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ARTICLE IN PRESSG ModelJHEH-12857; No. of Pages 13

International Journal of Hygiene and Environmental Health xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Hygiene andEnvironmental Health

jo u r n al homepage: www.elsev ier .com/ locate / i jheh

he effects of phthalate and nonylphenol exposure on body size andecondary sexual characteristics during puberty

ia-Woei Houa,b,1, Ching-Ling Linb,c,1, Yen-An Tsaid, Chia-Huang Changd, Kai-Wei Liaod,hing-Jung Yud, Winnie Yange, Ming-Jun Leee, Po-Chin Huangf, Chien-Wen Sung,in-Han Wangh, Fang-Ru Lin i, Wen-Chiu Wuj, Meng-Chih Leek, Wen-Harn Panl,m,ai-Hsiun Chenn,o, Ming-Tsang Wup,q,r, Chu-Chih Chenh, Shu-Li Wangg,hing-Chang Lees,t, Chao Agnes Hsiungu, Mei-Lien Chend,∗

Department of Pediatrics, Cathay General Hospital, Taipei, TaiwanSchool of Medicine, Fu Jen Catholic University, Taipei, TaiwanDepartment of Endocrinology & Metabolism, Cathay General Hospital, Taipei, TaiwanInstitute of Environmental and Occupational Health Sciences, School of Medicine, National Yang-Ming University, Taipei, TaiwanDepartment of Pediatric, Taipei City Hospital, TaiwanNational Environmental Health Research Center, National Health Research Institutes, Miaoli, TaiwanDivision of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, TaiwanDivision of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes, Miaoli, TaiwanDivision of Health Policy Translation, Institute of Population Health Sciences, National Health Research Institutes, Miaoli, TaiwanDepartment of Pediatrics, Taipei Hospital, Ministry of Health and Welfare, Taipei, TaiwanDepartment of Family Medicine, Taichung Hospital, Ministry of Health and Welfare, Taichung, TaiwanDivision of Preventive Medicine and Health Services Research, Institute of Population Health Sciences, National Health Research Institutes, Miaoli, TaiwanInstitute of Biomedical Sciences, Academia Sinica, Taipei, TaiwanDepartment of Laboratory Medicine and Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, TaiwanGraduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, TaiwanDepartment of Public Health, College of Health Sciences, Kaohsiung Medical University, Kaohsiung, TaiwanDepartment of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung, TaiwanCenter of Environmental and Occupational Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, TaiwanDepartment of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, TaiwanResearch Center of Environmental Trace Toxic Substance, National Cheng Kung University, Tainan, TaiwanInstitute of Population Health Sciences, National Health Research Institutes, Miaoli, Taiwan

r t i c l e i n f o

rticle history:eceived 12 February 2015eceived in revised form 27 April 2015ccepted 11 June 2015

eywords:hthalic acid estersonylphenolndocrine-disrupting chemicalsubertal maturitybesity

a b s t r a c t

Background: Some phthalic acid esters (PAEs) and nonylphenol (NP) are endocrine-disrupting chemicals(EDCs) that are widely used in consumer products. Consequently, the general population is exposedsimultaneously to both groups of chemicals.Objective: To investigate the single- and co-exposure effects of PAEs (DMP, DEP, DnBP, DiBP, BBzP, andDEHP) and NP on obesity and pubertal maturity to compare the body sizes of general adolescents withthe complainants of the phthalate-tainted foods scandal that occurred in Taiwan.Methods: This study included 270 general adolescents aged 6.5–15.0 years and 38 complainants aged6.5–8.5 years. Nine metabolites of the five PAEs and of NP were measured in urine. We used a ques-tionnaire to evaluate pubertal maturity, measured anthropometric indices (APs) to assess body size, andcollected urine samples to measure the two groups of chemicals.Results: We found that urinary PAE metabolite concentrations (specifically, metabolites of DEP,

DnBP, DiBP, and DEHP) were positively associated with the APs for abdominal obesity (includ-

Please cite this article in press as: Hou, J.-W., et al., The effects of phthalate and nonylphenol exposure on body size and secondarysexual characteristics during puberty. Int. J. Hyg. Environ. Health (2015), http://dx.doi.org/10.1016/j.ijheh.2015.06.004

ing skinfold thickness, waist circumference, waist-to-height ratio, and waist-to-hip) and indicated adose–response relationship. Mono-methyl phthalate (MMP) exposure was inversely associated withpubarche among boys. The daily intake of DEHP in general adolescents exceeded the reference doses(RfD-20 �g/kg bw/day) and tolerable daily intake (TDI-50 �g/kg bw/day) by 3.4% and 0.4%, respectively.No associations were observed between NP exposure or co-exposure and the APs or pubertal maturity.

∗ Corresponding author at: Institute of Environmental and Occupational Health Sciences, No. 155, Sec. 2, Linong Street, Taipei 112, Taiwan.E-mail address: mlchen@ym.edu.tw (M.-L. Chen).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.ijheh.2015.06.004438-4639/© 2015 Elsevier GmbH. All rights reserved.

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No significant differences were observed between general adolescents and the complainants with regardto weight, height, or BMI.Conclusions: The study suggests that PAE (specifically, DEP, DnBP, DiBP, and DEHP) exposure is associatedwith abdominal obesity in adolescents and that the APs for abdominal obesity are more sensitive thanBMI for measuring obesity among adolescents. We suggest that the RfD and TDI for PAEs should be revisedto provide sufficient protection.

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. Introduction

Phthalates, also called phthalic acid esters (PAEs) are ubiquitoushemicals in the environment. PAEs include both low moleculareight phthalates (LMW, MW <250 g/mol) such as dimethyl phtha-

ate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DnBP),nd di-iso-butyl phthalate (DiBP) and relatively high moleculareight phthalate metabolites (HMW, MW > 250 g/mol) such as

utyl benzyl phthalate (BBzP), and di-ethylhexyl phthalate (DEHP)Hoppin et al., 2013; Wolff et al., 2014). LMW PAEs are commonlysed as stabilizing agents in personal care products such perfumes,

otions, and cosmetics (Schettler, 2006). And HMW PAEs are used aslasticizers or softener in children’s toys, food packaging, and med-

cal devices (Hauser and Calafat, 2005). Several in vitro and vivotudies have suggested that DEP, DnBP, BBzP, and DEHP inhibitedstrogen receptor binding or had anti-androgenic effects (Grayt al., 2006; Harris et al., 1997; Jobling et al., 1995). These endocrine-isrupting chemicals (EDCs) adversely affect the endocrineystem.

On May 23, 2011, the Food and Drug Administration as wells the Ministry of Health and Welfare in Taiwan announced thatEHP and/or di-isononyl phthalate (DiNP) were adulterated as

clouding agent in food products (Li and Ko, 2012). A highoncentration (i.e., 600 ppm) of DEHP was detected in the rawaterials of probiotics. Following this announcement, the pub-

ic were nervous about potential adverse reproductive effects andalformations in infants or children caused by exposure to the

ainted foods. Many complainants sought help from clinics dur-ng this scandal. Wu et al. observed that serum thyroid-stimulatingormone (TSH) levels might be altered when children are exposedo high concentrations of phthalate-tainted food products (Wut al., 2013b).

PAEs are rapidly metabolized to their respective monoestersnd their oxidative products (Koch et al., 2012; Kurata et al.,012; Silva et al., 2003). These metabolites are partially glu-uronidated and excreted through urine and feces. The metabolitesf DMP, DEP, DiBP, DnBP, and BBzP are mono-methyl phthalateMMP), mono-ethyl phthalate (MEP), mono-iso-butyl phthalateMiBP), mono-n-butyl phthalate (MnBP), mono-benzyl phtha-ate (MBzP), respectively. Mono-(2-ethylhexyl) phthalate (MEHP)s formed primarily by the hydrolysis of DEHP and furtherxidized into secondary metabolites, including mono-(2-ethyl--oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-hydroxyhexyl)hthalate (MEHHP), and mono-(2-ethyl-5-carboxypentyl) phtha-

ate (MECPP) (Koch et al., 2005). These four metabolites areenerally measured to determine human exposure to DEHP.

Numerous studies have measured the metabolites of DMP, DEP,nBP, DiBP, BBzP, DEHP, and DiNP (Hatch et al., 2008; Hoppin et al.,013). Epidemiological studies have indicated that high detectionates (>90%) of PAE monoesters were found in DEP, DiBP, DnBP, andEHP among adolescents (Mieritz et al., 2012; Wang et al., 2013).

n Taiwan, MMP, MEP, MnBP, MiBP, MBzP, MEHP, MEOHP, MEHHP,nd metabolites of DiNP have been detected in the urine samples

Please cite this article in press as: Hou, J.-W., et al., The effects of phsexual characteristics during puberty. Int. J. Hyg. Environ. Health (201

f pregnant women and children (Lin et al., 2011). Except for DiNP,he detection rates of these metabolites were above 98.7%. Becausef the common exposure to several PAEs, we generally measured

© 2015 Elsevier GmbH. All rights reserved.

nine metabolites, including MMP, MEP, MnBP, MiBP, MBzP, MEHP,MEOHP, MEHHP, and MECPP simultaneously.

Puberty, a critical stage of human development, is defined asthe transition from childhood to adulthood. This period is markedby significant developmental changes such as cellular proliferationand a rapidly changing metabolism (Woodruff et al., 2010). The ageof pubertal onset has been declining in Western countries and inAsia over the past few decades (Herman-Giddens et al., 2001; Maet al., 2011, 2009; Susman et al., 2010). Early puberty may havelong-term effects such as reproductive cancers (Gail et al., 1989;McGlynn et al., 2007; Moorman et al., 2009), metabolic syndrome(Frontini et al., 2003), and psychological effects (Ge et al., 2003).

The rising prevalence of overweight and obesity has drawn greatattention globally, and these health concerns in children and ado-lescents from developed countries have increased between 1980and 2013. The prevalence (95% uncertainty intervals) of overweightor obesity was from 16.9 (16.1–17.7)% to 23.8 (22.9–24.7)% in boysand from 16.2 (15.5–17.1)% to 22.6 (21.7–23.6)% in girls (Ng et al.,2014). Moreover, Graversen et al. observed that the prevalence ofoverweight or obesity was consistently associated with a highbody mass index (BMI) during preschool years and with adult obe-sity, central obesity and the early onset of metabolic syndrome(Graversen et al., 2014). Overweight children most likely becomeobese adults. Many studies have reported that childhood obesityis a likely risk factor for metabolic syndrome (Lloyd et al., 2012),cardiovascular disease (Lloyd et al., 2010) and cancer (Berentzenet al., 2014; Gascon et al., 2004; Kitahara et al., 2014).

Abdominal obesity is also known as central obesity. The accu-mulation of abdominal fat increases waist size. Several studies havesuggested that abdominal obesity is associated with type 2 dia-betes (Heianza et al., 2014), cardiovascular disease (Shields et al.,2012), and metabolic syndrome (Wu et al., 2011). Furthermore,the Canadian Health Measures Survey found a positive associationbetween abdominal obesity and cardiovascular disease among menand women with normal BMI (Shields et al., 2012). Anthropomet-ric indices (APs) such as BMI (Kavak et al., 2014), hip circumference(HC) (Cameron et al., 2012), and waist circumference (WC) (Ronaet al., 2011) are used as screening tools to predict obesity amongadults and adolescents. However, BMI is age-dependent in adoles-cents (Mayer et al., 2014; Song et al., 2014), which might limit itsuse. Therefore, an index that is more sensitive is needed to detectabdominal obesity among adolescents.

NP is used in industrial processes, and its precursors, NPethoxylates (NPEs), are a nonionic surfactants used in detergents,emulsifiers, and other household products (Soares et al., 2008). Leeet al. (2013) showed that NP concentration in Taiwan river sedi-ments was relatively higher than that in other countries (Fu et al.,2007; Lee et al., 2013; Li et al., 2004). NP is an estrogen mimic thatcan affect the endocrine system by disrupting signal pathways andby interacting with estrogen receptors (Huang et al., 2010; Lawset al., 2000).

Numerous authors have explored the effects of exposure to spe-cific chemicals on body size and secondary sexual characteristics

thalate and nonylphenol exposure on body size and secondary5), http://dx.doi.org/10.1016/j.ijheh.2015.06.004

(SSCs). Epidemiological studies have shown positive associationsbetween MEHP and adipogenesis (Taxvig et al., 2012), LMWmetabolites and BMI (Trasande et al., 2013) and MEHP and MEP

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nd WC in Chinese school children (Wang et al., 2013). MBzP andEHP act as an obesogen by activating of peroxisome proliferator-

ctivated receptors (PPARs) (Hurst and Waxman, 2003). Thus, PAExposure may be associated with obesity. A case–control studyhowed that patients with precocious puberty had higher MnBPnd NP exposure levels in their plasma compared with a controlroup (Yum et al., 2013). Chen et al. noted that NP exposure mightegatively correlate with age at menarche (Chen et al., 2009). Notudy has been published regarding the interaction between PAEnd NP co-exposure and pubertal maturity.

The general population is exposed to numerous environmen-al chemicals simultaneously (Gascon et al., 2014; Shiue, 2014).s mentioned previously, both PAEs (specifically DEP, DnBP, BBzP,nd DEHP) and NP have estrogenic effects that might induce obe-ity. However, few researchers have studied the combined effectsf co-exposure to both groups of chemicals. The objectives of thistudy were to (1) investigate the single and combined effects ofAEs and NP on obesity and pubertal maturity among general ado-escents and (2) compare body size between general adolescentsnd complainants of phthalate-tainted food.

. Materials and methods

.1. Study subjects

This cross-sectional study included 270 adolescents and 38 com-laints and was conducted from May 2012 to February 2013. The70 general adolescents were between 6.5 and 15.0 years old andttended primary schools in Taipei, Taiwan. We recruited these par-icipants using stratified convenience sampling. Two classes fromach grade of the sampled primary schools (one through six) wereelected. All 537 students in the selected classes were invited toarticipate. Of the students invited, 346 students consented; theesponse rate was 64.6%. No significant differences were found withegard to the APs (e.g., weight, height, and BMI) or gender betweenarticipating and non-participating adolescents. Of these 346 par-icipants, 48 did not provide urine samples, 18 were not measuredor APs, and 11 were excluded because their urine creatinine levelsere below 30 mg/dL or higher than 300 mg/dL. Therefore, the data

f 270 participants were included for analysis.The Risk Assessment of Phthalate Incident in Taiwan (RAPIT) is a

rogram that includes studies designed to assess the health statusf the complainants. After the plasticizer contamination scandal,ome people complained to the Consumers’ Foundation as victimsf the plasticizer-tainted foods and filed a lawsuit for compen-ations. The complainants who sought help from the Consumerrotection Committee, Executive Yuan, Taiwan or other agenciese.g., the regional public health bureau) were transferred to spe-ialty clinics at three designated hospitals: the Taiwan’s Ministryf Health and Welfare hospitals in Taipei and Taichung and Kao-siung Medical University Hospital. The victims were invited toarticipate in the study during the clinic visit and encouraged tondergo follow-up health examinations. Thirty-eight complainantsged 6.5–8.5 years from northern, central, and southern Taiwanere included in this study.

The institutional review board of the Cathay General Hos-ital and the Ethics Committee of Taipei City Hospital, Taipeipproved this study. Informed consent was obtained from parentsr guardians and assent was obtained from participants.

.2. Questionnaire and urine sample collection

Please cite this article in press as: Hou, J.-W., et al., The effects of phsexual characteristics during puberty. Int. J. Hyg. Environ. Health (201

All general adolescents provided first morning urine samplesn a 30-mL brown glass bottles. After collection, the samples

ere stored in an ice bucket and transported to the laboratory

PRESSd Environmental Health xxx (2015) xxx–xxx 3

immediately, where they were stored at −20 ◦C until analysis. Thecomplainants’ urine samples were collected at the hospital day andstored at −80 ◦C until analysis.

All participants completed a structured questionnaire regardingsocio-demographic characteristics (age and gender), lifestyle(hours of watching television, using the computer, sleeping, andnumber of supplement taken), and SSCs. The puberty variablesincluded pubic hair for boys and girls and breast developmentand menstruation for girls. The researchers explained the term“secondary sexual characteristics” to participants with the aid ofTanner’s photographs (Marshall and Tanner, 1969, 1970). Partici-pants chose “present” if their SSCs were at a sexual maturity stage2 or above. The age of menarche was determined using the “statusquo method” in which each girl is asked about the date of her firstmenstrual period.

2.3. Anthropometric measurements

Well-trained researchers measured the APs (including weight,height, WC, HC, and skin fold thickness) following standard proce-dures.

The participants wore light clothes and stood straight and bare-foot while being measured. Weight and height were measuredusing an electronic measuring scale with an accuracy of 100 g.

Only general adolescents had WC, HC, and skinfold thicknessmeasurements taken. WC and HC were measured in centimetersusing a plastic tape measure. WC was measured at the midpointbetween the lowest rib and the iliac crest. HC was measured at thelargest point of the gluteal region. Skinfold thickness was measuredat the back of right upper arm using a Skinfold Slim Guide Caliper(Creative Health Products, MI, USA) to the nearest 1 mm.

BMI was calculated as weight (kg) divided by height squared(m2) and categorized according to the BMI-based age- andgender-specific criteria proposed by Taiwan’s Health PromotionAdministration and by the Ministry of Health and Welfare (Taiwan’sHealth Promotion Administration, 2014).

The waist-to-height ratio (WHeitR) was defined as the WCdivided by the height. The waist-to-hip ratio (WHiptR) was definedas the WC divided by the HC.

2.4. Urinary PAE metabolites, NP, and creatinine analysis

2.4.1. ReagentsMMP, MEP, MiBP, MnBP, MBzP, MEHP, MEOHP, MEHHP, MECPP

(>99%) and their 13C4 labeled internal standard (>99%) of thesenine phthalate monoesters were purchased from Cambridge Iso-tope Laboratories, Inc. (Andover, MA, USA). 4-n-Nonylphenol waspurchased from Fluka (Japan).

Ammonium acetate (>98%), acetonitrile (LC/MS grade), ethylacetate (>99.8%), hydrochloric acid (37%) and formic acid (>98%)were purchased from Merck (Darmstadt, Germany). NaH2PO4and �-glucuronidase/arylsulfatase were purchased from SigmaAldrich Laboratories, Inc. (St. Louis, MO, USA). Phosphoric acid(85%) was purchased from J.T. Baker (Phillipsburg, NJ, USA). �-glucuronidase (Escherichia coli-K12) was purchased from RocheBiomedical (Mannheim, Germany).

2.4.2. Urinary PAEs metabolites analysisNine metabolites including MMP, MEP, MiBP, MnBP, MBzP,

MEHP, MEOHP, MEHHP, and MECPP were measured in urine sam-ples. The analytical method was based on ultra-performance liquid

thalate and nonylphenol exposure on body size and secondary5), http://dx.doi.org/10.1016/j.ijheh.2015.06.004

chromatography–tandem mass spectrometry (UPLC–MS/MS) withisotope dilution for the quantitative detection of several phthalatemetabolites in human urine (Blount et al., 2000; Kato et al., 2005;Silva et al., 2007).

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ARTICLEJHEH-12857; No. of Pages 13

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In total, 200 �L of urine was thawed, vortex mixed, sonicated for min, and buffered with 250 �L ammonium acetate (1 M, pH 6.5).he urine was then spiked with 40 �L of a mixture of stable isotopeabeled (500 ng/mL) and 5 �L �-glucuronidase. After the additionf �-glucuronidase, the urine was incubated at 37 ◦C for 90 min forhe hydroly of glucuronide.

After incubation, the urine was diluted with 1 mL phosphateuffer (0.14 M NaH2PO4 in 0.85% of phosphoric acid). Then, therine was loaded onto a solid-phase extraction cartridge (SPE)OASIS-HLB 60 mg/3 mL, Nihon Waters Co. Ltd., Tokyo, Japan),hich was sequentially equilibrated with 1 mL acetonitrile (ACN),

mL H2O, and 1 mL phosphate buffer. After washing the cartridgeith 2 mL 0.1 M formic acid and 1 mL of H2O, monoesters were

luted with 2 mL ACN followed by 2 mL of ethyl acetate. This eluteas evaporated to dryness under dry nitrogen at 45 ◦C. Then, 1 mL

f H2O was added to the residue. Next, the solution was trans-erred to a glass vial, filtered through a 0.22 �m PTFE membranelter (Titin, USA) and analyzed using UPLC-MS/MS. In total, 5 �L ofach sample was injected into an ultra-performance liquid chro-atography system (UPLC; Acquity UPLC, Waters) coupled with

triple-quadruple mass spectrometer (Xevo TQ-S, Waters). Ana-ytes were separated on an ACQUITY UPLC BEH Phenyl Column1.7 �m, 150 mm × 2.1 mm, Waters, USA). Chromatography waserformed using a gradient program with mobile phase A (1 mLcetic acid in 1 L ACN) and mobile phase B (1 mL acetic acid in

L H2O).In addition to performing each urine analysis, the absolute

ecoveries were used for quality control by spiking a knownmount of PAE metabolites in human pooled urine. The aver-ge recoveries of MMP, MEP, MiBP, MnBP, MBzP, MEHP, MEOHP,EHHP and MECPP were 89–90%, 89–91%, 97–110%, 99–107%,

07–114%, 97–98%, 100–101%, 145–159%, and 99–102%, respec-ively, for PAE concentrations of 10–100 ng/mL. The detection limitsf these molecules were 0.39, 0.06, 0.25, 0.10, 0.19, 0.26, 0.42,.43, and 0.27 ng/mL, respectively. To minimize a potentially sys-ematic drift, all the urine samples analyses were completed inanuary 2014. The intra- and inter-day relative standard devia-ion (RSD) ranges were 0.78–10.49% and 3.57–12.23%, respectivelyTable S1).

.4.3. Urinary NP analysisThe analytical method employed was identical to the pre-

ious study (Chang et al., 2013). In brief, 10 mL of urineas thawed, adjusted to pH 5.5 and mixed it with 1 mL

mmonium acetate (1 M, pH 5.3). Urine samples were deconju-ated with 125 �L �-glucuronidase/arylsulfatase and incubatedt 37 ◦C for 15 h in a shaker bath. After incubation, the sam-les were acidified to pH 3. Deconjugated samples were cleanedith Supelco SPE (pH, 500 mg/3 mL, USA) and analyzed usingigh-performance liquid chromatography coupled with fluores-ent detection (HPLC-fluorescence; Hitachi, Tokyo, Japan). Theverage recoveries of NP ranged from 84 to 94% for NP con-entrations of 10–250 ng/mL. The detection limit of NP was.2 ng/mL.

.4.4. Urinary creatinine analysisThe urinary creatinine analytical method was based on the Hine-

ard and Tiderstrom’s modification of the Jaffe reaction. In brief,.1 mL of the10-fold diluted sample was added to 3 mL of 3.3 mM

Please cite this article in press as: Hou, J.-W., et al., The effects of phsexual characteristics during puberty. Int. J. Hyg. Environ. Health (201

icric acid and base reagent (0.17 M sodium hydroxide and 26 mModium tetraborate) mixed, and incubated at 37 ◦C for 15 min. Then,he sample was measured with a spectrophotometer at a wave-ength of 510 nm.

PRESSd Environmental Health xxx (2015) xxx–xxx

2.5. Estimation of daily phthalate intake

The average daily intake (DIENT) was estimated using the fol-lowing model described in the literature (Chen et al., 2008; Kochet al., 2003):

DIENT (�g/kg body weight/day)

= UE (�mol/g) × CE (g/day)Fue × BW (kg)

× MW

where UE (�mol/g creatinine) is the urinary concentration of themetabolites adjusted for creatinine; CE (g creatinine/day) is thedaily creatinine excretion rate; BW (kg) is the participant’s bodyweight; MW is the molecular weight of PAEs; and Fue is the excre-tion fractions based on studies conducted after oral intake of theparent phthalates, which included 69% of DnBP excreted in theurine as MnBP (Anderson et al., 2001), 70.3% of DiBP excreted asMiBP (Koch et al., 2012), 73% of BBzP excreted as MBzP (Andersonet al., 2001), and 44.2% of DEHP excreted as the sum of the DEHPmetabolites (Koch et al., 2005). The rate of DEP excreted as MEPin humans is unknown. Therefore, the value initially estimated forDnBP was selected for DEP (Soeborg et al., 2012; Wittassek et al.,2011).

The overall average daily intake (DIALL) of the complainants wasestimated by adding their intake of the tainted foods ascertainedusing a questionnaire and their DIENT. A panel of experts validatedthe questionnaire.

2.6. Statistical analyses

To describe participant characteristics, the data were reportedas the percentage and mean ± SD. The levels of APs and PAE metabo-lite concentrations were compared between general adolescents(aged 7–8 years) and the complainants using the Mann–WhitneyU test. We combined the phthalate metabolites in which the fourmetabolites of DEHP (MEHP, MEOHP, MEHHP, and MECPP) weresummed as total DEHP (�DEHP); LMW phthalate metabolites werethe sum of MMP, MEP, MiBP, and MnBP; HMW phthalate metabo-lites were the sum of �DEHP and MBzP, and �PAEs were the sumof the LMW and HMW metabolites (Hoppin et al., 2013; Wolff et al.,2014).

Multivariable linear or logistic regression models were used toinvestigate the relationships between the phthalate metabolite orNP concentrations and the APs and SSCs of general adolescentsafter adjusting for covariates. The APs covariates included age,gender and urinary creatinine; the SSCs covariates included ageat menarche, maternal age at menarche and urinary creatinine.Dose–response relationships were tested by dividing the phtha-late metabolites and NP concentrations in ng per mL into <25thpercentile, 25–75th percentile and <75th percentile by employ-ing multivariate linear or logistic regression models. Interactioneffects were tested by dividing the phthalate metabolites and NPconcentrations into the lower 50th percentile and upper 50thpercentile by employing multivariate linear or logistic regressionmodel.

The analyses were considered statistically significant whenp < 0.05. All analyses were performed using SPSS 17.0 (SPSS Inc.,Chicago, IL, USA).

3. Results

The mean age of the 270 general adolescents was 9.56 ± 1.81

thalate and nonylphenol exposure on body size and secondary5), http://dx.doi.org/10.1016/j.ijheh.2015.06.004

years, and 43.7% of the students were females (n = 118). The preva-lence rates of underweight, normal weight, and overweight were11.9%, 66.7%, and 21.5%, respectively. The mean ± SD for WC, HC,skinfold thickness, WHeitR, and WHiptR were 62.76 ± 9.43 cm,

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Table 1Demographic characteristics for general adolescents and the complainants.

Variables Generaladolescents(n = 270)

The complainants(n = 38)

p Valueb

n (%) n (%)

GenderMale 152 (56.3) 26 (68.4)Female 118 (43.7) 12 (31.6)

Agea 9.56 ± 1.81≤7 44 (16.3) 26 (68.4)8 48 (17.8) 12 (31.6)9 47 (17.4)10 38 (14.1)11 39 (14.4)≥12 54 (20.0)

Anthropometric indices (All)Weighta 34.03 ± 10.79Heighta 137.65 ± 12.27BMIa 17.58 ± 3.43

Under weight 32 (11.9)Normal 180 (66.7)Over weight 58 (21.5)

Waist circumference (cm)a 62.76 ± 9.43Hip circumference (cm)a 75.41 ± 9.37Waist-to-height ratioa 0.46 ± 0.05Waist-to-hip ratioa 0.83 ± 0.06Skinfold thickness (mm)a 15.23 ± 5.49

Anthropometric indices (7–8 age)Weighta 26.59 ± 7.00 25.60 ± 5.52 0.68Heighta 126.03 ± 6.28 124.74 ± 6.30 0.39BMIa 16.58 ± 3.24 16.32 ± 2.43 0.83

under weight 10 (11.2) 4 (10.5)normal 62 (69.7) 28 (73.7)over weight 17 (19.1) 6 (15.9)

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a Mean ± SD.b Compared anthropometric indices with general adolescents and the complaina

5.41 ± 9.37 cm, 15.23 ± 5.49 mm, 0.46 ± 0.05, and 0.83 ± 0.06,espectively. No significant differences between general adoles-ents and the complainants were found with regard to weight,eight, or BMI (Table 1).

Table 2 shows the distributions of urinary PAE metabolites andP concentrations (ng/mL) for participants (Tables S2 and S3 show

he distributions in �g/g creatinine and nmole/g creatinine, respec-ively). The lowest detection rate was 78.1% for MEHP, and the other

etabolites were detected at 94.4% (MBzP) or above. The geometriceans (ng/mL) from highest to lowest were MnBP (53.40 ng/mL),ECPP (49.12 ng/mL), MEHHP (35.68 ng/mL), MiBP (32.90 ng/mL),EP (25.07 ng/mL), MEOHP (23.78 ng/mL), MEHP (13.85 ng/mL),MP (8.17 ng/mL), MBzP (4.13 ng/mL), and NP (3.98 ng/mL). Fig. 1

ompares the distributions of log urinary PAE metabolite concen-rations between general adolescents and the complainants. Therinary PAE metabolite concentrations for the 7- to 8-year-olds inhe former group were not significantly different from those of theatter group.

Fig. 2 shows the association between PAE metabolites and thePs among general adolescents. With regard to PAE metabolite con-entrations below 25th percentile, the age- and gender-adjustedPs (excluding HC) significantly increased among general adoles-ents with 25–75th percentile PAEs and those with >75th percentileAEs in a dose–response relationship (p < 0.05). The detailed asso-iations are as follows: MEP, MiBP, MEOHP, MEHHP, MECPP, andMW were positively associated with WC; MEP, MEOHP, MEHHPnd LMW were positively associated with skinfold thickness; MEP,iBP, MEOHP, MEHHP, MECPP, LMW, and �PAEs were positively

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ssociated with WHeitR; and MEP, MiBP, MnBP, MEOHP, MEHHP,MW, and �PAEs were positively associated with WHiptR; MEPnd MEHHP were positively associated with BMI (Tables S3 and4: model 2).

o aged 7–8 years.

The association between PAE metabolites and SSCs by genderin general adolescents is shown in Fig. 3. The occurrence of pubichair among males from the general population with MMP con-centrations of >50th percentile was less likely than were thosewith concentrations of <50th percentile (odds ratio = 0.07, 95%CIs = 0.01–0.70).

No associations were found between urinary NP concentrationsand anthropometric indices or between these concentrations andSSCs (Figs. 2 and 3). We also did not find interactions between PAEand NP co-exposure and the anthropometric indices or betweenco-exposure and secondary sexual characteristics (data not shown).

Table 3 presents the estimated daily intake for the complainantsand general adolescents as well as the percentage of daily intakeexceeding reference doses (RfD) or tolerable daily intake (TDI). Themedian of DEHP daily intake for DIENT and DIall were 4.78 and16.96 �g/kg bw/day, respectively, among complainants. The medi-ans of DIENT for DEHP, DnBP, BBzP, and DEP were 6.06, 1.79, 1.91,and 1.44 �g/kg bw/day, respectively, for general adolescents. Thepercentages of DEHP DIENT that exceeded the RfD for the com-plainants and general adolescents were 2.6% and 3.4%, respectively.In total, 42.1% of the complainants exceeded RfD in DIall.

4. Discussion

In the present study, a dose–response relationship was foundbetween urinary PAE metabolite concentrations (specifically,metabolites of DEP, DnBP, DiBP, and DEHP) and APs (except HC)after adjusting for the confounders.

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Several in vitro or in vivo studies have suggested that PAE expo-sure promotes obesity by activating PPARs (Hurst and Waxman,2003), affecting the thyroid (Ishihara et al., 2003), or via an anti-androgenic effect (Gray et al., 2006). PPARs play a major role

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Table 2Distributions of urinary PAEs metabolites and NP concentrations.

Metabolites (ng/mL) Detection rate (%) Mean ± SD GM min Percentile max

25th 50th 75th

PAEsMMP 94.8 16.99 ± 27.28 8.17 0.12 4.95 9.51 18.27 242.70MEP 98.5 59.84 ± 142.19 25.07 0.12 11.96 23.31 48.43 1618.98MiBP 99.6 47.06 ± 61.64 32.90 0.12 21.46 31.14 46.67 527.42MnBP 99.6 75.42 ± 83.71 53.40 0.12 30.10 52.85 95.75 1064.23MBzP 94.4 11.58 ± 33.00 4.13 0.12 1.92 3.97 8.30 419.78MEHP 78.1 47.07 ± 42.41 13.85 0.18 10.04 35.42 87.08 219.03MEOHP 99.6 33.08 ± 29.94 23.78 0.12 16.43 27.03 41.00 333.84MEHHP 100.0 49.59 ± 49.64 35.68 0.23 23.49 37.72 60.30 604.70MECPP 100.0 63.99 ± 56.99 49.12 0.60 31.70 49.93 77.63 638.39DEHP 100.0 193.73 ± 153.63 152.55 2.59 100.74 168.87 237.19 1751.63LMW 100.0 199.31 ± 199.74 148.34 12.74 86.46 145.03 241.56 1705.04HMW 100.0 205.31 ± 161.95 161.72 2.83 107.72 181.49 253.65 1754.54�PAEs 100.0 404.62 ± 280.90 335.31 59.50 221.65 342.15 502.90 2010.48

NP 96.7 5.03 ± 3.75 3.98 0.30 2.87 3.91 6.30 28.96

Fig. 1. Box and whisker plots of log urinary PAE metabolite concentrations for general adolescents (aged 7–8 years) and the complainants. © minor outlier, � extreme outlier.

Table 3Estimated daily intake for the complainants and general adolescents and percentage of daily intake exceeded RfD or TDI.

Chemicals Distribution (�g/kg bw/day) >RfD (%) >TDI (%)

Mean ± SD Minimum Median Maximum 95th percentile

The complainants (n = 38)DIENT

DEHP 6.70 ± 5.05 2.10 4.78 22.97 19.02 2.6 0.0DIALL

DEHP 24.97 ± 28.61 3.00 16.96 159.40 97.18 42.1 7.9

General adolescents (n = 270)DIENT

DEHP 7.41 ± 6.78 0.03 6.06 83.58 16.35 3.4 0.4DnBP 2.53 ± 2.38 0.01 1.79 17.76 7.10 0.0 0.0DiBP 2.49 ± 2.34 0.01 1.76 17.43 6.97 – –BBzP 2.69 ± 2.53 0.01 1.91 18.84 7.53 0.0 0.0DEP 2.02 ± 1.90 0.01 1.44 14.18 5.67 0.0 0.0

RfD (Reference doses) for each chemical (�g/kg bw/day): DEHP-20, DnBP-100, BBzP-200, DEP-800, and DiBP-not applicable.TDI (Tolerable daily intake) for each chemical (�g/kg bw/day): DEHP-50, DnBP-10, BBzP-500, DEP- not applicable, and DiBP-not applicable.

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Fig. 2. Association between phthalate metabolites or NP concentration (The urinary concentrations were categorized by quartile into <25 the percentile, 25–75 the percentile,and >75 the percentile. The lowest concentration group was as reference) and anthropometric indices in general adolescents. Estimates in (A), (B), and (C) are from multivariablelinear regression models (beta, 95% CIs) adjusted for age, gender, and creatinine. Estimates in (D), (E), and (F) are from multivariable logistic regression models (ORs, 95% CIs)

adjusted for age, gender, and creatinine. ♦ beta or odds ratio (OR), represent 95% CIs, *p for trend <0.05.

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Fig. 2. (Continued ).

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Fig. 3. Association between phthalate metabolites or NP concentration (The urinary concentrations were categorized by median into <50 the percentile and >50 the percentile.The lowest concentration group was as reference) and secondary sexual characteristics by gender in general adolescents (♦ beta or odds ratio (OR), error bars represent95% CIs). (A) Estimates in breast development, pubic hair, and menstruation status in girls are from multivariable logistic regression models (ORs, 95% CIs) adjusted for age,age of menarche, and creatinine in mother. Estimates in age of menarche are from multivariable linear regression models (beta, 95% CIs) adjusted for age, age of menarche,and creatinine. (B) Estimates in pubic hair status in boys are from multivariable logistic regression models (ORs, 95% CIs) adjusted for age, age of menarche in mother, andcreatinine. *p < 0.05.

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n controlling adipocyte proliferation and differentiation as wells serve as metabolic sensors for lipophilic hormone, fatty acid,nd fatty acid metabolites (Hauser and Calafat, 2005). Three dif-erent PPAR isotypes, PPAR�, �, and �, have been identified inumans, rats, and chickens, respectively. Meeker and Ferguson2014) reported that DEHP monoesters are negatively associatedith serum testosterone levels in boy (6–12 years old) (Meeker

nd Ferguson, 2014). However, PAEs do not directly interact withndrogen receptors; their anti-androgen effects are mediated viaPAR�. In PPAR�-humanized mice, exposure to DEHP can increaseeight gain and exacerbate obesity (Feige et al., 2010).

Epidemiological studies have revealed evidence of a negativeelationship between testosterone and obesity among men (Brandt al., 2011). The anti-androgenic effects mediated through PPAR�ight be a mechanism of PAEs-induced obesity. PPAR� plays a

ey role in regulating the differentiation of adiopocytes and fattorage in the adipose tissue (Tontonoz and Spiegelman, 2008).he activation of PPAR� promotes the differentiation of preadiposeells into adipocytes (Feige et al., 2007). Taxvig et al. showed thatEHP caused increased adipogenesis (Taxvig et al., 2012). The adi-

ogenic effect of PAEs might occur through the activation of PPAR�r the inhibition of TSH. Thyroid function regulates energy bal-nce and metabolism. Recent studies have demonstrated possibleffects of metabolites of DEHP on the thyroid function of humansBoas et al., 2010; Wu et al., 2013b). Obesity is a proinflammatorytate caused when adipose tissues release and secrete proinflam-atory cytokines, adipokines, and free fatty acids (Huang et al.,

009). When a positive energy balance exists, excess free fattycids are primarily stored in adipose tissue. When adipose tissueeaches its maximal storage capacity, lipids begin to accumulate inctopic sites (e.g., visceral adipose tissue, renal sinus, and myocar-ial fat, and so on) (Slawik and Vidal-Puig, 2007). Thus, a consistentxposure to PAEs and abdominal obesity occur among general ado-escents.

In adolescent students, despite the low percentage of DIENT thatxceeded the RfD, we found a positive association between uri-ary PAE metabolite concentrations and the APs, particularly in WC,kinfold thickness, WHeitR and WHiptR. These results are consis-ent with previous studies (Wang et al., 2013) but differed fromhe finding of Hatch et al. (2008). Those authors found negativessociations between PAE metabolites in urine and BMI and WC inhe National Health and Nutrition Examination Survey (NHANES)999–2002. Although BMI is the most widely used index of obesity,

t does not provide information regarding the distribution of bodyat (Janssen et al., 2011). Conversely, abdominal obesity is a moreensitive index that includes measures such as WC, WHeitR, and

HiptR (Janssen et al., 2002; Zhang et al., 2014). These indices arelso better predictors of future cardiovascular risk among adults.ur study used WC, skinfold thickness, WHeitR, and WHiptR tovaluate abdominal obesity and found it to be a superior indicatorompared with BMI and HC.

Although 42.1% of DEHP DIall exceeded RfD because of thencreased exposure to tainted foods among the complainants, webserved that the concentration of urinary DEHP metabolites asell as the weight, height, and BMI of general adolescents didot significantly differ from those of the complainants 13 monthsfter the tainted foods scandal. Wu et al. studied 60 childrenho might have been exposed at a six-month follow-up assess-ent. These authors found that the levels of the DEHP metabolite

ecreased significantly (Wu et al., 2013a). The observed decreasen the DEHP metabolites at the a six-month follow-up assessment

ight be because of the short half-lives of the five major urinary

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EHP metabolites (MEHP, MEOHP, MEHHP, MECPP, and mono-(2-carboxymethyl)hexyl) phthalate (2cx-MMHP)), which are 5, 10,0, 12–15 and 24 h, respectively (Koch et al., 2004, 2005). Thus, weere unable to find a difference in the urinary PAE metabolites

PRESSd Environmental Health xxx (2015) xxx–xxx

between the complainants and general adolescents. No signifi-cant between-group differences in weight, height, or BMI wereobserved, which might be because of the limited sample size orlack of sensitivity among the indices with regard to predictingobesity. The residual effects of exposure to high concentrations ofPAE-tainted food products must be clarified.

We could not find that higher PAE exposure correlated with adecreased age at menarche. Several human studies have suggestedthat DEHP has estrogenic effect (Cobellis et al., 2003; Hokansonet al., 2006). PAEs have a weak estrogenic potency compared withestradiol and other estrogenic substances (Jobling et al., 1995).Jobling et al. examined the ability of PAEs to stimulate the transcrip-tional activity of the estrogen receptor. These authors used a lowerconcentration of 17 �-estradiol to compete for receptor binding.In the presence of estradiol, DBP and BBzP increased the transcrip-tional activity of the estrogen receptor (Jobling et al., 1995). Chenet al. showed that PAE metabolite (MEP, MBzP, MEHP, MEHHP, andMEOHP) concentrations in the urine were significantly higher inyouths with central precocious puberty than in a control group(Chen et al., 2013). One reason for this could be that sample size ofour subjects who presented menstruation was too small to reflectthis association.

Our data suggest that higher MMP exposure delays pubarche inboys. Many studies have found that PAEs have an anti-androgeniceffect. Wolff et al. reported that a higher exposure to LMW PAEswas associated with later pubic hair development (Wolff et al.,2010). These consistent findings suggest that PAEs may have ananti-androgenic effect.

Urine is an extensively used matrix for biomonitoring non-persistent chemical. The primary advantage of urine is that isconvenient to collection. However, the disadvantages of using spoturine samples such first-morning void or convenient samples forbiomonitoring include the variability in the urinary flow rate (UFR)and concentration, which is influenced by hydration status. Tocorrect the hydration status, the conventional methods are creati-nine correction. Several studies have been published regarding thedetermination of urinary creatinine.

In addition to creatinine excretion and urine flow being affectedby anthropometric parameters in children, water intake also has aprofound effect on creatinine concentrations and is a confounderof PAEs metabolites concentrations. Remer et al. showed that 24-h urinary creatinine excretion is positively associated with age(r = 0.86) and height (r = 0.93) (Remer et al., 2002). Mage et al.developed an equation to predict a healthy non-obese person’screatinine excretion (Mage et al., 2004, 2008). The parametersof this equation include weight, height, and age. Barr et al. sug-gested that a straightforward solution is to separate the urinarychemical concentration from the urinary creatinine level. In mul-tiple models, the chemical concentration and urinary creatininecould be included as covariates in model. Therefore, associationsbetween the health outcome and chemical concentration may notbe influenced by the correlation with creatinine (Barr et al., 2005).Thus, we stratified PAE metabolite concentrations into <25th per-centile, 25–75th percentile and <75th percentile, and performedthree regression models: unadjusted (model 1: in ng/mL), cre-atinine adjusted (model 2: creatinine as a model covariate), andcreatinine corrected (model 3: in �g/g creatinine). The results arepresented in Tables S4–S7. In model1, we could not address physio-logically “concentrated” or “dilute” urine. In model 3, when we usedthe unit of independent variable as �g/g creatinine, the creatinine-corrected urinary level may be a significant predictor of healthoutcome and indicate a creatinine-associated of health effect.

thalate and nonylphenol exposure on body size and secondary5), http://dx.doi.org/10.1016/j.ijheh.2015.06.004

The results regarding the findings for WC, HC, skinfold thick-ness, WHeitR, WHiptR and SSCs (breast development and pubichair among girls; pubic hair among boys) remained unchanged inthe three models. The only inconsistence found was regarding the

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ssociation between PAE metabolite levels and the age of menstru-tion. Models 1 and 2 did not suggest that DEHP has a significantffect on the menarcheal age. By contrast, significant associationsetween MEP and MEHHP and BMI were found in models 1 and

consistent with previous studies (Hays et al., 2015). Hays et al.ound that the urinary BPA concentration in adults increased sig-ificantly with BMI, but that this association disappears whenorrected for creatinine (Hays et al., 2015). Some studies haveentioned that creatinine excretion in children is considerably

nfluenced by development status, age, and height etc. These resultsuggested that the overadjustment of creatinine may affect thessociation.

The US Environmental Protection Agency established the RfDsf DEP, BBzP, DBP, and DEHP as 800, 200, 100, and 20 g/kg bw/day,espectively (US EPA, 2007a,b,c,d). The European Food Safetyuthority (EFSA) established the TDI of BBzP, DBP, and DEHP at00, 10 and 50 g/kg bw/day, respectively (EFSA, 2005a,b,c). In ourtudy, the percentages of general adolescents with DEHP DIENT thatxceeded the RfD or TDI were 3.4% and 0.4%, respectively. In spitef the low percentage of exceedance, we nevertheless found thessociations between urinary PAE metabolite concentrations andPs as well as between these concentrations and SSCs. These find-

ngs suggest that the RfD and TDI values of PAEs need to be revisedo provide a higher margin of protection.

Recent literatures have suggested that either PAE or NP pro-otes adipocyte differentiation and induces obesity in mice (Hao

t al., 2012a,b); furthermore, these chemicals may have estrogenicffects (Hokanson et al., 2006; Olsen et al., 2005). Muller et al. con-ucted a toxicokinetics study regarding NP exposure in human.hese authors found that NP bioavailability after oral applicationas approximately 20%. Approximately 10% of the applied dose was

xcreted as parent NP or NP resulting from cleavage of glucuronidend sulfate conjugates in urine (Muller et al., 1998). In our study,oth free NP and conjugates NP were determined. The biomarker ofP is reliable. The percentage of phthalate excreted as monoestershthalate ranged from 44.2% to 73%. The biomarkers of both groupsf chemicals highly correlated with exposure. However, few stud-es have examined the combined effect of NP and PAEs with regardo APs and SSCs. Hu et al. suggested that the combination of NP andnBP has an antagonistic effect on Sertoli cells and serum repro-uctive hormones (Hu et al., 2014). The high detection rates of PAEetabolites and NP in general adolescents indicate that adolescent

tudents are exposed to many chemicals simultaneously. The con-entrations of urinary NP were higher than were those reported inther studies (Calafat et al., 2005; Chen et al., 2009; Pirard et al.,012). The levels of PAE metabolites were 2- to 14-fold higher inhese studies than the NP levels in the present study (Table 2). How-ver, urinary PAE and NP concentrations did not correlated (dataot shown), thus indicating the different sources of these EDCs.herefore, we were unable to find an interaction between PAEs andP with regard to either the APs or SSCs.

This study has some limitations. First, because of its cross-ectional study, a causal relationship between PAE exposure andbesity cannot be determined. Second, we assumed that a singleorning spot urine sample would represent the long-term expo-

ure of adolescents to PAEs. Although several studies have indicatedhat a spot urine can represent of long-term exposure due to gen-ral unchanged daily habits (Preau et al., 2010; Watkins et al.,014), the variability of the urinary PAE concentrations might biashe results. Third, we used a structured questionnaire to collectata regarding secondary sexual characteristics; therefore, under-r over-reporting these characteristics could not be avoided. Sev-

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ral researchers have evaluated sexual maturity via pediatriciansxaminations; alternatively, well-trained examiners have usedhotos from Marshall and Tanner (1970) (Ma et al., 2009, 2011;usman et al., 2010). Our study found a consistent prevalence of

PRESSd Environmental Health xxx (2015) xxx–xxx 11

pubertal maturity among urban Chinese girls and boys (Ma et al.,2009, 2011).

5. Conclusions

Our results support the hypothesis that a dose–response rela-tionship exists between urinary PAE metabolite concentrations andskin fold thickness, WC, WHeitR, and WHiptR. In addition, MMPexposure affected pubertal development among boys. The DIENT ofgeneral adolescents exceeded the RfD and TDI by 3.4% and 0.4%,respectively. These low percentages, combined with the observedsignificant effects of PAEs exposure on the APs and SSCs, suggestthat the RfD and TDI of PAEs need to be revised to provide a highermargin of protection. The high detection rates of PAE metabolitesand NP among general adolescents indicate that youths are exposedto many chemicals simultaneously. The levels of PAE metaboliteswere 2- to 14-fold higher than were those of NP, and these EDCsdid not correlated, indicating that their pollution sources differed.No interaction of co-exposure to the two chemicals was observed.

Although 42.1% of DEHP DIall exceeded the RfD because of theadditional exposure caused by consuming tainted foods, signifi-cant differences were not observed between general adolescentsand the complainants with regard to weight, height, or BMI. Theseresults indicated that these indices may lack the sensitivity neededto measure obesity in adolescents.

Acknowledgments

This study was financially supported by the Ministry of Scienceand Technology of the Republic of China, Taiwan (NSC 99-2314-B-010-018-MY3, NSC 100-2314-B-281-001-MY3, NSC 101-2314-B-281-001-MY3) and by the Ministry of Health and Welfare of Taiwan.The authors thank Ms. Tai-Chen Tsai, Wei-Shun Chao, Zi-Yu Hsieh,Pei-Jung Chen and En-Yi Lin and Mr. Meng-Huan Yang for helpingto collect the interview data and to conduct the field work. Theauthors also thank the cooperation of the staff of the Pediatricsand Endocrinology & Metabolism Departments of Cathay GeneralHospital in Taipei. This manuscript has been edited by AmericanJournal Experts.

Appendix A. Supplementary data

Supplementary material related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijheh.2015.06.004

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