Science of the Total Environment 470–471 (2014) 726–732

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

Bisphenol A and cardiometabolic risk factors in obese children Naila Khalil a,⁎,1, James R. Ebert b,1, Lei Wang c, Scott Belcher d, Miryoung Lee e, Stefan A. Czerwinski f, Kurunthachalam Kannan c a

3123 Research Blvd, Suite #200, Center for Global Health, Department of Community Health, Boonshoft School of Medicine, Wright State, University, Dayton, OH, USA The Pediatric Lipid Clinic, the Children's Medical Center of Dayton, One Children's Plaza, Dayton, OH 45404, USA Wadsworth Center, New York State Department of Health and Department of Environmental Health Sciences, State University of New York, Albany, NY 12201-0509, USA d 231 Albert Sabin Way, University of Cincinnati, Cincinnati, OH 45267-0575, USA e Community Health and Pediatrics, Wright State University, 3171 Research Blvd. Dayton, OH 45420-4006, USA f Community Health, Wright State University, 3171 Research Blvd. Dayton, OH 45420, USA b c

H I G H L I G H T S • • • •

Cross sectional study of 39 obese and overweight children aged 3–8 years Urinary BPA (u-BPA) measured by liquid chromatography-tandem mass spectrometry Association between u-BPA and obesity analyzed by linear regression, spline analyses U-BPA concentration in male obese children was associated with adverse liver and metabolic effects and high diastolic blood pressure

a r t i c l e

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Article history: Received 11 July 2013 Received in revised form 26 September 2013 Accepted 26 September 2013 Available online 30 October 2013 Editor: Frank Riget Keywords: Bisphenol A Endocrine disruptor Non-monotonic dose response Childhood obesity Nonalcoholic fatty liver disease Spline analysis

a b s t r a c t Background and objective: Bisphenol-A (BPA) is an endocrine disruptor (ED) that has been associated with obesity and metabolic changes in liver in humans. Non-alcoholic fatty liver disease (NAFLD) affects 40% of all obese children in the United States. Association of BPA with NAFLD in children is poorly understood. We investigated if BPA might play a role. Methods: In a cross sectional study of 39 obese and overweight children aged 3–8 years enrolled from the Children Medical Center of Dayton, Ohio, anthropometric, clinical and biochemical assessment of serum samples were conducted. Urinary BPA was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was adjusted for urinary creatinine BPA (creatinine) using linear regression and spline analyses. Results: Higher urinary BPA (creatinine) concentration in overweight and obese children was associated with increasing free thyroxine. In male children BPA (creatinine) decreased with age, and was associated with elevated liver enzyme aspartate aminotransferase and diastolic blood pressure. The association of BPA (creatinine) persisted even after adjusting for age and ethnicity. Also in males, BPA concentration unadjusted for creatinine was significantly associated with serum fasting insulin and homeostasis model assessment for insulin resistance (HOMA-IR) showing non-monotonic exposure–response relationship. Conclusion: Urinary BPA in obese children, at least in males is associated with adverse liver and metabolic effects, and high diastolic blood pressure. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Obesity in children is a major public health concern. Early life obesity not only tracks to adulthood, increasing the risk of metabolic and cardiovascular disease (CVD) but also is associated with liver abnormalities

⁎ Corresponding author. Tel.: +1 937 258 5559; fax: +1 937 258 5544. E-mail addresses: [email protected] (N. Khalil), [email protected] (J.R. Ebert), [email protected] (S. Belcher), [email protected] (M. Lee), [email protected] (S.A. Czerwinski), [email protected] (K. Kannan). 1 Both authors contributed equally to the manuscript. 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.09.088

including non-alcoholic fatty liver disease (NAFLD). NAFLD affects 40% of obese children (Schwimmer et al., 2006). In addition to lifestyle factors, environmental chemicals acting as endocrine disruptors have been thought to play a role in childhood obesity (Newbold et al., 2009; DiVall, 2013) and NAFLD (Polyzos et al., 2012). Bisphenol A (BPA), a high production industrial chemical and component of polycarbonate plastics is ubiquitous in the environment (CDC, 2013). BPA is considered an endocrine-disrupting chemical (EDC) with estrogenic and thyroid hormone effects observed in experimental and epidemiological studies (Melzer et al., 2010; Moriyama et al., 2002; Vandenberg et al., 2009). According to 2003–2004 National

N. Khalil et al. / Science of the Total Environment 470–471 (2014) 726–732

Health and Nutrition Examination Survey (NHANES), 93% of the United States population sampled had measurable urinary BPA (Calafat et al., 2008), and children aged between 6 and 11 years had the highest urinary creatinine corrected BPA concentrations compared to other age categories (CDC, 2013). Furthermore, in other NHANES studies in children aged 6 to 19 years, urinary BPA concentration was associated with increased risk of obesity (Trasande et al., 2013a) and albuminuria (Trasande et al., 2013b). In children, major exposure to BPA occurs through food and water intake, although dental sealants, inhalation of house dust, and dermal absorption are considerable sources of BPA exposure (CDC, 2013). Epidemiological studies have linked BPA exposure in adults with obesity (Carwile and Michels, 2011; Shankar et al., 2012b) and insulin resistance (IR) (T. Wang et al., 2012), diabetes (Lang et al., 2008; Silver et al.), hypertension (Shankar and Teppala, 2012), peripheral artery disease (Shankar et al., 2012a), and risk of CVD (Melzer et al., 2010, 2012; Lang et al., 2008). Evidence is emerging regarding the underlying pathophysiology of BPA and metabolic dysregulation. Population based studies demonstrate that BPA is also associated with elevated serum liver enzymes in men (F. Wang et al., 2012) and with inflammatory biomarkers, fatty liver disease and IR in women (Tarantino et al., 2013). In particular both fatty liver disease and IR are thought to be related to BPA mediated inflammation (Ben-Jonathan et al., 2009; Hugo et al., 2008). In laboratory animals environmentally relevant doses of BPA influenced lipid metabolism in liver contributing to hepatic steatosis (Ronn et al., 2013). Kandaraki et al. (2011) described positive correlation between BPA and IR in women (Kandaraki et al., 2011). This association corroborates evidence from in vitro and in vivo experiments in which BPA exposure induced insulin secretion and IR (Alonso-Magdalena et al., 2006, 2010). Endocrine hormones and endocrine disruptors often exhibit non-monotonic dose response association (NMDR) (Vandenberg et al., 2012; Beausoleil et al., in press) as U-shaped, inverted U-shaped, or W-shaped curves reported in animal models (Cabaton et al., 2011; Markey et al., 2001) and limited epidemiological studies (reviewed in Vandenberg et al., 2012). The present exploratory study aimed to examine the relationship of urinary BPA, with anthropometric, clinical, hormonal and metabolic measures in a cohort of obese and overweight children. We hypothesized that urinary BPA will have a significant association with adiposity, serum lipids and metabolic panel. Further we hypothesized that association of BPA with the above parameters will differ by sex and exhibit non-linear exposure–response relationship. 2. Methods 2.1. Study population The study population comprised of 39 obese or overweight children aged 3–8 years (50% females, 62% Caucasians), who were enrolled from the Lipid Clinic at Children's Medical Center of Dayton (CMC) Ohio. The Lipid Clinic at CMC was established in 2001. It is a referral facility with expertise in diagnosing and managing children and adolescents presenting with obesity related metabolic diseases. The interdisciplinary team includes physicians, nurses, and dieticians. Children were invited to participate in this study from April to August 2012, if they were between 3 and 8 years of age, were not suffering from thyroid disease, diabetes (Type 1 or 2) or other chronic diseases (e.g., asthma). The study protocol was approved by the institutional review boards of the CMC, and Wright State University, and written informed consent was obtained from parents or guardians of all the participants. 2.2. Demographic and anthropometric measures Age and ethnicity of children were self-reported by parents. Anthropometric data included measurements of weight, stature, and waist circumference using a standardized protocol (Lohman et al., 1988).

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Weight was measured to nearest 0.1 kg on a Detecto-6550 balance scale in street clothes. Height in meters was measured without shoes using a stadiometer (SECA-216). BMI was calculated as weight/height2 (kg/m2). Age- and sex-standardized BMI z scores are calculated according to 2000 Centers for Disease Control and Prevention (CDC) year growth charts (Ogden et al., 2002). Overweight and obese were categorized as BMI z score of 1.036 or greater (85th percentile for age and sex) and 1.64 or greater (95th percentile), respectively (Trasande et al., 2012). In this group except one, all children were obese. Waist circumference (WC) was obtained from iliac crest to iliac crest with standard tape measure in standing position without any clothes covering the abdomen. Seated systolic (SBP) and diastolic blood pressure (DBP) in millimeters of mercury (mm Hg) were obtained using a sphygmomanometer (Dinamap Pro-100 model 400v2) using 2 cuffs sizes of 17–25 cm, or 23–33 cm based on the age of the child. 2.3. Biochemical assays Blood samples are obtained after an overnight fast by venipuncture into BPA free Vacutainer tubes. Biochemical data of interest included fasting insulin (FI), glucose, glycated hemoglobin (HbA1C), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG). Liver profile comprised of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Thyroid profile included thyroid stimulating hormone (TSH) and free thyroxine (FT4). The lipid and thyroid assays were performed on-site by the CMC laboratory, certified by the College of American Pathologists. Beckman Coulter DxC600i/Synchron was used for assay of plasma glucose, LDL-C, HDL-C, TC, TG, ALT and AST (normal range for both liver enzymes 0–45 IU/L). Serum insulin, TSH and FT4 were measured utilizing Beckman Coulter DxC600i/Access 2. HbA1C was measured by Siemens DCA Vantage Analyzer. Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated using the formula: fasting plasma glucose (mmol/l) × fasting insulin (m IU) / 22.5. 2.4. Bisphenol-A assay Spot sample of urine was collected in sterile polypropylene (PP), BPA-free urine cups. Ten milliliters of urine was transferred into BPA free glass tubes and frozen at −40 °C until shipment on dry ice to Wadsworth Center, Albany, New York for analysis (Fig. 1). Urine (500 μL) was transferred into a 15 mL PP tube (after thawing at room temperature for 30 min), and 10 ng of 13C12-bisphenol A (13C12-BPA) was added as an internal standard. Three hundred microliters of 1.0 M ammonium acetate containing 44 units of glucuronidase was added to each sample and the mixture was shaken in an orbital shaker at 37 °C for 12 h (Zhang et al., 2011). Then 0.5 mL of 1.0 M formic acid was added to stop the enzyme activity, and then 0.2 mL of Milli-Q water was added. The digested sample was extracted twice by ethyl acetate (5+4mL), and then the extracts were separated by centrifugation (5000 ×g, 5 minutes) and washed by 0.5 mL of Milli-Q water. The extracts were concentrated to near dryness by a gentle stream of N2, and then dissolved in 0.5 mL of methanol before analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyte levels in samples were quantified using a high-performance liquid chromatography (HPLC) coupled with API 2000 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS). The analytical method is similar to that described earlier (Kunisue et al., 2012). A 10 μL of the extract was injected onto an analytical column (Betasil® C18, 100 × 2.1 mm column; Thermo Electron Corporation, Waltham, MA), which was connected to a Javelin® guard column (Betasil® C18, 20 × 2.1 mm). The mobile phase was comprised of methanol and water containing 0.01 M ammonium acetate at a gradient starting from 25% methanol to 99% methanol in 4 minutes and held for

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a Mean Urinary BPA (ng/mL)

7

Urinary BPA (ng/mL) in Children by Sex p-value: 0.079

6 5 4 3 2 1 0

Male

Female

Mean Creatinine adjusted Urinary BPA (ug/g)

b Creatinine adjusted Urinary BPA (ug/g) in Children by Sex 8

p-value: 0.723

7 6 5 4 3 2 1 0 Male

Female

Fig. 1. a, b: Bisphenol analyte distribution in obese and overweight children by sex. a) BPA, b) Creatinine corrected urinary BPA.

10 minutes before it was reversed to initial condition. The flow rate and the column temperature were 300 μL/min and 25 °C respectively. To confirm that glass tubes were free from BPA contamination, and for quality assurance, procedural blanks were used and no background contamination was found. Quality assurance and quality control parameters also included validation of the method by spiking BPA into the sample matrices and passing through the entire analytical procedure to calculate recoveries of analytes through the analytical method. Reported concentrations were corrected for the recoveries of surrogate standard (isotopic dilution method). The BPA standards spiked to selected sample matrices and passed through the entire analytical procedure yielded a recovery of 98%. An external calibration curve was prepared by injecting 10 μL of 0.01, 0.02, 0.05, 0.10, 0.20, 0.5, 1, 2, 5, 10, and 20 ng/mL standards and regression coefficient was 0.99. For the analysis of creatinine, an aliquot of urine (10 μL) was diluted (160-fold) with Milli-Q water, and 800 ng of d3-creatinine was added. A mixture of methanol/Milli-Q water (50:50, v/v) containing 0.1% formic acid was used as the isocratic mobile phase. The positive ion MRM transitions monitored were 114 N 44 for creatinine and 117 N 47 for d3-creatinine. The MS/MS collision energy was 25 eV, ion source temperature was 400 °C, and cone voltage was 4500 V. The BPA limit of detection (LOD) was 0.1 ng/mL. For one child (n = 1 [2.5% of the sample]), urinary BPA concentrations below LOD, a value of 0.07 ng/mL was substituted for statistical analysis following usual practice (LOD divided by the square root of 2) (CDC, 2013). To correct for urinary dilution, all analyses were repeated with urinary creatinine adjusted BPA (BPA(creatinine)) concentration.

2.5. Statistical analysis The association of BPA with demographic, clinical and biochemical variables was analyzed in overall sample, as well as by sex. Assumption of normality was tested graphically and by using the Shapiro–Wilk statistic available in SAS as part of the basic descriptive analysis. The null hypothesis of a Shapiro–Wilk test explores if there is no significant departure from normality. When the p-value associated with goodnessof-fit statistic was more than 0.05, the null hypothesis was not rejected, concluding normal distribution. If the p-value was less than the predetermined critical value (b0.05), the null hypothesis was rejected, concluding non-normal distribution. Variables with non-normal distribution were log transformed. STATA program options “Gladder” (graphical representation of distribution) and “Ladder” (statistical test of distribution) procedures were used to choose the most appropriate of several possible transformations of each nonnormal variable. These STATA functions provide not only goodness-offit statistics upon which to evaluate transformations, but also superimpose plots of the transformed variable upon a normal curve to illustrate the goodness of fit. We chose the log transformation out of those with the best scores because it provided results that were easier for the audience to interpret. Sex differences were analyzed by t-tests and Mann– Whitney U tests. To assess univariate relationship of BPA (independent variable) with parameters of interest (dependent variable) linear regression was performed separately for each dependent variable. The association between log transformed BPA and metabolic variables HOMA-IR, FI and TSH was tested using cubic spline in regression models as reported in epidemiology literature for BPA (Braun et al., 2011) and other toxic environmental exposures including lead (Jemal et al., 2002), perfluoroalkyl surfactants (PFASs) (Lopez-Espinosa et al., 2011), atrazine, dioxin, and perchlorate (Vandenberg et al., 2012). Cubic polynomial splines allow the shape of the relationship between the exposure and outcome to be flexible and can show non-monotonic exposure–response relationship (Desquilbet and Mariotti, 2010). For 3 knot regression splines knots were located at 0.1, 0.5, and 0.9 percentile distribution of BPA. For 4 knot regression splines, knots were placed at 0.5, 0.35, 0.65, and 0.95 percentile distribution of BPA. Exposure–response association between BPA and metabolic outcome variables HOMA-IR, FI and TSH was tested with Wald test in separate regression models. Wald test was used to test if the slope of three segments of BPA was significantly different from zero. STATA software was used for spline analysis (Edition 9, StataCorp, College Station, Texas, USA). Other analyses were performed using SAS 9.2 (SAS Institute, Cary, NC, USA). In all analyses, a two-sided p-value of b 0.05 was considered statistically significant. 3. Results 3.1. Characteristics of participants The characteristics of participants overall, and by sex are presented in Table 1. Median (inter-quartile range) of urinary BPA concentration was 1.37 (1.2) ng/mL, and urinary BPA adjusted for creatinine (BPA(creatinine)) was 1.82 μg/g (2.6) (Table 1). Anthropometric, clinical and metabolic characteristics were comparable by sex with the exception of BMI z score which was significantly higher (p = 0.04) and serum FT4 concentration that was lower in males (p = 0.04) (Fig. 1). Neither urinary BPA nor BPA(creatinine), or urinary creatinine clearance was different in children across ethnicity (Table 1). However, in sex specific analysis only females (n = 9) of African American/other ethnicity had significantly higher urinary BPA compared to males (n = 6) of African American/other ethnicity. 3.2. Linear regression results Although separate models were run for all anthropometric, clinical and metabolic (dependent) variables shown in Table 1, only selected

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Table 1 Anthropometric, metabolic characteristics of study participants, overall and by sex. Characteristic, Mean (SD)

n

Overall

n

Female

n

Male

Age (year) Weight (kg) Height (cm) Waist circumference (cm) BMI (kg/m2) BMI z scoreb Diastolic BP (mm Hg) Systolic BP (mm Hg) LDL-C (mg/mL) HDL-C (mg/mL) Triglycerides (mg/mL)b Total Cholesterol (mg/mL) b AST (μ/L) ALT (μ/L)b Fasting insulin (Uiu/mL)b Fasting glucose (mg/mL) A1C (mmol/mol) HOMA (mmol/L)b FT4 (pmol/L)b TSH (μIU/mL)b Bisphenol A (ng/mL)b Caucasians Others BPA (creatinine) (μg/g)b Caucasians Others Urinary creatinine (μg/mL)b Caucasians Others

39 39 39 36 39 39 39 39 32 33 33 33 31 31 34 35 27 32 32 33 39 24 15 39 24 15 39 24 15

6.6 46.3 129 83.3 27.2 2.6 64.4 113.3 107 42 85 167 30.5 23 8.0 89.6 5.3 1.78 0.87 2.5 1.37 1.31d 1.40d 1.82 1.71e 2.26e 932 930f 930f

22 22 22 19 22 22 22 22 17 18 18 18 16 16 19 19 14 18 15 20 22 13 9 22 13 9 22 13 9

6.7 (1.3) 43.9 (12.8) 128 (10.9) 84.2 (10.8) 26.1 (5.0) 2.4 (0.8) 63.4 (6.7) 110 (12) 108 (30) 43.6 (11) 87 (108) 175 (61) 30.1 (5.5) 23 (9) 7.3 (7.9) 88 (7.4) 5.3 (0.4) 1.7 (2.2) 0.98 (0.4) 2.5 (1.9) 1.74 (3.3) 1.41 (2.9) 2.11(2.5) 2.52 (3.1) 1.90 (2.9) 3.45 (4.8) 871 (591) 857 (684) 931 (304)

17 17 17 17 17 17 17 17 15 15 15 15 15 15 15 16 13 14 17 13 17 11 6 17 11 6 17 11 6

6.4 49.5 130 82.2 28.7 2.7 65.7 116.2 106 40.1 85 163 30.9 23 8.0 91.5 5.3 1.8 0.80 2.5 1.12 1.19 0.90 1.20 1.23 1.05 1067 1283 757

(1.5) (14.7) (12.4) (11.1) (5.4) (0.7) (6.8) (11.0) (25.4) (11.6) (96) (52) (6.7) (9) (6.5) (8) (0.4) (1.5) (0.3) (1.6) (2.2) (2.5) (2.0) (2.6) (2.3) (6.2) (578) (822) (347)

p-Valuea (1.7) (16.8) (14.5) (11.6) (5.8) (0.6) (7) (11) (20) (12.8) (67) (47) (8.0) (17) (5.0) (8.4) (0.5) (1.2) (1.0) (1.4) (1.2) (1.4) (0.9) (1.9) (1.8) (1.0) (588) (799) (309)

0.58 0.24 0.66 0.60 0.15 0.04 0.32 0.15 0.81 0.39 0.68c 0.85c 0.74 0.80c 0.89c 0.20 0.83 0.72c 0.04c 0.87c 0.07c 0.78c 0.04c 0.06c 0.49c 0.11c 0.46c 0.23c 0.86c

a

p-Value tests mean/median differences between males and female. Median (Inter-quartile range). Mann–Whitney U test. d, e, f p-Value tests median overall analyte difference between participants of Caucasian and others ethnicities (d 0.785, e 0.466, f 0.502). b c

models that were significantly associated with BPA or BPA (creatinine) are presented in Table 2. 3.2.1. BPA overall and sex specific models In overall linear regression analysis and when analyzed by sex, in females BPA did not show any significant association with anthropometric and metabolic characteristics. However in males, BPA was significant in predicting decrease in serum FI (β Standard Error (SE)) = −0.36, = 0.10, p = 0.022 and HOMA-IR (β = −0.39 SE (0.10), p = 0.006). 3.2.2. In BPA (creatinine) and sex specific models In overall model serum FT4 was significantly associated with BPA(creatinine) (β (SE)=0.26, SE (0.1), p=0.028). In sex specific univariate regression analysis, in males BPA(creatinine) was a negative predictor

of age (β = −0.71, SE (0.2), p = 0.029), positive predictor of serum AST (β = 3.35, SE (1.4), p = 0.032) and diastolic blood pressure (β = 3.34, SE (1.1), p = 0.011). 3.2.3. Adjusted regression analysis for diastolic blood pressure Association between BPA(creatinine) and diastolic blood pressure (outcome variable) in males was significant when adjusted for age, and ethnicity (β = 4.01, SE (1.41), p b 0.014, adjusted R2 = 0.38, Table 3). 3.2.4. Spline analysis In spline analysis a non-monotonic exposure–response association between urinary BPA, serum TSH was noted. In addition, in males, urinary BPA concentration had significant non-linear relationship with HOMA-IR and FI (Fig. 2). Wald test was significant which indicated

Table 2 Simple linear regression between log transformed bisphenol A (BPA) and log transformed BPA (creatinine) and selected characteristics of study participants, overall and by sex. Characteristics

Age AST FI

a

HOMA-IR

a

Diastolic BP FT4a a b

Overall

BPA BPA BPA BPA BPA BPA BPA BPA BPA BPA BPA BPA

Cr Cr Cr Cr Cr Cr

Log transformed variables. SE: standard error.

Female

Male

β (SE)b

p-Value

R2

β (SE)

p-Value

R2

β (SE)

p-Value

R2

−0.01 (0.2) −0.37 (0.2) 1.95 (1.1) 1.48 (0.9) −0.11 (0.1) −0.04 (0.1) −0.13 (0.1) −0.05 (0.1) −0.61 (0.1) 1.05 (0.9) 0.15 (0.1) 0.26 (0.1)

0.629 0.051 0.262 0.116 0.407 0.696 0.345 0.656 0.543 0.238 0.290 0.028

0.0 0.10 0.04 0.08 0.02 0.00 0.30 0.00 0.01 0.04 0.04 0.18

−0.14 (0.2) −0.18 (0.2) 0.30 (1.3) −0.04 (1.2) 0.00 (0.2) −0.01 (0.2) −0.00 (0.2) −0.00 (0.2) −0.58 (0.1) −0.25 (1.2) 0.08 (0.2) 0.33 (0.2)

0.530 0.442 0.812 0.991 0.992 0.948 0.992 0.963 0.617 0.837 0.727 0.121

0.02 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.18

−0.12 (0.5) −0.71 (0.2) 5.05 (2.4) 3.35 (1.4) −0.36 (0.1) −0.08 (0.1) −0.39 (0.12) −0.08 (0.1) 0.20 (2.2) 3.34 (1.1) 0.06 (0.1) 0.04 (0.1)

0.815 0.029 0.058 0.032 0.022 0.501 0.006 0.439 0.927 0.011 0.318 0.931

0.00 0.28 0.25 0.31 0.34 0.04 0.48 0.05 0.00 0.36 0.10 0.00

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Table 3 Parameter estimates of simple (Model 1) and multivariable linear regression (Model 2) between log BPA (creatinine) and diastolic blood pressure in male obese and overweight children (n = 17). Parameter estimates Model 1, diastolic blood pressure Intercept Log BPA (creatinine), μg/g R2 = 0.36 Model 2, diastolic blood pressure Intercept Log BPA (creatinine), μg/g Age, years Ethnicity: Caucasian versus others R2 = 0.49, Adjusted R2 = 0.38.

β

SE

p

64.92 3.34

1.44 1.16

b0.001 0.011

64.37 4.01 0.67 −6.07

6.78 1.41 1.14 3.35

b0.001 0.014 0.566 0.093

that the slope of three segments of BPA was different from zero for FI (pb0.0001), HOMA-IR (pb0.006), TSH (pb0.0001) and showed variable patterns of increase and decrease at different exposure levels of BPA. 4. Discussion The results of this study suggest that higher urinary BPA(creatinine) concentration in overweight and obese children is associated with increasing FT4. In male children BPA(creatinine) decreased with age, and was associated with elevated liver enzyme AST and diastolic blood pressure. BPA concentration unadjusted for creatinine was associated with decreasing serum FI and HOMA-IR levels in linear regression and showed NMDR association. These results are suggestive that BPA exposure in obese children at least in males can lead to adverse liver, and metabolic effects and high diastolic blood pressure. The median urinary BPA concentration observed in obese children in the current study was lower than 2.8 ng/mL (4.4) as compared to that reported in US population based sample of children aged 6–19 years in 2009–2010 NHANES (Trasande et al., 2012). In the current analysis, even at comparatively lower BPA concentration, significant associations with several metabolic outcomes were observed. 4.1. BPA and thyroid hormone In this study, male obese children had significantly lower FT4 compared to female children. In overall linear regression BPA(creatinine) predicted higher FT4. In overall correlation analysis TSH was inversely associated with BPA which on spline analysis showed a NMDR association, suggesting that as concentration of BPA increased TSH hormones did not exhibit a linear pattern of increase. Thyroid hormone is essential for prenatal and post natal growth and brain development. BPA has been associated with potentially adverse effects on cognition and behavior in humans (Braun et al., 2011) which may be attributable to disruption of thyroid function. Limited human evidence shows that BPA exposure during pregnancy is inversely related to total T4 in pregnant women and decreased TSH in male infants (Chevrier et al., 2013). These associations may have implications in children during rapid growth phases of childhood and puberty. 4.2. BPA and age Prenatal growth, infancy, and childhood when rapid development occurs, are periods of highest vulnerability to adverse effects of BPA exposure (Chevrier et al., 2013). In the current study in male children urinary BPA concentration decreased significantly with age (only in BPA(creatinine)). As reported in 2009–2010 NHANES data in which children 6–11 years of age had significantly higher urinary BPA (2.25 ng/mL) concentration compared to ages 12–19 years (1.76 ng/mL) (p b 0.016) (Trasande et al., 2013b) (both sexes). According to 2003–2004 NHANES

Fig. 2. a, b, c: Non-linear association of BPA analytes with a) FI and b) HOMA-IR in males, and c) with TSH overall (all variables log transformed). p-Value for Wald test in A, B, C: b0.001.

children aged 6–11 years of age had the highest urinary BPA(creatinine) compared to other age categories (2012). In an epidemiological study of younger children aged 1, 2, and 3 years, median urinary BPA(creatinine) decreased from 18.0 ug/g, 9.6 ug/g, to 5.3 ug/g with age respectively (Braun et al., 2011). 4.3. BPA in males In the current analysis in male children serum FI and HOMA-IR showed a non-linear association with increasing urinary BPA concentration. This observation is consistent with evidence from in-vitro and invivo models, however human studies are non-existent. Estrogens (E2) and BPA have insulinotropic effects on pancreatic islets of Langerhans

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in male mice which indicate that E2, and BPA increases insulin content and secretion (Alonso-Magdalena et al., 2008). When Islets of Langerhans were cultured in the presence of glucose and increasing dosage of E2 (physiological dose of E2) for 48hours, insulin secretion increased in an inverted-U dose-dependent response pattern. When BPA was substituted for E2, increase of insulin secretion showed a similar inverted U-shaped dose response curve. This indicates that BPA as a xenoestrogen may mimic the effect of E2 on insulin secretion pathways, and increases pancreatic insulin content in a non-monotonic manner (Alonso-Magdalena et al., 2008). In animal models, prenatal BPA exposure disturbed pancreatic β-cell response and stimulated IR in male mice offspring (Alonso-Magdalena et al., 2006, 2010). On the contrary, female offspring displayed normal glucose and insulin parameters. Females are protected against IR more than are males, as in female presence of estrogens within the physiological range protect against diabetes in mice (Louet et al., 2004). The sexually dimorphic relationship suggests an association of BPA acting as an estrogen with obesity and glucose homeostasis in males but not in females (Mauvais-Jarvis, 2011). Further, data from animal studies suggest that males may not metabolize BPA as efficiently as females and may be at higher risk of BPA exposure because of delayed excretion and metabolism (Takeuchi et al., 2004).

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induced significant weight gain (Miyawaki et al., 2007). Various doses of BPA may function in distinctly different sometimes opposing manner. 4.6. BPA and diastolic blood pressure In this study, BPA was significantly related to diastolic blood pressure in male children even after adjusting for confounding effects of age and ethnicity. BPA at environmentally relevant doses inhibits the release of adiponectin, a key adipokine protective against hypertension and other components of metabolic syndrome (abdominal obesity, glucose intolerance, hyper-insulinemia, hyper-triglyceremia) (Hugo et al., 2008). As discussed above, in epidemiological studies, BPA exposure was associated with abnormal liver and thyroid function, higher levels of fasting glucose, estrogen-mimetic effects, IR, and HOMA-IR which are considered risk factors for hypertension. Furthermore, BPA can induce endothelial cell injury mediated through oxidative stress (Ooe et al., 2005) and elevations in lipids in animal models (Marmugi et al., 2012). Emerging evidence in human population also supports that association between BPA, hypertension (Shankar and Teppala, 2012) and peripheral arterial disease is plausible as noted in adults in NHANES 2003–2004 (Shankar et al., 2012a).

4.4. BPA and liver 4.7. Strengths and limitations Hepatotoxic effect of BPA has been described in animal models where BPA concentration lower than human protective dose No Observed Adverse Effect Level (NOAEL) can induce hepatic damage and mitochondrial dysfunction by increasing oxidative stress in the liver (Ronn et al., 2013). Based on hepatotoxic potential of BPA in rodent models, the U.S. Environmental Protection Agency derived a tolerable daily intake (TDI) of 50 μg/kg/day for humans after applying an uncertainty factor of 100 to the NOAEL of 5000 μg/kg/day (5000/100 = 50) (Marmugi et al., 2012). In rodents, BPA at TDI doses showed nonmonotonic dose response with liver enzymes that control fat synthesis (Marmugi et al., 2012). BPA may induce fat deposition in liver, either directly through a hepatotoxic effect and/or indirectly by promoting IR and inflammation (Moon et al., 2012) as shown in an increase in proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (Moon et al., 2012). In rats exposure to BPA at TDI dose, magnetic resonance imaging showed that liver fat content was significantly increased compared to controls (p = 0.04) (Ronn et al., 2013). In premenopausal women with poly cystic ovary syndrome (PCOS), serum BPA levels were related to HOMA-IR, markers of lowgrade inflammation including C-reactive protein (CRP), IL-6, and ultrasound quantification of hepatic steatosis (Tarantino et al., 2013). 4.5. BPA and non-monotonic exposure–response Endocrine hormones and endocrine disruptors such as BPA rarely display linear dose response association (Vandenberg et al., 2012). The reason being that after a hormone binds and saturates its receptor sites; excess hormone does not produce further response (Vandenberg et al., 2012). Alternatively, high doses can down regulate responses initiated at lower doses such that different effects can appear and disappear at different exposure concentrations. These non-monotonic exposure–response curves are commonly accepted in endocrinology (Vandenberg et al., 2012). BPA exposure has been associated with various health outcomes in a non-monotonic fashion displayed as U-shaped or inverted U-shaped, W-shaped curves. As reported in literature, when human fat explants was treated with BPA (0.1–10 nM) the adiponectin secretion response was a U-shaped curve; at low dose of BPA adiponectin secretion was lower, whereas at elevated BPA doses adiponectin secretion increased (Hugo et al., 2008). In animal experiments, when BPA was added in 1 or 10 μg BPA/L doses in drinking water during early development, 1 μg dose but not 10 μg of BPA/L

The main strengths of our study include its representation of young children from both sexes and the availability of extensive data on metabolic and hormonal parameters. To our knowledge this is the first study to report a non-monotonic exposure–response relationship between metabolic and hormonal effects of BPA in children. However there are several study limitations, including the small sample size, its cross-sectional design which does not allow establishing cause–effect associations. 5. Conclusions In obese and overweight male children BPA(creatinine) decreased with age, and was associated with elevated liver enzyme AST and diastolic blood pressure. BPA concentration unadjusted for creatinine showed non-linear association with decreasing serum FI and HOMA-IR. Further research to explore environmental exposure to BPA and obesity related health outcome in children is warranted. Grant support and institutional review for human subjects' research The work was not grant supported. The study protocol was approved by the institutional review boards of the Children's Medical Center of Dayton, and Wright State University. Written informed consent was obtained from parents or guardians of all the participants. The approval of research protocol is appended (Appendices 1–3). Conflict of interest statement All authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. We assure you that all of the authors have read and approved the paper. The content has not been published previously nor is it being considered by any other peer-reviewed journal. We have not used animals in this research. Written informed consent was obtained from the participants before the start of the study.

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