Nonylphenol in pregnant women and their matching fetuses: Placental transfer and potential risks of infants

Environmental Research 134 (2014) 143–148

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Nonylphenol in pregnant women and their matching fetuses: Placental transfer and potential risks of infants Yu-Fang Huang a,b,1, Pei-Wei Wang c,1, Li-Wei Huang d, Winnie Yang c, Ching-Jung Yu a, Shang-Han Yang a, Hsin-Hao Chiu a, Mei-Lien Chen a,n a

Institute of Environmental and Occupational Health Sciences, National Yang Ming University, No. 155, Sec. 2, Li-Nong St., Beitou, Taipei, Taiwan Department of Education and Research, Taipei City Hospital, Taipei, Taiwan c Department of Pediatrics, Taipei City Hospital, Taipei, Taiwan d Department of OBS & GYN, Taipei City Hospital, Women and Children's Campus, Taipei, Taiwan b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 March 2014 Received in revised form 23 June 2014 Accepted 11 July 2014 Available online 13 August 2014

As the predominant environmental biodegradation product of nonylphenol (NP) ethoxylates and with proven estrogenic effects, NP is formed during the alkylation process of phenols. The purposes of this study were (1) to examine maternal and prenatal exposure to NP in Taiwan, (2) to determine the level of placental protection against NP exposure as well as the level of NP in breast milk, and (3) to assess the potential risk for breastfed newborns exposed to NP through the milk. Thirty pairs of maternal and fetal blood samples, placenta, and breast milk during the 1st and the 3rd months of lactation were collected. External NP exposures of these specimens were then analyzed by using high-performance liquid chromatography coupling with fluorescence detection. Next, the sociodemographics, lifestyle, delivery method, dietary and work history were collected using a questionnaire. In addition, the daily intake of NP from consuming breast milk in the 1st and 3rd months for newborns was studied through deterministic and probabilistic risk assessment methods. The geometric means and geometric standard deviation of NP levels in maternal blood, fetal cord blood, placenta, and breast milk in the 1st and 3rd months were 14.6 (1.7) ng/ml, 18.8 (1.8) ng/ml, 19.8 (1.9) ng/g, 23.5 (3.2) ng/ml, and 57.3 (1.4) ng/ml, respectively. The probabilistic percentiles (50th, 75th, and 95th) of daily intake NP in breast milk were 4.33, 7.79, and 18.39 μg/kg-bw/day in the 1st month, respectively, and were 8.11, 10.78, 16.08 μg/kg-bw/day in the 3rd month, respectively. The probabilistic distributions (5th, 25th, and 50th) of risk for infants aged 1 month old were 0.27, 0.64, and 1.15, respectively, and that for infants aged 3 month old were 0.31, 0.46, and 0.62, respectively. Through repeated exposure from the dietary intake of expectant mothers, fetuses could encounter a high NP exposure level due to transplacental absorption, partitioning between the maternal and fetal compartments. Daily NP intake via breast milk in three month-old babies exceeded the tolerable daily intake (TDI) of 5 mg/kg bw/day indicated a potential risk for Taiwan infants. & 2014 Elsevier Inc. All rights reserved.

Keywords: Nonylphenol Mother Foetus Placenta Breast milk Risk

1. Introduction Alkylphenol polyethoxylates (APEOs), especially nonylphenol polyethoxylates (NPEOs) and octylphenol polyethoxylates (OPEOs), are a widely used class of non-ionic surfactants (Ying, 2006). Annual global production of APEOs is approximately 650,000 t (Guenther et al., 2002). NPEOs represent about 80% of APEOs, while OPEOs make up most of the remaining 20% (White et al., 1994). NPEOs are used as

n

Corresponding author. Fax: þ886 22827 8254. E-mail address: [email protected] (M.-L. Chen). 1 Yu-Fang Huang and Pei-Wei Wang are co-first authors.

http://dx.doi.org/10.1016/j.envres.2014.07.004 0013-9351/& 2014 Elsevier Inc. All rights reserved.

emulsifying, dispersing, wetting and foaming agents in various industries in Taiwan. Ding et al. indicated that NPE-type residues in Taiwanese rivers (0.6–2.4 μg/l) and sediments (250–8580 μg/kg dry wt) were higher than in other countries, owing to deficient municipal wastewater treatment in Taiwan (Ding and Fann, 2000; Ding and Tzing, 1998; Ding et al., 1999; Ding and Wu, 2000). Cheng and Ding (2002) also found NPEOs were detected in 41% of 90 household detergents at levels from 0.2% to 21% (Cheng and Ding, 2002). As an alkylphenol used mainly as intermediates in manufacturing, NP is also a degradation product of NPEOs used in industrial and institutional formulation. Mao et al. (2006) found that NP is ubiquitous in Taiwanese foods (Mao et al., 2006). In addition, biological monitoring revealed significant levels of NPs in both plasma and urine of pregnant

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women and newborns, pubertal students, as well as textile and housekeeping workers in Taiwan (Chang et al., 2013; Chen et al., 2008; Chen et al., 2009; Chen et al., 2005; Tsai et al., 2013). Attention has been drawn since Soto et al. found that NP induced cell proliferation and bound to estrogen receptor in human estrogensensitive MCF7 breast tumor cells (Soto et al., 1991). Laws et al. (2000) posited that NP can compete with estradiol or promegestone for estrogen and progesterone receptor binding (Laws et al., 2000). Xenoestrogen exposure may have serious consequences, especially for pregnant women, who are especially susceptible and vulnerable, and their fetuses. Notably, estrogen production plays an important role in promoting the expression of critical growth factors for placental villous angiogenesis and in regulating the processes of growth and development in the fetus (Albrecht and Pepe, 2010; Fujimoto et al., 2005). The adverse effects of NP include inhibition of gonadal development, low testis and epididymis mass, inhibition of ovarian development, and reduction in offspring viability (De Jager et al., 1999; Fan et al., 2001; Ferguson et al., 2000; Harris et al., 2001; Holdway et al., 2008; Jie et al., 2010; LeBlanc et al., 2000; Nagao et al., 2001; Yokota et al., 2001). NP exposure in utero may influence the weight of the infant's body and some reproductive organs, possibly inducing neurotoxic and reproductive toxic effects in the offspring (Kimura et al., 2006). Our previous studies demonstrated that a high maternal NP level was associated with small size for gestational age (SGA), decreased body length at birth, increased risk of low neonatal birth weight (Chang et al., 2013; Tsai et al., 2013), and disturbed pubertal development, especially in adolescent girls (Chen et al., 2009). As for their ubiquitous distribution, humans may be exposed to NP not only by drinking water, but also by other pathways, including ingestion of contaminated foods, such as seafood, fish, meat and milk; inhalation with air; and dermal absorption (Ahel et al., 1993; Clark et al., 1992). The question arises whether NPs circulating in an expectant mother's body pass through the placenta, eliciting possible estrogenic effects on developing fetuses. Previous studies identified 2,3,7,8-TCDD, PCBs, bisphenol A (BPA), NP, octylphenol, and phthalates in cord blood (Latini et al., 2003; Tan and Ali Mohd, 2003; Wang et al., 2004), indicating that almost all harmful chemicals in maternal circulation can pass to a fetus. In the pharmacokinetic study of NP in humans by Muller et al. (1998), the half-life of NP in blood and the bioavailability were found to be 2–3 h and 20% (Müller et al., 1998). The biological half-life of NP is quite short, suggesting that the accumulation of NP may be insignificant. However, the repeated exposure of a fetus to NP due to dietary intake and the increased amounts of NP taken by pregnant women is of concern if a placenta barrier to NP does not exist. The existence of NP in breast-milk has received increasing interest since human milk is the most important nourishment for newborns and reveals both parent and neonate exposure. As our current knowledge, the number of reports on NP levels in human milk is limited (Ademollo et al., 2008; Chen et al., 2010; Lin et al., 2009; Otaka et al., 2003), not to mention to study potential risk for NP of newborns through milk consumption. Therefore, in addition to examining maternal and prenatal exposure to NP in Taiwan, this study attempts to determine the level of placental protection against NP exposure as well as the level of NP in mother milk. The potential risk caused by exposure of NP to breastfed newborns one and three months-old through the milk is also assessed.

well as completed a structured questionnaire. The questionnaire collected data on sociodemographic characteristics (i.e. age, weight, height, and education level), lifestyle (i.e. frequency of using detergent and plastic products, consumption of healthy food and medications), and semi-quantitative measures of dietary consumption (i.e. intake of meat, vegetables, fruit, tea, and coffee). In total, 30 pregnant women from the TCH in metropolitan Taipei in northern Taiwan agreed to participate in this study from May to December 2013. Maternal venous blood was collected before delivery; fetal umbilicalcord blood and placenta were collected upon delivery at the hospital; and breast milk was collected during the 1st and 3rd month of lactation. All samples were immediately chilled and transported to the laboratory. Plasma was fractioned by centrifugation at 3000 rpm for 15 min and all samples were stored at  80 1C until further laboratory analysis.

2.2. Samples extraction and analysis 2.2.1. Reagents Analytical grade ammonium acetate (498%), acetic acid (100%), hydrochloric acid (37%), methanol (499.8%), acetonitrile (99.9%), n-hexane (498%), β-glucuronidase/ arylsulfatase (5.2 U/ml/ 2.1 U/ml) were purchased from Merck (Darmstadt, Germany) and 4-nonylphenol (p-isomers485%) were purchased from Fluka (Japan). Florisil (60/ 100 mesh) was purchased from Supelco (USA). A Millipore water purification device (Millipore, Bedford, MA) supplied ultrapure water. All water was prepared freshly before use and collected in a glass container.

2.2.2. Instrumentation A Hitachi (Tokyo) LC system was used for NP analysis. This system comprised an L-6200 intelligent pump, L-7200 auto-sampler, F-4010 fluorescence detector, L6100 interface for linking the detector, and D-6000 data management software. The software ran on a Copam computer (Taiwan) for online recording of the output.

2.2.3. Samples pretreatment and cleanup 2.2.3.1. Maternal and umbilical cord plasma. The plasma samples cleanup procedure of Inoue et al. (2000) (Inoue et al., 2000) and the enzymatic deconjugation of Muller et al. (1998) were adopted with some modifications (Chen et al., 2005). Briefly, 1 g of homogenized plasma was diluted with 9 ml of ultra pure water and then pH value was adjusted to 5.5 with acetic acid. Next, 1 ml of 1 M ammonium acetate solution (pH 5.3) and 125 μl β-glucuronidase/arylsulfatase were added. The mixture was incubated for 15 h at 37 1C in a shaker bath and acidified to pH 3 with 1 N hydrochloric acid (HCl). Following enzymatic deconjugation, samples were cleaned up with 3 ml Varian PH solid phase extraction (SPE) cartridges. The SPE cartridges were inserted with 2 cm silanized glasswool and were preconditioned with 20 ml methanol followed by 3 ml pure water acidified by HCl. After samples application, the cartridges were washed with 5 ml pure water. Finally, analytes were eluted with 3 ml methanol. To extend the lifespan of the HPLC column, all samples were filtered through a 5 μm PTFE membrane filter (Titin, USA).

2.2.3.2. Placenta. The placenta samples were prepared according to a procedure described in detail elsewhere (Wang et al., 2007). Briefly, a placenta sample of 5 g was dissolved in 60 ml of acetonitrile and extracted in acetonitrile/n-hexane, and pooled organic solvents were clean-up using packing column with 5 g of Florisil. After conditioning the column with 50 ml of n-hexane, the samples were passed through and then eluted with 40 ml of mixed n-hexane and acetone (7:3, v/v) solution. Finally, the eluate was dried with rotary vacuum evaporator in a water bath at 60 1C and was dissolved with 3 ml of methanol, followed by HPLC analysis.

2.2.3.3. Human breast milk. Breast milk samples were extracted following the same method as that for placenta samples except that a 10 g breast milk sample was analyzed.

2.2.4. Analytical conditions The reverse-phase column was a Luna C18-A (150  4.6 mm i.d.) with a particle size of 5 μm (Phenomenex, USA). The isocratic mobile phase was a mixture of acetonitrile: water (75:25, v/v) with a flow rate of 1.0 ml/min. The fluorescence detector was operated with an excitation wavelength of 275 nm and emission wavelength of 300 nm. The samples were injected in quantities of 50 μL.

2. Materials and methods 2.1. Study populations and data collection This study received approval from the Ethics Committee of Taipei City Hospital, Taipei (TCH). Before delivery, expectant mothers at the 3rd trimester of pregnancy provided written informed consent to participate in the study, agreed to provide venous blood, cord blood, placentas and breast milk in the 1st and 3rd months of lactation, as

2.2.5. QA/QC The method was validated by Chen et al. (2005) and Chen et al. (2008). No plastic product was used in the sample pretreatment. The average recoveries of blood, placenta, and breast-milk samples were 80–115%, 81–99%, and 98–105%, respectively, for NP concentrations of 5–500 ng/ml. The detection limit of the analytical method was 1.87 ng/ml, and the R2 for the standard curve was greater than 0.992.

Y.-F. Huang et al. / Environmental Research 134 (2014) 143–148

2.3. Exposure assessment and probabilistic analysis The daily intake of NP for breast milk consuming infants 1 and 3 months old was studied through both deterministic and probabilistic risk assessment methods. The daily intake of NP for breastfed infants was estimated by calculating the level of NP in breast milk, which was then multiplied with the quantity human milk taken daily, and divided by body weight. The equation was expressed as follows: daily intake of NP (μg/kg-bw/day)¼ (NP levels of breast milk in the 1st or 3rd month (μg/ml)  daily quantity of breast milk consumed by newborns at the age of 1st or 3rd month (ml/day))/ body weight (Kg). The average daily quantity of breast milk consuming infants 1 and 3 months old is approximately 165.0 and 142.5 ml/kg/day (W.H.O. and FAO, 2014). Probabilistic analysis (Monte Carlo analysis) was performed to characterize variability and uncertainty in the exposure assumptions, as well as quantify the range of theoretical exposure dose. Probability density functions for each parameter were log-normally distributed. In addition, the boundaries of input values randomly selected in Monte Carlo simulations were defined using the mean point estimates. Monte Carlo simulations were run 5000 times using Microsoft Excel™ 2007 and Oracle Crystal Ball, Fusion Edition, Release 11.1.2.1.000 software (Oracle Corporation, USA).

2.4. Risk characterization Potential risks of infants one and three months-old derived from the consumption of mother's breast milk were assessed by comparing the tolerable daily intake (TDI) of 5 μg/kg-bw/day, as proposed by the Danish Institute of Safety and Toxicology, with the percentile exposures (50th, 75th, and 95th) of NP. The equation was calculated as follows: potential risk ¼ TDI/ average daily exposure. A situation in which this value is greater than one suggests that exposure to NP is less likely to pose a significant health risk to the infants. However, a potential risk may exist if a risk less than one indicates that the exposure level exceeds TDI.

2.5. Statistical analysis The levels of NP in maternal and fetal blood, placenta, and breast milk samples were skewed. Thus all measurements were subjected to log transformation prior to statistical analyses. The significance of differences in NP level between motherfetus pairs was then investigated using the Wilcoxon matched–pairs signed-ranks test. Next, the relationship between NP levels for mothers and their fetus was examined using the Spearman correlation coefficient. Statistical analysis was performed using SPSS 17.0.

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the NP levels in mother breast milk in the 3rd month and fetal cord blood (p ¼0.002, Wilcoxon matched–pairs signed-ranks test). Meanwhile, the NP concentrations significantly differed in maternal blood and breast milk in the 1st and 3rd months (p¼ 0.041 and 0.001), and in placenta and breast milk in the 3rd month (p ¼0.001), respectively. Similar results was also observed for NP concentrations in breast milk between the 1st and 3rd months (p ¼0.0011). Based on the average level of NP in the 1st and 3rd months of breast-milk feeding, the daily intake of NP by infants one and three months-old was estimated as 6.1 and 8.6 μg/kg-bw/day, respectively. Assuming the average daily breast milk NP levels in the 1st and 3rd months were 36.8 and 60.5 ng/ml, respectively, and the average daily quantity of breast milk consumed by infants one and three months old were 702.1 and 825.2 ml/day (WHO and FAO, 2014), given that body weight in one and three months-old infants were 4.3 and 5.8 kg, respectively, then the daily intake X¼ (36.8  10–3 μg/ml  702.1 ml/day)/4.3 kg and X¼ (60.5  10  3 μg/ml  825.2 ml/day)/5.8 kg, we obtain a daily intake, X¼ 6.1 and 8.6 μg. In order to avoid an underestimation of exposure, a Monte Carlo analysis was performed to quantify the range of theoretical exposure dose of NP. Fig. S1 shows the cumulative distribution from describing the daily intake of NP through the consumption of mother's breast milk for infants one and three months-old. The probabilistic percentiles (50th, 75th, and 95th) of daily intake NP in breast milk were 4.33, 7.79, and 18.39 μg/kg-bw/day in the 1st month, respectively, and were 8.11, 10.78, 16.08 μg/kg-bw/day in the 3rd month, respectively. Health risks in infants associated with exposure to NP through the consumption of mother's breast milk were assessed using point and probabilistic analysis. The health risk was calculated as TDI of 5 μg/kg-bw/day divided with average daily exposure to NP. Here, the exposure point estimate and probabilistic distributions (5th, 25th, and 50th) of risk for infants one month-old were 0.82, 0.27, 0.64, and 1.15, respectively, and that for infants three monthold were 0.58, 0.31, 0.46, and 0.62, respectively.

3. Results Table 1 shows the characteristics of expectant mothers and newborns. The pregnant women had a mean age of 33.8 years old and an average body mass index (BMI) at delivery of 25.7 Kg/m2. Most pregnant women (27/30, 70%) had a bachelor's degree or higher. No women smoked cigarettes or drank alcohol during gestation. Of the 32 newborns, 12 were male and 20 were female. Average birth weight in the 1st and 3rd month, length, and circumference were 4255 7496 g, 5791 71101 g, 49 72 cm, and 34 71 cm, respectively. All newborns had Apgar scores≧7 in five minutes. To examine whether maternal BMI affect NP exposure in pregnant women, the association between maternal BMI and NP levels in maternal blood, placenta, and human milk were analyzed using Spearman correlation. It was found that maternal BMI showed inverse relationship to NP levels in maternal blood (r ¼  0.18), placenta (r ¼ 0.06), and human milk (r ¼  0.08 and 0.05 for the 1st and the 3rd month, respectively). Table 2 shows the median, geometric means (GM), geometric standard deviation (GSD), mean 7SD, range, and positive detection of NP levels in maternal and fetal blood, placenta, and human breast milk. NPs were detectable in most of all samples, except for human milk in the 1st month with a detection rate of 93.7%. Higher GM (GSD) levels of NP were found in maternal breast milk in the 3rd month than that in breast milk in the 1st month, placenta, fetal blood, and maternal blood with levels of 57.3 (1.4) ng/ml, 23.5 (3.2) ng/ml, 19.8 (1.9) ng/g, 18.8 (1.8) ng/ml, and, 14.6 (1.7) ng/ml, respectively. There was significant difference between

Table 1 General characteristics of pregnant women and their newborns. n

(%)

Mean 7 SD

Mother (n ¼30) Age (yrs) 33.873.4 o 30 5 16.7 30–34 14 46.7 4 34 11 36.7 2 Maternal BMI at delivery (Kg/m ) 25.7 74.3 18.5–24.9 16 50 ≧25 16 50 Educational level Senior high 3 10.0 College 19 63.3 Graduate 8 26.7 Weeks of gestation (wks) 39 w71 a Newborn (n¼ 32) Gender Male 12 37.5 Female 20 62.5 Birth weight (g) 1st month 4255 7496 3rd month 5791 71101 Birth body length (cm) 49 72 Birth circumference (cm) 34 71 Apgar score 5 min o7 0 0 5 min≧7 32 100 a

2 twins

Range

29–43

19.7–37.2

37wþ 2–41wþ 3

3600–5200 4700–7800 46–54 32–36

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Table 2 Comparisons of NP levels in specimens. Sample

N

Median

GM (GSD)

Mean 7 SD

Range

%4DL

Maternal blood (ng/ml) Fetal cord blood (ng/ml) Placenta (ng/g) Human milk (ng/ml) 1st month 3rd month

28 29 30

14.6 17.9 20.7

14.6 (1.7) 18.8 (1.8) 19.8 (1.9)

16.5 7 8.4 22.4 7 13.8 23.6 7 13.2

4.5–33.4 4.4–57.6 5.4–54.4

100 100 100

16 15

22.0 57.2

23.5 (3.2) 57.3 (1.4)

36.8 7 38.1 60.5 7 20.2

ND-160.1 27.2–104.8

93.7 100

ND: not-detectable for measurements below 1.87 (ng/ml) DL: detection limit

Table 3 NP levels in other studies pertaining to three samples specimen types- mother blood, cord blood, and human milk. Biological matrix

Population/country

NP levels

4 LOD (%)

References

Human blood Human plasma Human plasma

Cord blood (n¼ 180) , Malaysia Housekeeping workers, Taiwan A. Cord blood North Taiwan(n¼50) Central Taiwan(n¼ 124) B. Paired mother-newborn Maternal plasma(n¼42) Cord plasma(n¼ 42) Maternal blood(n ¼201) Cord blood(n¼ 201), China in Shanghai

Range (ng/ml) o 0.05–15.17 Mean 7 SD (ng/g) 53.21 7 49.74 GM (GSD), range (ng/g) 12.9 (42.4), o1.82–211 2.7 (6.6), o 1.82–182 GM (GSD), range (ng/g) 5.8 (8.4) , o 1.82–268 3.4 (6.2), o 1.82–100 Range, 95th level (ng/ml) 60–5580, 1630 20–1280, 910 Mean (μg/kg) 0.3 Mean, range (ng/g) 1.0, 0.65–1.40 Mean 7SD, range (ng/ml) 327 16.2, 13.4–56.3

86

Tan and Ali Mohd (2003) Chen et al. (2005) Chen et al. (2005)

Human serum

Human milk Human milk Human milk

Breast milk (n ¼2), Germany Breast milk (n ¼3), Japan Breast milk (n ¼10), Italy

4. Discussion Our previous study verified that NP is one of the most common contaminants in Taiwan, more than other AP compounds such as 4-tert-octylphenol, 2,4-di-tert-butylphenol, nonylphenol monoethoxylate, and p-n-nonylphenol diethoxylate (Chen et al., 2005; Lu et al., 2007). Exposure of pregnant women and the fetuses to NP appears to be critical, because NP may cause prenatal and/or postnatal reproductive and development effects. Results of this study demonstrated that NP levels are the highest among human milk, followed by placenta, maternal blood, and fetal cord blood, indicating prenatal and postnatal NP exposure. Through the repeated exposure from mothers’ dietary intake, fetuses could encounter a high NP exposure level due to breast milk or/and trans-placental absorption, partitioning between the maternal and fetal compartments. Daily NP intake of three month-old babies via breast milk exceeded the TDI of 5 mg/kg bw/day indicated a potential risk for Taiwan infants. The pregnant and lactating mothers and their infants in this study were clearly exposed to NP. The GM NP level in maternal blood was 14.6 ng/ml (range 4.5–33.4 ng/ml), while that in umbilical cord blood was 18.8 ng/ml (range 4.4–57.6 ng/ml). These levels were higher than the cord blood level (rangeo0.05–15.17 ng/ml) that was previously reported in Malaysia (Tan and Ali Mohd, 2003). However, the NP level in mother-infant dyads was lower than that in a Chinese study, in which the maternal and cord blood levels were 60–5580 ng/ml and 20–1280 ng/ml; it was also lower than that in a study of individuals with an occupational NP exposure in plasma of 53.2749.7 ng/g (Chen et al., 2005) (Table 3). Generally, wastewater treatment plants are not able to degrade alkylphenols. Chen et al. (2013) found high levels of NP in raw water (118–361 ng/l). The levels of NP were still high in treated water (Chen et al., 2013). The AP levels in Taiwanese rivers and sediments were higher than those in other countries such as Germany and Japan (Cheng et al., 2006). Inputs of APs into the aquatic environment may account for their presence in acquatic products

76 26 52 52 66

Li et al. (2013)

Guenther et al. (2002) Otaka et al. (2003) Ademollo et al. (2008)

such as fish and shellfish. Alkylphenols persist in the environment and enter the food chain, eventually accumulating in the lipid enriched matrix. The everyday unrestricted and intensive use of NPEO detergents and the contamination of the environment and foods from animal sources through bioaccumulation may contribute to the ubiquitously high levels of NP in Taiwan. However, Shanghai, China, which is a bustling metropolitan area with numerous industrial plants, NP pollution is a serious threat. Furthermore, the mean milk NP levels in the 1st and 3rd months (36.8 and 60.5 ng/ml) were higher than in other countries, based on previous studies, which have found levels of 0.3 μg/kg in Germany (Guenther et al., 2002), 1.0 ng/g in Japan (Otaka et al., 2003), and 32 μg/l in Italy (Ademollo et al., 2008) (Table 3). Those results suggest that infants in Taiwan are exposed to relatively high levels of NP through breast feeding. In addition to human milk, infant formula is another main form of nourishment for newborns. Ferrer et al. (2011) reported that NP was present in 50% (4/8) of infant formula samples at levels from o0.005 (LOD) to 0.69 mg/kg from Italian and Spanish markets. These levels are of the same order of magnitude as that found in our study. This study elucidates the factors that influence NP levels in human milk by examining the association between the dietary habits (dietary behavior and food frequency) of pregnant women and NP levels. The association between milk NP level in the 3rd month and the frequency of total food consumption was analyzed using Spearman correlation. However, a non-significant correlation was found between milk NP level and the frequency of total food intake (r¼0.23, p¼0.41). Herein, only the trend in frequency of total food consumption (rice and noodle, milk, eggs, vegetables, fruits, seafood, as well as fish) increased with the NP level in the 3rd month (Fig. 1 in Supplementary data). This relationship accords with the findings of Lu et al. (2007), in which rice is the most commonly consumed source of NP (21.5%) in Taiwanese foods, followed by acquatic products (17.9%), livestock (17.4%), vegetables and fruits. However, according to our results, fish or seafood consumption does not increase NP levels in human milk, possibly owing to the small sample size.

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In addition to helping newborns and infants receive essential nutrients, breastfeeding is associated with immunological benefits conferred to newborn children, including fewer respiratory and ear infections, reduced child mortality and other health advantages which extend into adulthood (W.H.O. and FAO, 2007). The daily intakes of NP and potential risks for breastfed infants are estimated using the probabilistic Monte Carlo analysis. Results of this study demonstrates that the probabilistic percentiles (50th, 75th, and 95th) of daily intake NP in breast milk are 4.33, 7.79, and 18.39 μg/kg-bw/day in the 1st month, respectively, and are 8.11, 10.78, 16.08 μg/kg-bw/day in the 3rd month, respectively. In addition, the probabilistic distributions (5th, 25th, and 50th) of risk for infants one-month old were 0.27, 0.64, and 1.15, respectively, and that for infants three-month old are 0.31, 0.46, and 0.62, respectively. The average NP daily intakes via breast milk for one and three month-old infants are 6.1 and 8.6 μg/kg-bw/day, respectively. There are 31.2% (5/16) and 93.3% (14/15) of breast milk fed infants in the 1st and 3rd months exceeded TDI of 5 ug/kg-bw/day. These results are higher than those obtained in studies in Italy and central Taiwan, which yielded 2.2 and 0.625 μg/kg-bw/day, respectively (Ademollo et al., 2008; Chen et al., 2010). The probable reason is the variation of dietary habits and types of food among countries and geography. The subjects in our study were all from metropolitan Taipei. The higher daily intake of NP in this study is consistent with our previous studies, which revealed that such subject had a much higher internal NP level than subjects from other regions of Taiwan (Chen et al., 2008). The fact that the daily NP intake of three monthold babies via breast milk exceeded the TDI of 5 mg/kg bw/day indicated a potential risk for Taiwan infants. The benefits of breast milk versus the adverse effects of exposure to pollutants are still contentious (Mead, 2008). The American Academy of Pediatrics has reaffirmed the benefits of breastfeeding to the infant, mother, and community by recommending an exclusive duration of about 6 months, followed by continued breastfeeding for 1 year or longer as complementary foods are introduced, as mutually desired by the mother and infant (Eidelman et al., 2012). Breastfeeding should thus not be discouraged. Rather, the sources of NP exposure in the population should be identified and eliminated. NP exposure in the placenta of vulnerable groups has seldom been assessed. To our knowledge, this study presents the placental NP level in the range of 5.4–54.4 (GM, 19.8) ng/g in humans for the first time. This finding suggests that fetuses are exposed to NP due to trans-placental absorption, partitioning between the maternal and fetal compartments. This finding also correlates with the observation of Chen et al. (2008). It is expected that the placenta is an effective barrier against fetal exposure to certain harmful proteins and also protects the developing embryo against some hormones, including estrogen, circulating in maternal blood that adversely affect fetal development. The fetus may be exposed to chemicals through trans-placental absorption by the diffusion, facilitated diffusion and active transport. Thus, of priority concern is knowing at what rate a chemical crosses the placenta and whether the fetal chemical levels is higher than the maternal plasma levels. General chemicals with both lipophilic and hydrophilic properties (log Kow¼  0.9 to 5) move readily across the placental membrane to the fetus, compared to more lipophilic chemicals. Owing to its relatively low log Kow of 3.8 to 4.8 (4.5), NP can be relatively easily across the placenta (California EPA, 2009). Balakrishnan et al. (2011) demonstrated that 4-NP can across the human placenta at the transfer percentage and transfer index in 22.7% and 0.8 in a dual ex-vivo re-circulation model of placental perfusion. Paired sample analysis is another method to test the potential transfer of NP through the placenta. Among our mother-infant pairs, 60.7% (17/28) of the fetal plasma was higher than that in maternal plasma; similar results were found in the studies of Osamu and Shinshi (2000) and Lee et al. (2008). Apart from paired

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maternal blood-cord blood samples, a broad type of maternalfetus paired samples (e.g., maternal blood-placenta, cord bloodmilk, placenta-cord blood, and placenta-milk samples) has been used to test the NP transfer to fetus. Results of this study demonstrates significant differences between the NP levels in breast milk in the 3rd month and cord blood (Wilcoxon matched–pairs signed-ranks test, p ¼0.002), in maternal blood and breast milk in the 1st and 3rd months (p ¼0.041 and 0.001). This study explored the prenatal and postnatal exposure of NP. Surprising, according to our results, the NP levels in maternal specimens were not related to those in fetal specimens (data not shown). Studies on trans-placental absorption of NP, BPA, and diethylstilbestrol (DES), one of the most potent non-steroidal synthetic estrogens, had similar findings (Osamu and Shinshi, 2000; Chen et al., 2008; Lee et al., 2008). Based on Müller's pharmacokinetic study, the half-life of NP in blood is 2–3 h, and the bioactivity of NP is 20% after ingested orally and intravenously (Muller et al., 1998). Even with rapid metabolism of NP, it can be detected in mother-fetus dyads samples. This finding implies that pregnant women are repetitively and persistently exposed to NP. Through the repeated exposure from mothers’ dietary intake, fetuses could encounter a high NP exposure level. Despite its contributions, this study has certain limitations. The first limitation is the relatively small sample size resulting from the challenge of establishing a cohort of pregnant women. Second, the demographic characteristics of pregnant women, such as age and parity, as well as the dietary pattern may affect the milk NP level. Kunisue et al. (2006) reported that higher levels of PCB and dioxins in milk are observed in primiparas than in secundiparas and in even older mothers during their first pregnancy. 5. Conclusion This study demonstrates for the first time the existence of NP level in placenta and also identifies prenatal and postnatal exposure to NP. In addition, through the repeated exposure from mothers’ dietary intake, fetuses are exposed to NP both during gestation via transplacental absorption and after during lactation through breast milk. Moreover, the daily NP intake of three month-old babies via breast milk exceeded the TDI of 5 mg/kg bw/day indicated a potential risk for Taiwan infants. We recommend larger population studies to provide good suggestions regarding food choices to lactating mothers in order to prevent excess exposure to NP in their newborns. Further studies with a larger study group are necessary to shed light on the mechanisms and kinetics on placental transfer of NP and its health effects on a vulnerable population.

Acknowledgments The authors would like to thank the National Science Council of the Republic of China, Taiwan (Contract no. NSC 99-2314-B-010018-MY3) and Taipei City Government Department of Health (Contract no. TCHIRB-1010303) for financially supporting this research. Ted Knoy is appreciated for his editorial assistance.

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