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Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years
Environmental Pollution xxx (2016) 1e8
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Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years* Jianqiu Guo a, Chunhua Wu a, *, Dasheng Lu a, b, Shuai Jiang a, b, Weijiu Liang c, Xiuli Chang a, Hao Xu c, Guoquan Wang b, Zhijun Zhou a a
School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Collaborative Innovation Center of Social Risks Governance in Health, Fudan University, No.130 Dong'an Road, Shanghai 200032, China Shanghai Municipal Center for Disease Control and Prevention, No. 1380 Zhongshan West Road, Shanghai 200336, China c Shanghai Center for Disease Control and Prevention, No.39 Yunwushan Road, Changning District, Shanghai 200051, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 May 2016 Received in revised form 15 December 2016 Accepted 16 December 2016 Available online xxx
Parabens, known as ubiquitous preservatives, have been linked to adverse health outcomes in humans. This study aimed to examine urinary paraben concentrations of children at 3 years of age and evaluate their associations with anthropometric parameters. Urinary parabens including methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP) and benzylparaben (BeP) were measured among 436 children in a birth cohort using gas chromatography with tandem mass spectrometry. Generalized linear models were performed to evaluate associations of paraben exposures with age- and sex-specific z scores, including weight, height, weight for height and body mass index. MeP, EtP and PrP were the dominant parabens in urinary samples, with the median concentrations of 6.03 mg/L, 3.17 mg/L, 2.40 mg/L, respectively. The median values of estimated daily intake (EDIurine) of five urinary paraben concentrations were 12.10, 5.68, 4.50, 0.06 and 0.17 mg/kg-body weight/day, respectively. Urinary EtP concentrations were positively associated with weight z scores [regression coefficient b ¼ 0.16, 95% confidence interval (CI): 0.04, 0.29; p ¼ 0.01] and height z scores (b ¼ 0.15, 95% CI: 0.03, 0.27; p ¼ 0.01). Positive associations were found between the sum of molar concentrations of five parabens and height z scores among all children (b ¼ 0.24, 95% CI: 0.04, 0.45; p ¼ 0.02). These significant associations were only observed in boys. Our findings suggest that exposure to parabens may be adversely associated with physical growth in 3-year-old boy children. Further prospective studies are warranted to understand the toxicological mechanisms of paraben exposures and potential risk of children. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Parabens Exposure assessment Anthropometric measures Urine Child health
1. Introduction Parabens are a class of para-hydroxybenzoic acid with alkyl and aryl esters, which are the most extensively used preservatives in foodstuffs, pharmaceuticals, and personal care products worldwide (Błe˛ dzka et al., 2014). Commercially available parabens include methylparaben (MP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP) and benzylparaben (BeP). Human exposure to parabens through topical contaction with or ingestion of products containing parabens (Guo et al., 2014; Liao et al., 2013). Recently, parabens have been reported to be present in water, especially at
*
This paper has been recommended for acceptance by David Carpenter. * Corresponding author. E-mail address: [email protected] (C. Wu).
maximum concentrations of 3.14 mg/L in the Pearl River Delta and of 1.65 mg/L in urban surface water in Beijing, China, respectively (Li et al., 2016; Yu et al., 2011). The serious human exposure to parabens has led to extensive distribution in various human biological samples, including urine, serum, breast milk, placental tissue and amniotic fluid (Hines et al., 2015; Philippat et al., 2013; Valle-Sistac et al., 2016). Additionally, parabens in fatty components of human body tissues were also found, which suggested that bioaccumulation could potentially impact fat deposition (Wang et al., 2015). Previously regarded as safe (Soni et al., 2005), parabens have recently raised concern for the evidence of potential endocrine disruptors, with a lower estrogen receptor binding affinity than 17 b-estradiol (Boberg et al., 2010). Epidemiological studies have shown associations between urinary parabens and adverse health outcomes, including reproductive toxicity (Meeker et al., 2011),
http://dx.doi.org/10.1016/j.envpol.2016.12.040 0269-7491/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Guo, J., et al., Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.040
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J. Guo et al. / Environmental Pollution xxx (2016) 1e8
oxidative stress (Watkins et al., 2015), immune modulation (Savage et al., 2012) and even breast cancer (Pan et al., 2015). Moreover, parabens may also interfere with thyroid hormones and activate the peroxisome proliferator-activated receptorg(PPARg), which was a nuclear receptor superfamily playing a key role in adipogenesis and lipid accumulation (Pereira-Fernandes et al., 2013). Furthermore, a study had reported that parabens could promote development and differentiation of multipotent cells into mature adipocyte cells (Hu et al., 2013), indicating parabens may play a role in obesity epidemic. The long-term exposure to low-dose of parabens seemed alarming susceptibility for young children because of their immaturity of physiological functions, behaviors and potentially enhanced target organ sensitivity (SCCS, 2011). Limited epidemiological researches suggested inconsistent results that exposure to parabens was associated with adverse effects on body growth. Specifically, associations of gestational exposure to parabens with increase in birth weight (Philippat et al., 2014) and with reduction in birth weight and length (Geer et al., 2016) were found. Compared to prenatal exposure to parabens, relatively scarce data are available regarding the effects of postnatal exposure on body growth. Only one study revealed non-significant association of paraben exposure with childhood obesity, but a positive association between the paraben metabolite of 3,4-dihydroxy benzoic acid (3,4DHB) and obesity (Xue et al., 2015). Given the widespread exposure to parabens and possible endocrine disruption, it is imperative to examine the potential risk of exposure to these pollutants in relation to childhood obesity. Generally, parabens are rapidly absorbed, metabolized, and excreted in urine from the body (Moos et al., 2015a). Urinary paraben concentrations have been known to be valid biomarkers of exposure (Ye et al., 2006). Thus, the objectives of the present study are to assess parabens exposure of children and its potential associations with body growth at 3 years old in a birth cohort from an agricultural region in Jiangsu Province, China.
2.2. Urinary measurement A spot urine sample was collected from each child in the Health Care Centre and was transferred to the high-density polypropylene centrifuge tubes (Corning Incorporated, USA). All samples were immediately stored at 20 C, then frozen shipped to the laboratory and kept at 80 C until analysis. As previously described by Lu et al. (2015), urinary parabens including MeP, EtP, PrP, BuP and BeP were measured by largevolume-injection gas chromatography with tandem mass spectrometry (LVI-GC-MS/MS). Briefly, the urine samples were prepared by hydrochloric acid hydrolysis, liquid-liquid extraction, and solid-phase extraction clean up. After derivatization, the samples were analyzed by LVI-GC-MS/MS. The limits of detection (LODs) for metabolites were 0.01 mg/L, which was defined as a signal-to-noise ratio of three. To account for variability in urinary dilution, creatinine concentrations and specific gravity (SG) of urine samples for individuals were determined. Creatinine concentration was determined by ELx800 Universal Microplate Reader (wavelength 340e750 nm; BIO-TEK, USA), and SG was measured using a handheld refractometer (Atago PAL 10-S, Tokyo, Japan). 2.3. Estimated daily intake of parabens Estimated daily intake (EDIurine) of parabens was calculated based on the measured urinary concentrations of parabens and a simple steady-state toxicokinetic model. The EDIurine of parabens for children was performed using the formula (1) as described by Ma et al. (2013).
EDIurine ¼
50 C V BW
(1)
2. Materials and methods
where EDIurine (mg/kg bw/day) is the estimated daily intake of individual paraben; C (mg/L) is the measured concentration of urinary paraben; V (L/day) is daily urinary excretion rate as 0.6 L/day in this study (Pei and Wen, 2004). BW (kg) is children's body weight of the child.
2.1. Study participants and questionnaires
2.4. Child anthropometry
During July 2012eApril 2013, 498 children were recruited in our study when they visited Sheyang Maternal and Child Health Care Centre. All children's mothers had previously participated in our longitudinal cohort study during pregnancy at hospital. Questionnaire survey was administered to each participated child's caregiver by trained interviewers. Data covering child's sociodemographics, living environment and lifestyles was collected. Information regarding pregnancy and maternal health was obtained from medical records and questionnaires previously (Qi et al., 2012). Each caregiver had signed an informed consent form and agreed to donate child's urine sample. This study was carried out with the permission of the Ethics Committees of Fudan University. Of the 498 children, we excluded 33 children who had no or inadequate urine samples, 20 children who did not complete the questionnaires, two twins, two children who had congenital anomalies at birth, two children whose mothers suffered from serious pregnancy complication, and one child without body growth measures. Finally, 436 children at 3 years of age were enrolled in the present study. These children did not differ significantly from the initially recruited subjects on all attributes of interests, including demographic characteristics, parental and socioeconomic information (data not shown).
Anthropometry measurements of children were required without jacket and shoes by pediatric physicians who were blind to this research. Body weight was measured using a digital scale and rounded to 0.1 kg. Body height was measured to the nearest 0.1 cm using metal column height-measuring stands. The children were all stand straight against metal column. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Anthropometric outcomes were compared with sexspecific World Health Organization (WHO) child growth standards. Correspondingly, age- and sex-standardized z scores were generated using WHO child growth standards (WHO , 2006). Therefore, the final measures of body sizes were z scores for weight, height, weight for height and BMI. 2.5. Statistical analysis Generalized linear models were used to examine associations between SG-adjusted paraben concentrations and body growth outcomes. Individual paraben concentration and the sum molar P concentration of the five urinary paraben ( paraben) adjusted for SG were examined with the models. Furthermore, analyses for P quartiles of parabens were conducted separately to investigate potential non-monotonic exposure-response relationships. To
Please cite this article in press as: Guo, J., et al., Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.040
J. Guo et al. / Environmental Pollution xxx (2016) 1e8
explore the possibility whether associations were different between sexes, an interaction term (sex log transformed paraben concentration) was introduced into the models and a stratified analysis was further conducted. Due to skewed distribution of urinary paraben concentrations, log transformed values were used to perform univariate and multivariate analyses. The differences in paraben levels between children's sociodemographic characteristics were examined using Wilcoxon rank-sum test or Kruskal-Wallis rank sum test. Correlations between log transformed concentrations of urinary parabens were assessed using Pearson correlation coefficients. Paraben levels below the LOD were substituted by the value of LOD divided by the square root of two. Paraben concentrations were corrected for SG using the formula (2):
Pc ¼ P½ð1:016 1Þ=ðSG 1Þ
(2)
where Pc is SG-adjusted paraben concentrations (mg/L); P is unadjusted paraben levels (mg/L); SG is the specific gravity of the urine sample; 1.016 is the median level in the population of this study. Creatinine excretion is dependent on individual physiological factors including muscle mass and physical growth. Thereby SGadjusted concentrations were used, as recommended by Smith et al. (2013). Covariates were selected for the final statistical model in terms of sociodemographic and biological considerations, and in relation to concentrations of urinary parabens or measured body size of children (p < 0.1). The following covariates were included: maternal BMI, paternal BMI, child's sex, maternal education (
3
listed in Table 1. For children at 3 years of age, the mean body weight (mean ± standard deviation) was 16.2 ± 2.2 kg; the mean height was 99.7 ± 4.1 cm and BMI was 16.3 ± 1.4 kg/m2. Additionally, 69.7% of maternal education levels were less than high school, and 39.4% of the children were living in countryside. For body size measures adjusted for age and sex, no significant differences were observed between children's body size of and WHO reference parameters (data not shown). 3.2. Urinary paraben concentrations and estimated daily intake Table 2 shows the distribution of urinary paraben concentrations adjusted for both creatinine and SG among 436 children. MeP, EtP, PrP and BuP were found in over 94% urine samples, whereas BeP was only presented in 80.73% of samples. The geometric mean (GM) concentration (range) of MeP was 5.27 mg/L (
3. Results 3.1. Population characteristics The sociodemographic characteristics of mother-child pairs are
When prenatal concentration of parabens in maternal urine was adjusted, association of postnatal exposure to parabens with body size measures was virtually unchanged. Namely, all changes in the estimated regression coefficients were less than 5%. The inclusion
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J. Guo et al. / Environmental Pollution xxx (2016) 1e8 Table 1 Characteristics of mothers-child pairs at birth and follow-up.
Child characteristics Gender of child Male Female Birth weight (kg) Birth length (cm) Weight (kg) Height (cm) BMIa (kg/m2) BMI z scoreb Household characteristics Maternal pre-pregnancy BMI (kg/m2) Parental BMI (kg/m2) Maternal education level
1100 mother-newborn pairs (n% or mean ± SDc)
436 mother-child pairs (n % or mean ± SD)
589 (53.5) 511 (46.5) 3.5 ± 0.4 51.3 ± 2.3
221 (50.7) 215 (49.3) 3.4 ± 0.6 49.7 ± 8.7 16.2 ± 2.2 99.7 ± 4.1 16.3 ± 1.4 0.6 ± 1.0
21.4 ± 2.8 24.0 ± 3.9
21.4 ± 3.0 24.0 ± 3.9
702 (63.9) 397 (36.1)
304 (69.7) 131 (30.0)
398 (36.4) 227 (20.7) 470 (42.9)
130 (29.8) 134 (30.7) 172 (39.4)
Notes: missing values: weight (n ¼ 1), height (n ¼ 1), maternal education level (n ¼ 1). a BMI: body mass index (weight in kilogram divided by squared height in meter). b z score defined as growth indictors for age and sex according to the WHO growth reference. c SD: standard deviation.
Table 2 Distributions of urinary paraben concentrations in 436 children. Analyte
Unit
>LOD (%)
GM
GSD
Range
mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mmol/L mmol/mol Cre. mmol/LSG
97.73
5.27 12.59 6.26 3.32 7.94 4.03 2.98 7.13 3.58 0.04 0.09 0.05 0.10 2.25 0.13 0.16 43.11 0.19
7.41 7.94 7.83 9.33 10.00 9.03 6.03 6.30 6.20 3.63 3.98 3.65 7.59 7.94 7.87 3.71 3.89 3.63
Selected percentiles P25
P50
P75
P95
1.55 6.03 22.46 82.13 3.91 16.84 55.62 213.93 2.12 8.13 27.62 102.89 EtP 94.56 1.05 3.17 13.71 106.38 2.33 8.24 31.67 352.13 1.29 4.31 15.95 127.16 PrP 98.64 0.94 2.40 10.07 66.27 2.29 6.42 25.34 176.59 1.19 3.02 13.04 70.41 BuP 97.51 0.02 0.03 0.09 0.42 0.03 0.08 0.23 1.08 0.02 0.04 0.11 0.47 BeP 80.73 0.02 0.09 0.39 3.66 0.05 0.23 0.96 10.57 0.02 0.12 0.45 5.87 P PBs 0.06 0.16 0.41 1.43 17.92 41.86 105.50 455.17 0.08 0.19 0.45 1.59 P Abbreviations: MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; PBs: the sum of molar paraben concentrations; Cre.: creatinine; SG: specific gravity; LOD: limit of detection; GM: geometric mean; GSD: geometric standard deviation. MeP
Table 3 EDIurine (mg/kg-bw/day) of parabens calculated from urinary concentrations.
Male
Female
Total
GM 5th Percentile Median 95th Percentile GM 5th Percentile Median 95th Percentile GM 5th Percentile Median 95th Percentile
MeP
EtP
PrP
BuP
BeP
9.56 0.26 12.26 193.4 10.17 0.56 12.02 143.9 9.86 0.36 12.10 150.8
7.52 0.02 7.19 244.6 5.32 0.32 5.03 126.1 6.34 0.02 5.68 187.1
5.23 3.91 0.40 122.8 6.09 0.40 5.82 124.0 5.64 0.41 4.50 118.2
0.07 0.01 0.06 0.53 0.08 0.01 0.06 0.98 0.07 0.01 0.06 0.64
0.19 0.01 0.17 6.73 0.19 0.01 0.17 8.91 0.20 0.01 0.17 7.06
Abbreviations: EDIurine: estimated daily intake based on urinary paraben concentrations; MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; GM: geometric mean.
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J. Guo et al. / Environmental Pollution xxx (2016) 1e8
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Table 4 Associations between urinary paraben concentrations and changes in body size measures in 436 children. Weight z score
Height z score
Weight for height z score
BMI z score
b (95% CI)
p
b (95% CI)
p
b (95% CI)
p
b (95% CI)
p
0.08 (0.07, 0.23) 0.16 (0.04, 0.29) 0.01 (0.18, 0.16) 0.12 (0.12, 0.30) 0.07 (0.21, 0.07) 0.16 (0.05, 0.38)
0.32 0.01 0.92 0.42 0.34 0.14
0.09 0.15 0.02 0.12 0.05 0.24
(0.07, 0.23) (0.03, 0.27) (0.13, 0.18) (0.17, 0.32) (0.08, 0.19) (0.04, 0.45)
0.17 0.01 0.76 0.23 0.44 0.02
0.06 (0.26, 0.15) 0.13 (0.05, 0.26) 0.04 (0.21, 0.13) 0.03 (0.18, 0.25) 0.15 (0.29, 0.01) 0.06 (0.16, 0.28)
0.60 0.06 0.64 0.77 0.05 0.29
0.04 (0.13, 0.20) 0.12 (0.02, 0.26) 0.03 (0.20, 0.15) 0.04 (0.19, 0.26) 0.15 (0.30, 0.01) 0.05 (0.18, 0.28)
0.58 0.09 0.74 0.76 0.06 0.66
0.08 (0.06, 0.23) 0.16 (0.03, 0.28) 0.00 (0.16, 0.17) 0.12 (0.09, 0.32) 0.04 (0.18, 0.10) 0.17 (0.04, 0.39)
0.25 0.01 0.99 0.28 0.56 0.11
0.11 0.15 0.05 0.14 0.08 0.23
(0.02, 0.26) (0.03, 0.27) (0.11, 0.21) (0.06, 0.34) (0.06, 0.21) (0.03, 0.43)
0.11 0.01 0.56 0.16 0.28 0.02
0.04 (0.12, 0.19) 0.12 (0.01, 0.26) 0.05 (0.22, 0.13) 0.06 (0.15, 0.28) 0.12 (0.27, 0.02) 0.06 (0.16, 0.28)
0.65 0.06 0.58 0.56 0.10 0.58
0.04 (0.12, 0.19) 0.12 (0.02, 0.25) 0.04 (0.22, 0.14) 0.06 (0.15, 0.29) 0.12 (0.27, 0.03) 0.06 (0.17, 0.28)
0.64 0.09 0.66 0.56 0.11 0.62
0.00 (0.14, 0.14) 0.08 (0.07, 0.22) 0.07 (0.24, 0.09) 0.20 (0.46, 0.06) 0.12 (0.27, 0.03) 0.01 (0.23, 0.26)
0.95 0.30 0.38 0.13 0.13 0.91
0.03 (0.09, 0.16) 0.11 (0.02, 0.24) 0.04 (0.11, 0.19) 0.17 (0.41, 0.07) 0.08 (0.23, 0.05) 0.18 (0.05, 0.42)
0.65 0.10 0.63 0.16 0.22 0.13
0.02 (0.16, 0.13) 0.01 (0.14, 0.15) 0.15 (0.31, 0.02) 0.14 (0.41, 0.12) 0.09 (0.24, 0.06) 0.11 (0.34, 0.14)
0.84 0.90 0.08 0.30 0.23 0.38
0.02 0.01 0.15 0.14 0.09 0.14
0.79 0.95 0.07 0.31 0.22 0.30
a
Total MeP EtP PrP BuP BeP P PBs Maleb MeP EtP PrP BuP BeP P PBs Femaleb MeP EtP PrP BuP BeP P PBs
(0.17, (0.15, (0.32, (0.41, (0.25, (0.39,
0.13) 0.14) 0.02) 0.13) 0.06) 0.12)
P Abbreviations: MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; PBs: the sum of molar paraben concentrations; z score defined as growth indictors for age and sex according to the WHO growth reference; CI: confidence interval. a Models were adjusted for maternal body mass index, paternal body mass index, child's gender, maternal education, family annual income, inhabitation, feeding pattern, smoking status, time spent playing outdoors, sampling season, child's sex log (each paraben) and birth outcome measures (weight, length or ponderal index). b Child's sex and child's sex log (each paraben) were excluded from models.
of several confounding factors such as urinary phenols did not alter the significantly positive associations between paraben exposures and anthropometric measures (data not shown). Additionally, positive associations were observed between urinary paraben concentrations and anthropometric indicators, no matter whether adjusted for SG or not. After exclusion of preterm births and low birth weight, the results did not change substantially (data not shown). 4. Discussion In our study, children were extensively exposed to five parabens with the wide range of EDIurine, especially for EDIurine values of MeP ranging from 0.01 to 1571.42 mg/kg-bw/day. We found that urinary EtP concentrations were positively associated with weight and height parameters of children. Moreover, the positive associations P between urinary paraben concentrations and anthropometric outcomes was significantly observed among boys. 4.1. Exposure assessment of parabens Parabens were widely concerned in urine samples. MeP, EtP and PrP were the dominant parabens, which was consistent with those reported in environmental monitoring and dietary exposure in China (Guo et al., 2014; Li et al., 2016). Parabens exposure in our study was comparable to those reported in some studies, except for EtP slightly higher than others (USCDC, 2015; Frederiksen et al., 2013; Larsson et al., 2014; Myridakis et al., 2015a,b; Wang et al., 2013) (seeing Supplemental Material, Table S2). However, there were a contrast cases. For example, the unadjusted MeP levels (150.0 mg/L) among 4 years old children in Spain were 25-fold higher than ours (Casas et al., 2011). The difference may be due to actual different exposure across disparate geographical regions and population lifestyles, as well as different socioeconomic characteristics and education levels or even the combination of all of these factors. Therefore, making comparisons across studies should be
cautioned because of variation from exposure assessment methods to study designs. Total EDIurine values of children in the present study were similar to those in Chinese young adults (Ma et al., 2013), and lower than those in Korean adults (Kang et al., 2016). In 1974, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) allocated the total acceptable daily intake (ADI) of 0e10 mg/kg-bw/day for the sum of MeP, EtP and PrP (JECFA, 1974). While in 2007, the JECFA excluded PrP from the preservative parabens used in food because of potential adverse effects (JECFA, 2007). The use of PrP and BuP was banned in young children's cosmetic products in Denmark due to their inherent endocrine properties (SCCS, 2011). Recently, a study in China regarding the estrogenicity of MeP and EtP has shown that exposure to high levels of MeP and EtP may have a high burden of estrogenicity-related diseases, and suggested that ADI of parabens established by the JECFA for protecting human health might be too high (Sun et al., 2016). Therefore, potential adverse health effects of paraben exposures might still exist for 3-year-old children in the present study. Similar to previous studies (Moos et al., 2015b; Wang et al., 2013), we found positive correlation between paraben levels. In particular, MeP and PrP were moderately correlated (r ¼ 0.40; p < 0.05), the both are the most common parabens and often used in combination in personal care products (Soni et al., 2005). Meanwhile, slightly weaker correlations between EtP and BuP concentrations suggested potential common sources of their exposure. Because parabens were often combined in use for a stronger antimicrobial activity, these correlations indicated simultaneous and concurrent exposures (Moos et al., 2015b).
4.2. Paraben exposures and anthropometric measures Scarce evidences on associations between exposure to parabens and anthropometric measures were available and the existing results were uncertain. For prenatal exposure, considering effects on promoting adipocyte differentiation, a birth cohort study found
Please cite this article in press as: Guo, J., et al., Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.12.040
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positive association between maternal urinary paraben concentrations and birth weight in 520 French boys (Philippat et al., 2014). Due to differences across subpopulations and exposure assessment methods, no associations of prenatal exposure to parabens with offspring size at birth among boys were observed in a smaller cohort (n ¼ 191) (Philippat et al., 2012). However, decreases in birth size measures were found in associations with maternal exposure to parabens, suggesting potential reproductive toxicity (Geer et al., 2016). The adverse effects of prenatal and postnatal exposures on child growth could be discrepant, thus our findings cannot be compared directly with previous studies. We also cannot exclude prenatal paraben exposures as possible contributors to these associations. Although both prenatal paraben exposure and birth size were simultaneously introduced to the models predicting postnatal body size may be overcorrecting, sensitive analysis showed no significant change was observed, which suggested that association between postnatal paraben exposures and body size measures still existed in consideration of prenatal exposure to parabens. Multiple environmental phenolic compounds have been verified to increase risk of weight gain by interfering with lipid metabolism. For example, bisphenol A exposure has been examined to be associated with obesity prevalence in children (Trasande et al., 2012). Consistent with numerous research findings about the environmental pollutants, we found that children's EtP exposure was positively associated with weight z score. Like reported by Xue et al. (2015), 3,4-DHB metabolite of parabens was shown to be associated with childhood obesity, but no association between paraben exposure and increase in BMI was obtained, maybe overweight or obesity elicited by parabens could be observed during later life or in a larger sample. For parabens, the effects on adipocyte differentiation, which was one of the most contributing process to obesity were still unclear in human. However, recent studies have shown that parabens could promote adipocyte differentiation by activating the glucocorticoid receptor or the PPARg in rodent cells, which were known as major mechanisms suggesting potential link to the development of weight gain (Hu et al., 2013; Taxvig et al., 2012; Pereira-Fernandes et al., 2013). Additionally, potential adipogenic effects of parabens are known to increase with an increase in the length of linear alkyl chain in vitro (Hu et al., 2013). It is possible that biological activity and mechanisms of action differ among the parabens, while associations between EtP (rather than other parabens) and body size measures may largely attribute to higher EtP concentrations in our study compared with EtP values in other studies (Xue et al., 2015; Geer et al., 2016). In addition to weight gain, our study also observed exposure to P EtP and parabens were positively associated with height z scores. So far, the underlying mechanisms of these associations have not been well demonstrated. Increased height with paraben exposures may most strongly rest on estrogenic effects, and partly on their disruption of thyroid function. Parabens are weakly estrogenic with a potency ranging from 104 to 107fold compared to the activity of 17 b-estradiol (Watanabe et al., 2013), which have been examined to stimulate bone growth even at low levels in experimental animal studies (Albin et al., 2012; Cutler, 1997). The positive associations in our study population might reflect the effects of exposure to parabens on height gain. The significant associations were found only for length and body weight but not BMI may be due to various reasons, including exposure dose, child's sex, exposure time window and timing of outcomes. Bisphenol A, as typical phenolic endocrine disrupting chemical, in animal and human studies, have been revealed its associations with postnatal weight gain and adiposity may be not apparent until sexual maturity (Harley et al., 2013; Wei et al., 2011). Parabens have also been considered as potential endocrine
disruptors with a lower estrogen receptor binding affinity (Boberg et al., 2010). In consideration of estrogenic action of parabens, adverse association between paraben exposure and body size may be notable with children grow up. Based on our results, adverse effects regarding proceeding development of obesity still need to be tracked during later life. 4.3. Influence of sex on paraben exposures and body sizes Parabens are potential endocrine disruptors with estrogen-like effects on body size may differ by sex (Shaw and DeCatanzaro, 2009). Similarly, we found positive associations between paraben exposures and growth outcomes in boys but not in girls. Previous studies indicated that biosynthesis and function of 17 b-estradiol, along with tissue distribution of estrogen receptors may vary by sex (Gillies and McArthur, 2010). Moreover, adverse effects on reproduction and development of parabens maybe selectively varied by gender in human and animals (Dodge et al., 2015; Meeker et al., 2011). In our study, the total EDIurine of EtP value was statistically significantly greater for males than for females (7.52 mg/kg-bw/day vs. 5.32 mg/kg-bw/day). These results collectively indicated that environmentally relevant levels of paraben exposures during childhood may produce developmental toxicity in endocrinesensitive targets differed by gender. 4.4. Strengths and limitations A great strength is that the participants in the current study were from a birth cohort with a large sample size, which minimize the bias from potential confounding factors compared to retrospective cross-sectional studies. We observed an increase in body size measures associated with parabens exposure among in 3-yearold children, and the associations differed by gender. However, some limitations in our study should also be noted. Although parabens in spot urine sample may reasonably represent individual exposure over several months (Smith et al., 2012), using spot urine samples may also cause exposure misclassification. A large sample size may partly minimize the error to draw a relatively accurate conclusion. Given environmental exposure levels to paraben for the general population are relatively stable, spot urine samples could represent the recent exposure for environmental parabens with short half-lives (Ye et al., 2006). Additionally, specific diet information was not sufficiently obtained in this study. Paraben concentrations may be a surrogate for lifestyles and dietary factors which are predictive of weight gain. The increased weights and heights of boys may be due to greater consumption of nutrients instead of the paraben in the food which would covary with consumption of paraben bearing foods. It is also possible that obese children metabolize paraben differently than normal weight children. 5. Conclusions We found exposure to parabens were associated with increase in anthropometric parameters in 3-year-old children. The present study strengthens the new evidence on associations of postnatal exposure to parabens with children's body sizes. Adverse effects of parabens on child growth during critical development time window deserve additional attention. Further researches are required regarding the safety of the paraben chemicals and underlying mechanisms of interfering with body growth. Conflicts of interest The authors declare that they have no actual or potential
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competing interests. Acknowledgments We are also very grateful for the participants in our birth cohort and the colleagues in Sheyang Maternal and Child Health Care Centre, Jiangsu Province (China). The authors acknowledge Dr. Yang Cao (Karolinska Institutet) for his assistance with revision and polishing the manuscript. This study was partly funded by Projects of International Cooperation and Exchange of the National Natural Science Foundation of China (NSFC-STINT 81611130087) and also supported by grants from Shanghai Project “3-Year Action” (GWIV27.3). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2016.12.040. References Albin, A., Niklasson, A., Westgren, U., Norjavaara, E., 2012. Estradiol and pubertal growth in girls. Horm. Res. Paediatr. 78, 218e225. Boberg, J., Taxvig, C., Christiansen, S., Hass, U., 2010. Possible endocrine disrupting effects of parabens and their metabolites. Reprod. Toxicol. 30, 301e312. ska, J., Wa˛ sowicz, W., 2014. Parabens. From environmental Błe˛ dzka, D., Gromadzin studies to human health. Environ. Int. 67, 27e42. ndez, M.F., Llop, S., Guxens, M., Ballester, F., Olea, N., Irurzun, M.B., Casas, L., Ferna ~ o, I., Tardo n, A., Vrijheid, M., Calafat, A.M., Sunyer, J., 2011. Rodríguez, L.S.M., Rian Urinary concentrations of phthalates and phenols in a population of Spanish pregnant women and children. Environ. Int. 37, 858e866. Cutler, G.J., 1997. The role of estrogen in bone growth and maturation during childhood and adolescence. J. Steroid Biochem. Mol. Biol. 61, 141e144. Dodge, L.E., Williams, P.L., Williams, M.A., Missmer, S.A., Toth, T.L., Calafat, A.M., Hauser, R., 2015. Paternal urinary concentrations of parabens and other phenols in relation to reproductive outcomes among couples from a fertility clinic. Environ. Health Perspect. 123, 665e671. Euser, A.M., de Wit, C.C., Finken, M.J.J., Rijken, M., Wit, J.M., 2008. Growth of preterm born children. Horm. Res. 70, 319e328. Frederiksen, H., Nielsen, J.K.S., Mørck, T.A., Hansen, P.W., Jensen, J.F., Nielsen, O., Andersson, A., Knudsen, L.E., 2013. Urinary excretion of phthalate metabolites, phenols and parabens in rural and urban Danish mother-child pairs. Int. J. Hyg. Environ. Health 216, 772e783. Geer, L.A., Pycke, B.F., Waxenbaum, J., Sherer, D.M., Abulafia, O., Halden, R.U., 2016. Association of birth outcomes with fetal exposure to parabens, triclosan and triclocarban in an immigrant population in Brooklyn, New York. J. Hazard Mater. http://dx.doi.org/10.1016/j.jhazmat.2016.03.028. Gillies, G.E., McArthur, S., 2010. Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol. Rev. 62, 155e198. Guo, Y., Wang, L., Kannan, K., 2014. Phthalates and parabens in personal care products from China: concentrations and human exposure. Arch. Environ. Contam. Toxicol. 66, 113e119. Harley, K.G., Aguilar, S.R., Chevrier, J., Tyler, K., Aguirre, H., Bradman, A., Holland, N.T., Lustig, R.H., Calafat, A.M., Eskenazi, B., 2013. Prenatal and postnatal bisphenol A exposure and body mass index in childhood in the CHAMACOS cohort. Environ. Health Perspect. 121, 514e520. Hines, E.P., Mendola, P., von Ehrenstein, O.S., Ye, X., Calafat, A.M., Fenton, S.E., 2015. Concentrations of environmental phenols and parabens in milk, urine and serum of lactating North Carolina women. Reprod. Toxicol. 54, 120e128. Hu, P., Chen, X., Whitener, R.J., Boder, E.T., Jones, J.O., Porollo, A., Chen, J., Zhao, L., 2013. Effects of parabens on adipocyte differentiation. Toxicol. Sci. 131, 56e70. JECFA (Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives), 1974. World Health Organization Technical Report Series. 17th Report of the Joint FAO/WHO Expert Committee on Food Additives, vol. 539. JECFA (Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives), 2007. World Health Organization Technical Report Series. 67th Report of the Joint FAO/WHO Expert Committee on Food Additives, vol. 940. Kang, H., Kyung, M., Ko, A., Park, J., Hwang, M., Kwon, J., Suh, J., Lee, H., Moon, G.I., Hong, J., Hwang, I.G., 2016. Urinary concentrations of parabens and their association with demographic factors: a population-based cross-sectional study. Environ. Res. 146, 245e251. €rklund, K., Palm, B., Wennberg, M., Kaj, L., Lindh, C.H., Larsson, K., Ljung Bjo €nsson, B.A.G., Berglund, M., 2014. Exposure determinants of phthalates, parJo abens, bisphenol A and triclosan in Swedish mothers and their children. Environ. Int. 73, 323e333.
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Urinary paraben concentrations and their associations with anthropometric measures of children aged 3 years* Jianqiu Guo a, Chunhua Wu a, *, Dasheng Lu a, b, Shuai Jiang a, b, Weijiu Liang c, Xiuli Chang a, Hao Xu c, Guoquan Wang b, Zhijun Zhou a a
School of Public Health, Key Laboratory of Public Health Safety of Ministry of Education, Collaborative Innovation Center of Social Risks Governance in Health, Fudan University, No.130 Dong'an Road, Shanghai 200032, China Shanghai Municipal Center for Disease Control and Prevention, No. 1380 Zhongshan West Road, Shanghai 200336, China c Shanghai Center for Disease Control and Prevention, No.39 Yunwushan Road, Changning District, Shanghai 200051, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 May 2016 Received in revised form 15 December 2016 Accepted 16 December 2016 Available online xxx
Parabens, known as ubiquitous preservatives, have been linked to adverse health outcomes in humans. This study aimed to examine urinary paraben concentrations of children at 3 years of age and evaluate their associations with anthropometric parameters. Urinary parabens including methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP) and benzylparaben (BeP) were measured among 436 children in a birth cohort using gas chromatography with tandem mass spectrometry. Generalized linear models were performed to evaluate associations of paraben exposures with age- and sex-specific z scores, including weight, height, weight for height and body mass index. MeP, EtP and PrP were the dominant parabens in urinary samples, with the median concentrations of 6.03 mg/L, 3.17 mg/L, 2.40 mg/L, respectively. The median values of estimated daily intake (EDIurine) of five urinary paraben concentrations were 12.10, 5.68, 4.50, 0.06 and 0.17 mg/kg-body weight/day, respectively. Urinary EtP concentrations were positively associated with weight z scores [regression coefficient b ¼ 0.16, 95% confidence interval (CI): 0.04, 0.29; p ¼ 0.01] and height z scores (b ¼ 0.15, 95% CI: 0.03, 0.27; p ¼ 0.01). Positive associations were found between the sum of molar concentrations of five parabens and height z scores among all children (b ¼ 0.24, 95% CI: 0.04, 0.45; p ¼ 0.02). These significant associations were only observed in boys. Our findings suggest that exposure to parabens may be adversely associated with physical growth in 3-year-old boy children. Further prospective studies are warranted to understand the toxicological mechanisms of paraben exposures and potential risk of children. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Parabens Exposure assessment Anthropometric measures Urine Child health
1. Introduction Parabens are a class of para-hydroxybenzoic acid with alkyl and aryl esters, which are the most extensively used preservatives in foodstuffs, pharmaceuticals, and personal care products worldwide (Błe˛ dzka et al., 2014). Commercially available parabens include methylparaben (MP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP) and benzylparaben (BeP). Human exposure to parabens through topical contaction with or ingestion of products containing parabens (Guo et al., 2014; Liao et al., 2013). Recently, parabens have been reported to be present in water, especially at
*
This paper has been recommended for acceptance by David Carpenter. * Corresponding author. E-mail address: [email protected] (C. Wu).
maximum concentrations of 3.14 mg/L in the Pearl River Delta and of 1.65 mg/L in urban surface water in Beijing, China, respectively (Li et al., 2016; Yu et al., 2011). The serious human exposure to parabens has led to extensive distribution in various human biological samples, including urine, serum, breast milk, placental tissue and amniotic fluid (Hines et al., 2015; Philippat et al., 2013; Valle-Sistac et al., 2016). Additionally, parabens in fatty components of human body tissues were also found, which suggested that bioaccumulation could potentially impact fat deposition (Wang et al., 2015). Previously regarded as safe (Soni et al., 2005), parabens have recently raised concern for the evidence of potential endocrine disruptors, with a lower estrogen receptor binding affinity than 17 b-estradiol (Boberg et al., 2010). Epidemiological studies have shown associations between urinary parabens and adverse health outcomes, including reproductive toxicity (Meeker et al., 2011),
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oxidative stress (Watkins et al., 2015), immune modulation (Savage et al., 2012) and even breast cancer (Pan et al., 2015). Moreover, parabens may also interfere with thyroid hormones and activate the peroxisome proliferator-activated receptorg(PPARg), which was a nuclear receptor superfamily playing a key role in adipogenesis and lipid accumulation (Pereira-Fernandes et al., 2013). Furthermore, a study had reported that parabens could promote development and differentiation of multipotent cells into mature adipocyte cells (Hu et al., 2013), indicating parabens may play a role in obesity epidemic. The long-term exposure to low-dose of parabens seemed alarming susceptibility for young children because of their immaturity of physiological functions, behaviors and potentially enhanced target organ sensitivity (SCCS, 2011). Limited epidemiological researches suggested inconsistent results that exposure to parabens was associated with adverse effects on body growth. Specifically, associations of gestational exposure to parabens with increase in birth weight (Philippat et al., 2014) and with reduction in birth weight and length (Geer et al., 2016) were found. Compared to prenatal exposure to parabens, relatively scarce data are available regarding the effects of postnatal exposure on body growth. Only one study revealed non-significant association of paraben exposure with childhood obesity, but a positive association between the paraben metabolite of 3,4-dihydroxy benzoic acid (3,4DHB) and obesity (Xue et al., 2015). Given the widespread exposure to parabens and possible endocrine disruption, it is imperative to examine the potential risk of exposure to these pollutants in relation to childhood obesity. Generally, parabens are rapidly absorbed, metabolized, and excreted in urine from the body (Moos et al., 2015a). Urinary paraben concentrations have been known to be valid biomarkers of exposure (Ye et al., 2006). Thus, the objectives of the present study are to assess parabens exposure of children and its potential associations with body growth at 3 years old in a birth cohort from an agricultural region in Jiangsu Province, China.
2.2. Urinary measurement A spot urine sample was collected from each child in the Health Care Centre and was transferred to the high-density polypropylene centrifuge tubes (Corning Incorporated, USA). All samples were immediately stored at 20 C, then frozen shipped to the laboratory and kept at 80 C until analysis. As previously described by Lu et al. (2015), urinary parabens including MeP, EtP, PrP, BuP and BeP were measured by largevolume-injection gas chromatography with tandem mass spectrometry (LVI-GC-MS/MS). Briefly, the urine samples were prepared by hydrochloric acid hydrolysis, liquid-liquid extraction, and solid-phase extraction clean up. After derivatization, the samples were analyzed by LVI-GC-MS/MS. The limits of detection (LODs) for metabolites were 0.01 mg/L, which was defined as a signal-to-noise ratio of three. To account for variability in urinary dilution, creatinine concentrations and specific gravity (SG) of urine samples for individuals were determined. Creatinine concentration was determined by ELx800 Universal Microplate Reader (wavelength 340e750 nm; BIO-TEK, USA), and SG was measured using a handheld refractometer (Atago PAL 10-S, Tokyo, Japan). 2.3. Estimated daily intake of parabens Estimated daily intake (EDIurine) of parabens was calculated based on the measured urinary concentrations of parabens and a simple steady-state toxicokinetic model. The EDIurine of parabens for children was performed using the formula (1) as described by Ma et al. (2013).
EDIurine ¼
50 C V BW
(1)
2. Materials and methods
where EDIurine (mg/kg bw/day) is the estimated daily intake of individual paraben; C (mg/L) is the measured concentration of urinary paraben; V (L/day) is daily urinary excretion rate as 0.6 L/day in this study (Pei and Wen, 2004). BW (kg) is children's body weight of the child.
2.1. Study participants and questionnaires
2.4. Child anthropometry
During July 2012eApril 2013, 498 children were recruited in our study when they visited Sheyang Maternal and Child Health Care Centre. All children's mothers had previously participated in our longitudinal cohort study during pregnancy at hospital. Questionnaire survey was administered to each participated child's caregiver by trained interviewers. Data covering child's sociodemographics, living environment and lifestyles was collected. Information regarding pregnancy and maternal health was obtained from medical records and questionnaires previously (Qi et al., 2012). Each caregiver had signed an informed consent form and agreed to donate child's urine sample. This study was carried out with the permission of the Ethics Committees of Fudan University. Of the 498 children, we excluded 33 children who had no or inadequate urine samples, 20 children who did not complete the questionnaires, two twins, two children who had congenital anomalies at birth, two children whose mothers suffered from serious pregnancy complication, and one child without body growth measures. Finally, 436 children at 3 years of age were enrolled in the present study. These children did not differ significantly from the initially recruited subjects on all attributes of interests, including demographic characteristics, parental and socioeconomic information (data not shown).
Anthropometry measurements of children were required without jacket and shoes by pediatric physicians who were blind to this research. Body weight was measured using a digital scale and rounded to 0.1 kg. Body height was measured to the nearest 0.1 cm using metal column height-measuring stands. The children were all stand straight against metal column. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Anthropometric outcomes were compared with sexspecific World Health Organization (WHO) child growth standards. Correspondingly, age- and sex-standardized z scores were generated using WHO child growth standards (WHO , 2006). Therefore, the final measures of body sizes were z scores for weight, height, weight for height and BMI. 2.5. Statistical analysis Generalized linear models were used to examine associations between SG-adjusted paraben concentrations and body growth outcomes. Individual paraben concentration and the sum molar P concentration of the five urinary paraben ( paraben) adjusted for SG were examined with the models. Furthermore, analyses for P quartiles of parabens were conducted separately to investigate potential non-monotonic exposure-response relationships. To
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explore the possibility whether associations were different between sexes, an interaction term (sex log transformed paraben concentration) was introduced into the models and a stratified analysis was further conducted. Due to skewed distribution of urinary paraben concentrations, log transformed values were used to perform univariate and multivariate analyses. The differences in paraben levels between children's sociodemographic characteristics were examined using Wilcoxon rank-sum test or Kruskal-Wallis rank sum test. Correlations between log transformed concentrations of urinary parabens were assessed using Pearson correlation coefficients. Paraben levels below the LOD were substituted by the value of LOD divided by the square root of two. Paraben concentrations were corrected for SG using the formula (2):
Pc ¼ P½ð1:016 1Þ=ðSG 1Þ
(2)
where Pc is SG-adjusted paraben concentrations (mg/L); P is unadjusted paraben levels (mg/L); SG is the specific gravity of the urine sample; 1.016 is the median level in the population of this study. Creatinine excretion is dependent on individual physiological factors including muscle mass and physical growth. Thereby SGadjusted concentrations were used, as recommended by Smith et al. (2013). Covariates were selected for the final statistical model in terms of sociodemographic and biological considerations, and in relation to concentrations of urinary parabens or measured body size of children (p < 0.1). The following covariates were included: maternal BMI, paternal BMI, child's sex, maternal education (
3
listed in Table 1. For children at 3 years of age, the mean body weight (mean ± standard deviation) was 16.2 ± 2.2 kg; the mean height was 99.7 ± 4.1 cm and BMI was 16.3 ± 1.4 kg/m2. Additionally, 69.7% of maternal education levels were less than high school, and 39.4% of the children were living in countryside. For body size measures adjusted for age and sex, no significant differences were observed between children's body size of and WHO reference parameters (data not shown). 3.2. Urinary paraben concentrations and estimated daily intake Table 2 shows the distribution of urinary paraben concentrations adjusted for both creatinine and SG among 436 children. MeP, EtP, PrP and BuP were found in over 94% urine samples, whereas BeP was only presented in 80.73% of samples. The geometric mean (GM) concentration (range) of MeP was 5.27 mg/L (
3. Results 3.1. Population characteristics The sociodemographic characteristics of mother-child pairs are
When prenatal concentration of parabens in maternal urine was adjusted, association of postnatal exposure to parabens with body size measures was virtually unchanged. Namely, all changes in the estimated regression coefficients were less than 5%. The inclusion
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J. Guo et al. / Environmental Pollution xxx (2016) 1e8 Table 1 Characteristics of mothers-child pairs at birth and follow-up.
Child characteristics Gender of child Male Female Birth weight (kg) Birth length (cm) Weight (kg) Height (cm) BMIa (kg/m2) BMI z scoreb Household characteristics Maternal pre-pregnancy BMI (kg/m2) Parental BMI (kg/m2) Maternal education level
1100 mother-newborn pairs (n% or mean ± SDc)
436 mother-child pairs (n % or mean ± SD)
589 (53.5) 511 (46.5) 3.5 ± 0.4 51.3 ± 2.3
221 (50.7) 215 (49.3) 3.4 ± 0.6 49.7 ± 8.7 16.2 ± 2.2 99.7 ± 4.1 16.3 ± 1.4 0.6 ± 1.0
21.4 ± 2.8 24.0 ± 3.9
21.4 ± 3.0 24.0 ± 3.9
702 (63.9) 397 (36.1)
304 (69.7) 131 (30.0)
398 (36.4) 227 (20.7) 470 (42.9)
130 (29.8) 134 (30.7) 172 (39.4)
Notes: missing values: weight (n ¼ 1), height (n ¼ 1), maternal education level (n ¼ 1). a BMI: body mass index (weight in kilogram divided by squared height in meter). b z score defined as growth indictors for age and sex according to the WHO growth reference. c SD: standard deviation.
Table 2 Distributions of urinary paraben concentrations in 436 children. Analyte
Unit
>LOD (%)
GM
GSD
Range
mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mg/L mg/g Cre. mg/L SG mmol/L mmol/mol Cre. mmol/LSG
97.73
5.27 12.59 6.26 3.32 7.94 4.03 2.98 7.13 3.58 0.04 0.09 0.05 0.10 2.25 0.13 0.16 43.11 0.19
7.41 7.94 7.83 9.33 10.00 9.03 6.03 6.30 6.20 3.63 3.98 3.65 7.59 7.94 7.87 3.71 3.89 3.63
Selected percentiles P25
P50
P75
P95
1.55 6.03 22.46 82.13 3.91 16.84 55.62 213.93 2.12 8.13 27.62 102.89 EtP 94.56 1.05 3.17 13.71 106.38 2.33 8.24 31.67 352.13 1.29 4.31 15.95 127.16 PrP 98.64 0.94 2.40 10.07 66.27 2.29 6.42 25.34 176.59 1.19 3.02 13.04 70.41 BuP 97.51 0.02 0.03 0.09 0.42 0.03 0.08 0.23 1.08 0.02 0.04 0.11 0.47 BeP 80.73 0.02 0.09 0.39 3.66 0.05 0.23 0.96 10.57 0.02 0.12 0.45 5.87 P PBs 0.06 0.16 0.41 1.43 17.92 41.86 105.50 455.17 0.08 0.19 0.45 1.59 P Abbreviations: MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; PBs: the sum of molar paraben concentrations; Cre.: creatinine; SG: specific gravity; LOD: limit of detection; GM: geometric mean; GSD: geometric standard deviation. MeP
Table 3 EDIurine (mg/kg-bw/day) of parabens calculated from urinary concentrations.
Male
Female
Total
GM 5th Percentile Median 95th Percentile GM 5th Percentile Median 95th Percentile GM 5th Percentile Median 95th Percentile
MeP
EtP
PrP
BuP
BeP
9.56 0.26 12.26 193.4 10.17 0.56 12.02 143.9 9.86 0.36 12.10 150.8
7.52 0.02 7.19 244.6 5.32 0.32 5.03 126.1 6.34 0.02 5.68 187.1
5.23 3.91 0.40 122.8 6.09 0.40 5.82 124.0 5.64 0.41 4.50 118.2
0.07 0.01 0.06 0.53 0.08 0.01 0.06 0.98 0.07 0.01 0.06 0.64
0.19 0.01 0.17 6.73 0.19 0.01 0.17 8.91 0.20 0.01 0.17 7.06
Abbreviations: EDIurine: estimated daily intake based on urinary paraben concentrations; MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; GM: geometric mean.
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Table 4 Associations between urinary paraben concentrations and changes in body size measures in 436 children. Weight z score
Height z score
Weight for height z score
BMI z score
b (95% CI)
p
b (95% CI)
p
b (95% CI)
p
b (95% CI)
p
0.08 (0.07, 0.23) 0.16 (0.04, 0.29) 0.01 (0.18, 0.16) 0.12 (0.12, 0.30) 0.07 (0.21, 0.07) 0.16 (0.05, 0.38)
0.32 0.01 0.92 0.42 0.34 0.14
0.09 0.15 0.02 0.12 0.05 0.24
(0.07, 0.23) (0.03, 0.27) (0.13, 0.18) (0.17, 0.32) (0.08, 0.19) (0.04, 0.45)
0.17 0.01 0.76 0.23 0.44 0.02
0.06 (0.26, 0.15) 0.13 (0.05, 0.26) 0.04 (0.21, 0.13) 0.03 (0.18, 0.25) 0.15 (0.29, 0.01) 0.06 (0.16, 0.28)
0.60 0.06 0.64 0.77 0.05 0.29
0.04 (0.13, 0.20) 0.12 (0.02, 0.26) 0.03 (0.20, 0.15) 0.04 (0.19, 0.26) 0.15 (0.30, 0.01) 0.05 (0.18, 0.28)
0.58 0.09 0.74 0.76 0.06 0.66
0.08 (0.06, 0.23) 0.16 (0.03, 0.28) 0.00 (0.16, 0.17) 0.12 (0.09, 0.32) 0.04 (0.18, 0.10) 0.17 (0.04, 0.39)
0.25 0.01 0.99 0.28 0.56 0.11
0.11 0.15 0.05 0.14 0.08 0.23
(0.02, 0.26) (0.03, 0.27) (0.11, 0.21) (0.06, 0.34) (0.06, 0.21) (0.03, 0.43)
0.11 0.01 0.56 0.16 0.28 0.02
0.04 (0.12, 0.19) 0.12 (0.01, 0.26) 0.05 (0.22, 0.13) 0.06 (0.15, 0.28) 0.12 (0.27, 0.02) 0.06 (0.16, 0.28)
0.65 0.06 0.58 0.56 0.10 0.58
0.04 (0.12, 0.19) 0.12 (0.02, 0.25) 0.04 (0.22, 0.14) 0.06 (0.15, 0.29) 0.12 (0.27, 0.03) 0.06 (0.17, 0.28)
0.64 0.09 0.66 0.56 0.11 0.62
0.00 (0.14, 0.14) 0.08 (0.07, 0.22) 0.07 (0.24, 0.09) 0.20 (0.46, 0.06) 0.12 (0.27, 0.03) 0.01 (0.23, 0.26)
0.95 0.30 0.38 0.13 0.13 0.91
0.03 (0.09, 0.16) 0.11 (0.02, 0.24) 0.04 (0.11, 0.19) 0.17 (0.41, 0.07) 0.08 (0.23, 0.05) 0.18 (0.05, 0.42)
0.65 0.10 0.63 0.16 0.22 0.13
0.02 (0.16, 0.13) 0.01 (0.14, 0.15) 0.15 (0.31, 0.02) 0.14 (0.41, 0.12) 0.09 (0.24, 0.06) 0.11 (0.34, 0.14)
0.84 0.90 0.08 0.30 0.23 0.38
0.02 0.01 0.15 0.14 0.09 0.14
0.79 0.95 0.07 0.31 0.22 0.30
a
Total MeP EtP PrP BuP BeP P PBs Maleb MeP EtP PrP BuP BeP P PBs Femaleb MeP EtP PrP BuP BeP P PBs
(0.17, (0.15, (0.32, (0.41, (0.25, (0.39,
0.13) 0.14) 0.02) 0.13) 0.06) 0.12)
P Abbreviations: MeP: methylparaben; EtP: ethylparaben; PrP: propylparaben; BuP: butylparaben; BeP: benzylparaben; PBs: the sum of molar paraben concentrations; z score defined as growth indictors for age and sex according to the WHO growth reference; CI: confidence interval. a Models were adjusted for maternal body mass index, paternal body mass index, child's gender, maternal education, family annual income, inhabitation, feeding pattern, smoking status, time spent playing outdoors, sampling season, child's sex log (each paraben) and birth outcome measures (weight, length or ponderal index). b Child's sex and child's sex log (each paraben) were excluded from models.
of several confounding factors such as urinary phenols did not alter the significantly positive associations between paraben exposures and anthropometric measures (data not shown). Additionally, positive associations were observed between urinary paraben concentrations and anthropometric indicators, no matter whether adjusted for SG or not. After exclusion of preterm births and low birth weight, the results did not change substantially (data not shown). 4. Discussion In our study, children were extensively exposed to five parabens with the wide range of EDIurine, especially for EDIurine values of MeP ranging from 0.01 to 1571.42 mg/kg-bw/day. We found that urinary EtP concentrations were positively associated with weight and height parameters of children. Moreover, the positive associations P between urinary paraben concentrations and anthropometric outcomes was significantly observed among boys. 4.1. Exposure assessment of parabens Parabens were widely concerned in urine samples. MeP, EtP and PrP were the dominant parabens, which was consistent with those reported in environmental monitoring and dietary exposure in China (Guo et al., 2014; Li et al., 2016). Parabens exposure in our study was comparable to those reported in some studies, except for EtP slightly higher than others (USCDC, 2015; Frederiksen et al., 2013; Larsson et al., 2014; Myridakis et al., 2015a,b; Wang et al., 2013) (seeing Supplemental Material, Table S2). However, there were a contrast cases. For example, the unadjusted MeP levels (150.0 mg/L) among 4 years old children in Spain were 25-fold higher than ours (Casas et al., 2011). The difference may be due to actual different exposure across disparate geographical regions and population lifestyles, as well as different socioeconomic characteristics and education levels or even the combination of all of these factors. Therefore, making comparisons across studies should be
cautioned because of variation from exposure assessment methods to study designs. Total EDIurine values of children in the present study were similar to those in Chinese young adults (Ma et al., 2013), and lower than those in Korean adults (Kang et al., 2016). In 1974, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) allocated the total acceptable daily intake (ADI) of 0e10 mg/kg-bw/day for the sum of MeP, EtP and PrP (JECFA, 1974). While in 2007, the JECFA excluded PrP from the preservative parabens used in food because of potential adverse effects (JECFA, 2007). The use of PrP and BuP was banned in young children's cosmetic products in Denmark due to their inherent endocrine properties (SCCS, 2011). Recently, a study in China regarding the estrogenicity of MeP and EtP has shown that exposure to high levels of MeP and EtP may have a high burden of estrogenicity-related diseases, and suggested that ADI of parabens established by the JECFA for protecting human health might be too high (Sun et al., 2016). Therefore, potential adverse health effects of paraben exposures might still exist for 3-year-old children in the present study. Similar to previous studies (Moos et al., 2015b; Wang et al., 2013), we found positive correlation between paraben levels. In particular, MeP and PrP were moderately correlated (r ¼ 0.40; p < 0.05), the both are the most common parabens and often used in combination in personal care products (Soni et al., 2005). Meanwhile, slightly weaker correlations between EtP and BuP concentrations suggested potential common sources of their exposure. Because parabens were often combined in use for a stronger antimicrobial activity, these correlations indicated simultaneous and concurrent exposures (Moos et al., 2015b).
4.2. Paraben exposures and anthropometric measures Scarce evidences on associations between exposure to parabens and anthropometric measures were available and the existing results were uncertain. For prenatal exposure, considering effects on promoting adipocyte differentiation, a birth cohort study found
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positive association between maternal urinary paraben concentrations and birth weight in 520 French boys (Philippat et al., 2014). Due to differences across subpopulations and exposure assessment methods, no associations of prenatal exposure to parabens with offspring size at birth among boys were observed in a smaller cohort (n ¼ 191) (Philippat et al., 2012). However, decreases in birth size measures were found in associations with maternal exposure to parabens, suggesting potential reproductive toxicity (Geer et al., 2016). The adverse effects of prenatal and postnatal exposures on child growth could be discrepant, thus our findings cannot be compared directly with previous studies. We also cannot exclude prenatal paraben exposures as possible contributors to these associations. Although both prenatal paraben exposure and birth size were simultaneously introduced to the models predicting postnatal body size may be overcorrecting, sensitive analysis showed no significant change was observed, which suggested that association between postnatal paraben exposures and body size measures still existed in consideration of prenatal exposure to parabens. Multiple environmental phenolic compounds have been verified to increase risk of weight gain by interfering with lipid metabolism. For example, bisphenol A exposure has been examined to be associated with obesity prevalence in children (Trasande et al., 2012). Consistent with numerous research findings about the environmental pollutants, we found that children's EtP exposure was positively associated with weight z score. Like reported by Xue et al. (2015), 3,4-DHB metabolite of parabens was shown to be associated with childhood obesity, but no association between paraben exposure and increase in BMI was obtained, maybe overweight or obesity elicited by parabens could be observed during later life or in a larger sample. For parabens, the effects on adipocyte differentiation, which was one of the most contributing process to obesity were still unclear in human. However, recent studies have shown that parabens could promote adipocyte differentiation by activating the glucocorticoid receptor or the PPARg in rodent cells, which were known as major mechanisms suggesting potential link to the development of weight gain (Hu et al., 2013; Taxvig et al., 2012; Pereira-Fernandes et al., 2013). Additionally, potential adipogenic effects of parabens are known to increase with an increase in the length of linear alkyl chain in vitro (Hu et al., 2013). It is possible that biological activity and mechanisms of action differ among the parabens, while associations between EtP (rather than other parabens) and body size measures may largely attribute to higher EtP concentrations in our study compared with EtP values in other studies (Xue et al., 2015; Geer et al., 2016). In addition to weight gain, our study also observed exposure to P EtP and parabens were positively associated with height z scores. So far, the underlying mechanisms of these associations have not been well demonstrated. Increased height with paraben exposures may most strongly rest on estrogenic effects, and partly on their disruption of thyroid function. Parabens are weakly estrogenic with a potency ranging from 104 to 107fold compared to the activity of 17 b-estradiol (Watanabe et al., 2013), which have been examined to stimulate bone growth even at low levels in experimental animal studies (Albin et al., 2012; Cutler, 1997). The positive associations in our study population might reflect the effects of exposure to parabens on height gain. The significant associations were found only for length and body weight but not BMI may be due to various reasons, including exposure dose, child's sex, exposure time window and timing of outcomes. Bisphenol A, as typical phenolic endocrine disrupting chemical, in animal and human studies, have been revealed its associations with postnatal weight gain and adiposity may be not apparent until sexual maturity (Harley et al., 2013; Wei et al., 2011). Parabens have also been considered as potential endocrine
disruptors with a lower estrogen receptor binding affinity (Boberg et al., 2010). In consideration of estrogenic action of parabens, adverse association between paraben exposure and body size may be notable with children grow up. Based on our results, adverse effects regarding proceeding development of obesity still need to be tracked during later life. 4.3. Influence of sex on paraben exposures and body sizes Parabens are potential endocrine disruptors with estrogen-like effects on body size may differ by sex (Shaw and DeCatanzaro, 2009). Similarly, we found positive associations between paraben exposures and growth outcomes in boys but not in girls. Previous studies indicated that biosynthesis and function of 17 b-estradiol, along with tissue distribution of estrogen receptors may vary by sex (Gillies and McArthur, 2010). Moreover, adverse effects on reproduction and development of parabens maybe selectively varied by gender in human and animals (Dodge et al., 2015; Meeker et al., 2011). In our study, the total EDIurine of EtP value was statistically significantly greater for males than for females (7.52 mg/kg-bw/day vs. 5.32 mg/kg-bw/day). These results collectively indicated that environmentally relevant levels of paraben exposures during childhood may produce developmental toxicity in endocrinesensitive targets differed by gender. 4.4. Strengths and limitations A great strength is that the participants in the current study were from a birth cohort with a large sample size, which minimize the bias from potential confounding factors compared to retrospective cross-sectional studies. We observed an increase in body size measures associated with parabens exposure among in 3-yearold children, and the associations differed by gender. However, some limitations in our study should also be noted. Although parabens in spot urine sample may reasonably represent individual exposure over several months (Smith et al., 2012), using spot urine samples may also cause exposure misclassification. A large sample size may partly minimize the error to draw a relatively accurate conclusion. Given environmental exposure levels to paraben for the general population are relatively stable, spot urine samples could represent the recent exposure for environmental parabens with short half-lives (Ye et al., 2006). Additionally, specific diet information was not sufficiently obtained in this study. Paraben concentrations may be a surrogate for lifestyles and dietary factors which are predictive of weight gain. The increased weights and heights of boys may be due to greater consumption of nutrients instead of the paraben in the food which would covary with consumption of paraben bearing foods. It is also possible that obese children metabolize paraben differently than normal weight children. 5. Conclusions We found exposure to parabens were associated with increase in anthropometric parameters in 3-year-old children. The present study strengthens the new evidence on associations of postnatal exposure to parabens with children's body sizes. Adverse effects of parabens on child growth during critical development time window deserve additional attention. Further researches are required regarding the safety of the paraben chemicals and underlying mechanisms of interfering with body growth. Conflicts of interest The authors declare that they have no actual or potential
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competing interests. Acknowledgments We are also very grateful for the participants in our birth cohort and the colleagues in Sheyang Maternal and Child Health Care Centre, Jiangsu Province (China). The authors acknowledge Dr. Yang Cao (Karolinska Institutet) for his assistance with revision and polishing the manuscript. This study was partly funded by Projects of International Cooperation and Exchange of the National Natural Science Foundation of China (NSFC-STINT 81611130087) and also supported by grants from Shanghai Project “3-Year Action” (GWIV27.3). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2016.12.040. References Albin, A., Niklasson, A., Westgren, U., Norjavaara, E., 2012. Estradiol and pubertal growth in girls. Horm. Res. Paediatr. 78, 218e225. Boberg, J., Taxvig, C., Christiansen, S., Hass, U., 2010. Possible endocrine disrupting effects of parabens and their metabolites. Reprod. Toxicol. 30, 301e312. ska, J., Wa˛ sowicz, W., 2014. Parabens. From environmental Błe˛ dzka, D., Gromadzin studies to human health. Environ. Int. 67, 27e42. ndez, M.F., Llop, S., Guxens, M., Ballester, F., Olea, N., Irurzun, M.B., Casas, L., Ferna ~ o, I., Tardo n, A., Vrijheid, M., Calafat, A.M., Sunyer, J., 2011. Rodríguez, L.S.M., Rian Urinary concentrations of phthalates and phenols in a population of Spanish pregnant women and children. Environ. Int. 37, 858e866. Cutler, G.J., 1997. The role of estrogen in bone growth and maturation during childhood and adolescence. J. Steroid Biochem. Mol. Biol. 61, 141e144. Dodge, L.E., Williams, P.L., Williams, M.A., Missmer, S.A., Toth, T.L., Calafat, A.M., Hauser, R., 2015. Paternal urinary concentrations of parabens and other phenols in relation to reproductive outcomes among couples from a fertility clinic. Environ. Health Perspect. 123, 665e671. Euser, A.M., de Wit, C.C., Finken, M.J.J., Rijken, M., Wit, J.M., 2008. Growth of preterm born children. Horm. Res. 70, 319e328. Frederiksen, H., Nielsen, J.K.S., Mørck, T.A., Hansen, P.W., Jensen, J.F., Nielsen, O., Andersson, A., Knudsen, L.E., 2013. Urinary excretion of phthalate metabolites, phenols and parabens in rural and urban Danish mother-child pairs. Int. J. Hyg. Environ. Health 216, 772e783. Geer, L.A., Pycke, B.F., Waxenbaum, J., Sherer, D.M., Abulafia, O., Halden, R.U., 2016. Association of birth outcomes with fetal exposure to parabens, triclosan and triclocarban in an immigrant population in Brooklyn, New York. J. Hazard Mater. http://dx.doi.org/10.1016/j.jhazmat.2016.03.028. Gillies, G.E., McArthur, S., 2010. Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol. Rev. 62, 155e198. Guo, Y., Wang, L., Kannan, K., 2014. Phthalates and parabens in personal care products from China: concentrations and human exposure. Arch. Environ. Contam. Toxicol. 66, 113e119. Harley, K.G., Aguilar, S.R., Chevrier, J., Tyler, K., Aguirre, H., Bradman, A., Holland, N.T., Lustig, R.H., Calafat, A.M., Eskenazi, B., 2013. Prenatal and postnatal bisphenol A exposure and body mass index in childhood in the CHAMACOS cohort. Environ. Health Perspect. 121, 514e520. Hines, E.P., Mendola, P., von Ehrenstein, O.S., Ye, X., Calafat, A.M., Fenton, S.E., 2015. Concentrations of environmental phenols and parabens in milk, urine and serum of lactating North Carolina women. Reprod. Toxicol. 54, 120e128. Hu, P., Chen, X., Whitener, R.J., Boder, E.T., Jones, J.O., Porollo, A., Chen, J., Zhao, L., 2013. Effects of parabens on adipocyte differentiation. Toxicol. Sci. 131, 56e70. JECFA (Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives), 1974. World Health Organization Technical Report Series. 17th Report of the Joint FAO/WHO Expert Committee on Food Additives, vol. 539. JECFA (Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives), 2007. World Health Organization Technical Report Series. 67th Report of the Joint FAO/WHO Expert Committee on Food Additives, vol. 940. Kang, H., Kyung, M., Ko, A., Park, J., Hwang, M., Kwon, J., Suh, J., Lee, H., Moon, G.I., Hong, J., Hwang, I.G., 2016. Urinary concentrations of parabens and their association with demographic factors: a population-based cross-sectional study. Environ. Res. 146, 245e251. €rklund, K., Palm, B., Wennberg, M., Kaj, L., Lindh, C.H., Larsson, K., Ljung Bjo €nsson, B.A.G., Berglund, M., 2014. Exposure determinants of phthalates, parJo abens, bisphenol A and triclosan in Swedish mothers and their children. Environ. Int. 73, 323e333.
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