Some exceptions were observed for mEP and mIPeP, which were found at higher concentrations in the Northeast, while mBP and mBzP were higher in the South. The median concentrations of phthalate metabolites were lowest in the Southeast region of Brazil (Table 4). One possible explanation for the higher concentrations of DEHP metabolites in the Central-west and mBP in the South is the difference in food consumption patterns among the regions of Brazil. Meat and rice are consumed more in the Central-west (g day−1) than in other regions of the country, whereas milk and dairy products are consumed more in the South than in other regions (IBGE, 2011). Several studies have associated the use of PCPs with the high concentrations of mEP in urine (Cutanda et al., 2015; Guo and Kannan, 2013; Guo et al., 2014a; Hartmann et al., 2015; Heudorf et al., 2007; Koniecki et al., 2011; Mariana et al., 2016). Brazil is one of the global leaders in the consumption of PCPs and the second largest market for

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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Table 3 Median urinary concentrations of phthalate metabolites (ng mL−1) in Brazilian children and their relations to age and gender. Group

N

mEP

mECPP

mCMHP

mEOHP

mEHHP

∑4DEHP

mIBP

mBP

mMP

mCHP

mBzP

mCPP

mCOP

mCNP

mIPeP

mIPrP

mPrP

6–10 11–14 P-value Female Male P-value

134 166

46.6 65.8 0.137 67.7 47.5 0.048

53.0 51.5 0.572 45.4 56.8 0.027

12.9 13.2 0.907 12.5 13.3 0.136

15.2 17.7 0.102 14.8 20.3 0.182

22.1 26.6 0.123 22.9 26.4 0.263

108 112 0.701 94.7 115 0.072

30.4 57.3 0.020 47.9 41.6 0.862

30.0 49.7 0.020 43.6 41.2 0.767

8.21 8.62 0.407 8.15 8.25 0.991

0.91 0.94 0.918 0.83 1.10 0.088

1.92 1.79 0.635 1.73 2.17 0.125

1.31 1.28 0.653 1.15 1.37 0.317

2.33 2.14 0.692 1.78 2.48 0.242

0.89 0.91 0.678 0.88 0.98 0.227

4.32 7.92 0.007 5.92 7.11 0.791

0.12 0.12 0.885 0.14 0.10 0.505

0.59 0.71 0.325 0.69 0.64 0.170

151 149

*bold: significance level is P b 0.05.

children's PCPs. Brazil is the largest consumer market for fragrances and deodorants in the world (Euromonitor, 2016a, 2016b; Oetterer, 2015; Oetterer and Nunes, 2015). High urinary concentrations of mEP can be attributed to the usage of PCPs among the Brazilian population. Higher concentrations of mEP found in the Northeast and Northern regions of Brazil may be related to the cultural habit of using large amounts of fragrances in those regions (Euromonitor, 2016a).

3.3. Principal component analysis (PCA) For PCA analysis, we used the concentrations of ∑4DEHP, sum of mIBP and mBP, and mEP in order to create the principal components, PC1 and PC2. We selected only these three variables since their detection rates were 100% and these three were the major compounds found in urine. It was necessary to decrease the number of variables to a few linear combinations of the data set that correspond to a specific principal component. Two principal components (PC1 and PC2) were extracted, and the score plot that was generated accounted for 85.9% of the variabilities. The results of the PCA suggest differences in exposure patterns in the Central-west and Southern regions and between the Northeast and Northern regions of Brazil (Fig. 3). The points that are proximal in the loading plot (Fig. 3), ∑4DEHP and sum of mIBP and mBP, denote similar exposure patterns. Previous studies have revealed that food is the most important source of DEHP (Schecter et al., 2013; Schettler, 2006; Serrano et al., 2014), DBP, and DIBP (Cao et al., 2015; Cheng et al., 2016; Fierens et al., 2014; Ji et al., 2014; Schecter et al., 2013; Serrano et al., 2014). On the contrary, mEP was not in

proximity to ∑4DEHP and the sum of mIBP and mBP in the loading plot. This can be explained by the source of mEP, which is PCPs (Cutanda et al., 2015; Guo and Kannan, 2013; Hartmann et al., 2015; Heudorf et al., 2007; Koniecki et al., 2011; Mariana et al., 2016).

3.4. Estimated daily intakes The EDIs (median, range, and 95th percentile) of phthalates by Brazilian children stratified by age, gender, and demographic region are presented in Table S13. The daily intake values for the creatininebased model are shown in Table S14. The highest EDI values were found for DEHP, followed by DEP, DIBP, DBP, and DMP. The median EDIs of DEHP, DEP, DIBP, DBP, and DMP were 7.16, 2.14, 1.75, 1.70, and 0.29 μg/kg-bw/day, respectively. The EDI for DEHP in Brazilian children was higher than the values reported for children from Denmark (4.0 μg/kg-bw/day; Frederiksen et al., 2011), China (3.7 μg/kg-bw/day; Wang et al., 2015), Belgium (3.4 μg/kg-bw/day; Dewalque et al., 2014), and Austria (1.6 μg/kg-bw/day; Hartmann et al., 2015) and lower than that reported for children from North America (creatinine-based model; 6.0 μg/kg-bw/day; Christensen et al., 2014). The EDI values of DEP in children from Denmark, China, Belgium, and Austria were 1.1, 0.7, 1.5 and 0.7 μg/kg-bw/day, respectively (Dewalque et al., 2014; Frederiksen et al., 2011; Hartmann et al., 2015; Wang et al., 2015), which were lower than the values found for Brazilian children. The EDIs for DIBP (1.75 μg/kg-bw/day), DBP (1.70 μg/kg-bw/day). and DMP (0.29 μg/kg-bw/day) calculated for the

Fig. 2. Composition of phthalate metabolites in urine from Brazilian children in five geographic regions of Brazil (‘others’ represents the sum of phthalate metabolites that were not presented).

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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Table 4 Geographic variations in median urinary concentrations of phthalate metabolites (ng mL−1) in Brazilian children. Region

N

mEP

mECPP

mCMHP

mEOHP

mEHHP

∑4DEHP

mIBP

mBP

mMP

mCHP

mBzP

mCPP

mCOP

mCNP

mIPeP

mIPrP

mPrP

Southeast South Central-west Northeast North P-value

109 45 31 87 28

40.2 65.0 42.3 109 92.1 0.031

34.9 73.6 117 52.5 50.7 b0.001

9.32 15.9 28.9 11.3 14.5 b0.001

2.83 30.8 47.1 19.7 21.5 b0.001

4.54 41.8 72.4 26.2 29.4 b0.001

59.6 165 281 109 115 b0.000

9.51 72.6 79.2 49.3 60.9 b0.001

10.0 93.1 76.1 50.5 49.5 b0.001

7.28 9.31 10.3 8.42 8.12 0.335

0.65 1.11 1.18 0.69 0.93 0.039

1.30 2.65 1.66 2.04 2.11 0.050

1.12 1.86 2.06 1.02 1.22 0.001

1.15 3.62 5.78 2.26 1.34 b0.001

0.71 1.42 2.81 0.92 0.73 b0.001

0.70 7.81 9.02 10.2 7.08 b0.001

0.085 0.115 0.310 0.120 0.129 0.006

0.43 0.62 0.82 0.91 0.85 b0.001

*bold: significance level is P b 0.05.

present study population were similar to the EDIs reported for Chinese children (1.5, 1.9, and 0.3 μg/kg-bw/day, respectively). Our findings suggest that DEHP, DBP, and DIBP exposures are of particular concern for Brazilian children. The EDIs for DEHP found for 52 Brazilian children were above the USEPA RfD of 20 μg/kg-bw/day. Moreover, the EDI values for 28 and 14 children exceeded the TDI of 10 μg/kg-bw/day recommended for DBP and DIBP, respectively.

3.5. Risk assessment To quantitatively assess the risk of chemicals, a ratio between the actual exposure dose and an acceptable exposure dose is used. This ratio is referred to as the hazard quotient (HQ). A HQ with a value greater than one indicates a potential risk from exposure (Søeborg et al., 2012). One approach that allows the assumption of adverse effects resulting from simultaneous exposures is a dose-addition model that uses the hazard index (HI) for several chemicals (Christensen et al., 2014; Hartmann et al., 2015; Søeborg et al., 2012). In this study, a cumulative risk of exposure to phthalates was carried out by the HI approach, which is the sum of different HQs based on similar toxicological end points (Dewalque et al., 2014; Gao et al., 2016; Hartmann et al., 2015; Koch et al., 2011; Søeborg et al., 2012; Wang et al., 2015). The selected reference values were the TDI for DnBP, BBzP and DEHP based on impairment of germ cells and spermatozoa as endpoints as developed by the EFSA (EFSA, 2005a, 2005b, 2005c), and the RfD AA for DnBP, DIBP and, DEHP reported by Kortenkamp and Faust (2010) based on anti-androgenic endpoints, such as suppression of fetal testosterone production. The RfD values of DEP, DEHP, DIBP and DBP were mainly based on altered organ weight and increased mortality (for DEHP) in animal studies other than androgenic effects (USEPA, 1990, 1993a, 1993b, 1993c). Since the U.S. EPA's RfDs for phthalates were not developed on the basis of anti-androgenic effects, HI values were only calculated according to the TDIs and RfD AAs (HQ of DEHP, DIBP and, DBP). The individual HQTDI and HQRfD AA values for phthalates were summed to obtain HITDI and HIRfD AA, respectively. HQ is the ratio of exposure to no adverse effect level, whereas HI includes the assumption of adverse health effects that result from simultaneous exposures based on similar toxicological

effects (Dewalque et al., 2014; Gao et al., 2016; Hartmann et al., 2015; Koch et al., 2011; Søeborg et al., 2012; Wang et al., 2015). The HQs and HIs for Brazilian children are shown in Table 5. Our results showed that 17 (5.7%), 14 (4.7%), and 28 (9.3%) out of 300 children exceed the TDI of 50 μg/kg-bw/day for DEHP, 10 μg/kg-bw/day for DIBP, and 10 μg/kg-bw/day for DBP, respectively. It should be noted that 98 out of 300 Brazilian children showed HITDI values higher than 1. This finding suggests that approximately one-third of Brazilian children may have a potential risk for adverse effects from exposure to phthalates. It should also be noted that the cumulative risk of phthalate exposures may be underestimated because only three phthalates (DEHP, DIBP, and DBP) were included in the calculation of HITDI due to the lack of reference values for DEP, DMP and DIPeP. This risk assessment is to develop baseline data that needs further development. The results of HQs and HIs for Brazilian children stratified by age, gender, and region are shown in Table S15. 3.6. Association between phthalates and 8OHDG Several studies have shown a link between phthalate exposure and adverse human health outcomes (Bamai et al., 2016; Ejaredar et al., 2015; Hauser and Calafat, 2005; Johns et al., 2015; Mariana et al., 2016). Although the mechanisms for these health effects have not been strongly established, there is accumulating experimental and epidemiologic evidence that phthalate exposures contribute to oxidative stress (Aly et al., 2016; Asimakopoulos et al., 2016; Ferguson et al., 2011; Ferguson et al., 2012; Ferguson et al., 2015; Ferguson et al., 2016; Guo et al., 2014b; Holland et al., 2016; Kim et al., 2014; Wang et al., 2011; Wu et al., 2017). Oxidative stress is an imbalance between the endogenous formation of reactive oxygen species (ROS), and the organism's capacity to detoxify or eliminate the ROS or to repair damage caused by the ROS. Phthalates disrupt the oxidative balance in a cell by activating peroxisomes and by inducing cytochrome P450 enzyme activity (Mathieu-Denoncourt et al., 2015a, 2015b). Oxidized DNA repair products are excreted in urine, and therefore urinary 8OHDG is considered to be an important marker of oxidative stress (Angerer et al., 2007; Asimakopoulos et al., 2016; Ferguson et al., 2012; Ferguson et al.,

Fig. 3. Principal component analysis of urinary phthalate metabolite concentrations in Brazilian children, stratified by geographic regions of Brazil.

Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193

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Table 5 Hazard quotients (HQ) and Hazard Index (HI) of phthalate exposures based on TDI (EFSA), RfD (U.S.EPA) and, RfD AA (Kortenkamp and Faust, 2010) for Brazilian children. Phthalates

DEP DEHP DIBP DBP HI

HQTDI

HQRfD

HQRfD AA

GM

Median

95th

N N 1 (%)

GM

Median

95th

N N 1 (%)

GM

Median

95th

N N 1 (%)

– 0.145 0.175 0.126 0.526

– 0.143 0.126 0.170 0.595

– 1.160 0.990 1.910 3.470

– 17 (5.7) 14 (4.7) 28 (9.3) 98 (32.7)

0.003 0.361 0.013 0.013 –

0.003 0.358 0.018 0.017 –

0.042 2.900 0.099 0.191 –

0 (0.0) 52 (17.3) 0 (0.0) 1 (0.3) –

– 0.241 0.006 0.013 0.283

– 0.239 0.008 0.017 0.280

– 1.93 0.050 0.191 2.10

– 30 (10.0) 0 (0.0) 1 (0.3) 39 (13.0)

2015; Kim et al., 2014; Guo et al., 2014b; Wang et al., 2011; Zhang et al., 2013). In this study, 8OHDG was found in 94.6% of children's urine samples, with concentrations that ranged from 0.40 to 29.5 ng mL−1 (median: 4.40 ng mL− 1) (Table 1). No statistically significant differences were observed in the urinary concentrations of 8OHDG among various demographic groups (gender, age, and region). Recent studies have suggested strong positive associations between urinary concentrations of phthalate metabolites and biomarkers of oxidative stress, including 8OHDG, in different populations (Asimakopoulos et al., 2014b, 2016; Ferguson et al., 2011; Ferguson et al., 2012; Ferguson et al., 2015; Ferguson et al., 2016; Guo et al., 2014b; Holland et al., 2016; Kim et al., 2014; Mathieu-Denoncourt et al., 2015a, 2015b; Wang et al., 2011; Wu et al., 2017). In line with those results, our results suggest that there were significant positive relationships (r = 0.143–0.363, p b 0.05, Table 1) between the log-transformed concentrations of 8OHDG with the sum concentrations of phthalates (∑25Pht), sum of DEHP metabolites (∑4DEHP), mECPP, mCMHP, mEP, mIBP, mBP, mMP, mCPP, mBzP, mCOP, mCNP, mIPep, and mPrP. Additionally, in animal studies, phthalate-induced oxidative stress has been reported (Abdul-Ghani et al., 2012; Aly et al., 2016; Du et al., 2015; Mathieu-Denoncourt et al., 2015a; Mathieu-Denoncourt et al., 2015b; Shono and Taguchi, 2014). This study reiterates that phthalate exposure is associated with oxidative stress in Brazilian children populations. 4. Conclusions This was the first study to assess the occurrence and profiles of urinary concentrations of phthalate metabolites in a Brazilian population. The results suggest that Brazilian children are exposed to higher levels of mEP and ∑4DEHP than those previously reported in other populations. Significant differences in phthalate levels were found among the sub-groups of the population (age, gender, and region). PCA analysis showed two potential exposure sources: one dominated by mEP (PCPs) and the second dominated by the secondary metabolites of DEHP, mBP, and mIBP (diet). A risk assessment from phthalate exposures in children showed that more than one-fifth of the Brazilian children are exposed to unsafe levels of phthalates. Positive correlations were found between the urinary concentrations of 8OHDG and several phthalate metabolites. Our findings suggest that phthalate exposure contributes to oxidative stress. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank all Brazilian children for providing urine samples for this study. This research was supported in part by São Paulo Research Foundation (FAPESP, grant numbers 2015/20928-3 and 2013/23710-3). The sample analysis was conducted at Wadsworth Center, New York State Department of Health.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.01.193.

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Please cite this article as: Rocha, B.A., et al., Urinary concentrations of 25 phthalate metabolites in Brazilian children and their association with oxidative DNA damage, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.01.193