triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation

triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation

Accepted Manuscript Triclosan/triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation Ling Wei, Pen...

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Accepted Manuscript Triclosan/triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation

Ling Wei, Pengyun Qiao, Ying Shi, Yan Ruan, Jie Yin, Qingqing Wu, Bing Shao PII: DOI: Reference:

S0009-8981(16)30528-9 doi: 10.1016/j.cca.2016.12.024 CCA 14610

To appear in:

Clinica Chimica Acta

Received date: Revised date: Accepted date:

11 December 2016 22 December 2016 22 December 2016

Please cite this article as: Ling Wei, Pengyun Qiao, Ying Shi, Yan Ruan, Jie Yin, Qingqing Wu, Bing Shao , Triclosan/triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cca(2016), doi: 10.1016/ j.cca.2016.12.024

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ACCEPTED MANUSCRIPT Triclosan/Triclocarban Levels in Maternal and Umbilical Blood Samples and their Association with Fetal Malformation

Ling Wei1*, Pengyun Qiao2*, Ying Shi3, Yan Ruan1, Jie Yin4, Qingqing Wu5§, Bing Shao4§, 1

Department of Obstetrics, Beijing Obstetrics and Gynecology Hospital, Capital

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Medical University, Beijing, China 2

Xiangheyuan Supervision Station, The Institute of Inspection and Supervision, National Health and

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3

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Clinical Center of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang 261031, China

Family Planning Commission in Chaoyang District of Beijing, China Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing

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5

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Centre for Disease Control and Prevention, Beijing, China

Department of Ultrasound, Beijing Obstetrics and Gynecology Hospital, Capital Medical University,

Correspondence to:Qingqing Wu, 5Department of Ultrasound, Beijing Obstetrics and Gynecology

Hospital,

Capital

[email protected]

Medical

University,

Beijing,

China.

Tel.:(+86)13641211037

Email:

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§

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*These authors contributed eaqually

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Beijing, China

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Bing Shao, Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Centre for Disease Control and Prevention, Beijing, China. Tel.: 8613381081679 Email: [email protected] §

These correspond authors contributed eaqually to the work

ACCEPTED MANUSCRIPT Abstract Triclosan (TCS) and triclocarban (TCC) are widely used as antimicrobial compounds in consumer products. TCS and TCC are frequently found in waste water and sewage. In this study, we investigate the potential impact of exposure to triclosan (TCS) and triclocarban (TCC) on fetal abnormalities. We measured TCS and TCC levels in maternal and umbilical cord blood samples from 39 pregnant women

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diagnosed with fetal or post-birth abnormalities at Beijing Obstetrics and Gynecology Hospital. 52

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pregnant women who gave birth to healthy neonates during the same period of time were included as

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controls. Applying ultra-performance liquid chromatography-tandem mass spectrometry, TCS and TCC concentrations were measured in maternal and fetal sera. Significantly increased levels of TCS were

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detected in maternal sera from mothers with abnormal births. Similar levels of TCS or TCC were found in maternal and cord sera in control group. The concentrations of TCS or TCC in maternal sera

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correlated with those in umbilical cord sera (r=0.649, P<0.01). These observations suggest that maternal blood test could be a useful assay for detecting fetal exposure to TCS and TCC, and high exposure to

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TCS may be potentially associated with increased risk for fetal malformations.

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Key Words: Fetal Abnormalities; Triclosan; Triclocarban;Umbilical Cord Blood

ACCEPTED MANUSCRIPT Introduction Fetal anomalies/defects or birth defects include all abnormalities in the structure, function and/or metabolism of the fetus that develop prior to birth. The etiology of birth defects is generally thought to be a multifactorial process. Hereditary, environmental factors, or a combination of the two are often involved. In China alone fetal anomalies were found in 5.6% of all live births in 2012, with a total of

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900,000 infants affected [1].

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Triclosan (TCS) and triclocarban (TCC) are analogous in chemical structure but independent of each other. These chemicals have been widely used for more than 50 years as an antimicrobial component in

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consumer products such as toothpastes, soaps, shampoos, detergents, and medical disinfectants, for personal hygiene care, treatment of textiles and manufacturing of consumer plastics. TCS and TCC are

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also increasingly identified as contaminants in waste water and sewage material. Untreated or incompletely treated waste water often contains detergent metabolites, pharmaceuticals, and plasticizers

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that pollute environments, which can adversely affect agriculture and different ecosystems [2-4].

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TCS/TCC may act alone or interact with other contaminants to exert biological impacts on human health,

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especially on human reproduction [5].

Humans exposure to TCS and TCC usually occurs by dermal absorption or ingestion [6], or other

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environment exposures such as inhalation of contaminated indoor or outdoor air [7]. TCS has been detected in human breast milk [8], human plasma [9], urine [6], amniotic fluid [10], and umbilical cord blood [11]. Although these compounds are thought to be of low systemic toxicity in mammals, studies indicated that thyroxine homeostasis in weaning rats [12] and sheep could be disrupted by these compounds. Additionally it is thought that estrone sulfonation is inhibited by TCS and TCC [13]. These compounds were also found to interfere with blastocyst implantation in mice [14].

ACCEPTED MANUSCRIPT Although studies in animal models indicated that TCS and TCC could be endocrine disruptors, their impact on fetal health in human has not been determined. In this study, we measured the TCS and TCC levels in umbilical cord serum samples and analyzed their associations with fetal anomalies. Delineation of the association between fetal abnormalities and in utero exposure to TCS and TCC will help us to assess the risk of birth defects caused by these compounds, and build a foundation for the design of

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preventive procedures against these factors.

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Materials and Methods Reagents and standards

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Standard TCS (99.5%) and TCC (99%) were purchased from Dr. Ehrenstorfer Gmbh (Augsburg, Germany) and Toronto Research Chemicals (North York, Ontario, Canada). 13

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C12-TCS (99%) and

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C6-TCC (99%) were obtained from Cambridge Isotope Laboratories (Andover, MA) and used as the

internal standards. LC-MS grade methanol, acetonitrile, and water were purchased from Sigma-Aldrich

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(St. Louis, MO). Analytical-grade sodium acetate, and acetic acid (99.5%) were obtained from Beijing

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Chemical Works (Beijing, China). β-glucuronidase/arylsulfatase (Helix pomatia, 100,000 units/mL) was

Sample collection

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purchased from Roche Diagnostic GmbH (Mannheim, Germany).

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Two groups (fetal anomaly: N=39; controls: N=53) were included in this study. The fetal anomaly group comprised pregnant women who were diagnosed with fetal abnormalities during gestation or delivery of babies with malformations. Controls were pregnant women with normal fetus free of malformation and adverse neonatal outcomes at birth during the same period of time (form March 2013 to February 2014). Maternal venous (2mL) and umbilical cord blood (2mL) from all cases were obtained after patients’ admission to the Beijing Obstetrics and Gynecology Hospital. The study protocol was approved by the Institutional Review Board of Beijing Obstetrics and Gynecology Hospital. All study subjects have

ACCEPTED MANUSCRIPT signed informed consent documents before participation.

Sample preparation and storage Blood samples were centrifuged at 900 × g for 5 min and sera were transferred to polypropylene tubes. Serum samples were frozen and sent to Beijing Key Laboratory of Diagnostic and Traceability

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Technologies for Food Poisoning (Beijing Center for Disease Control and Prevention) where the

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samples were stored at −20ºC until measurement. For each sample (2 mL), concentrations of total (free

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and conjugated) TCS and TCC were measured using automated on-line solid phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (on-line SPE Enzyme

solutions

were

prepared

daily

by

diluting

500

μL

of

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UHPLC-MS/MS).

β-glucuronidase/arylsulfatase into 10 mL of a 0.2-mol/L sodium acetate buffer (pH 5.4). To hydrolyze

solution (40 μg/L of

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conjugated target chemicals, a 100-μL aliquot of serum was mixed with 50 μL of the internal standard C12-TCS and 4 μg/L of

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C6-TCC) and 100 μL of the enzyme solution. After

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incubation overnight at 37ºC, 300 μL of acetonitrile was added to the sample. Following vigorous

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vortexing, the mixture was centrifuged at 14,000 rpm for 15 min. 400 μL of the supernatant was diluted

Instrument analyses

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with 400 μL water, and subjected to on-line SPE UHPLC-MS/MS analysis.

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Chromatographic analyses of samples were performed using a Dionex Ultimate 3000 UHPLC system (Dunnyvale, CA) that consists of two binary pumps with degassers, an autosampler, and a column compartment with a 6-port switching valve. The samples (500 μL) were injected, and the analytes were extracted using a CAPCELL PAK MF C8 column (4.6 mm I.D. × 50 mm, 5 μm particle size, 8 nm pore size, Shiseido, Japan) operating at 2.0 mL/min. By switching the 6-port valve, the chemicals were back-flushed by a solvent stream of 0.3 mL/min into a Waters Acquity UPLCTM HSS T3 column (2.1 × 50 mm, 1.8 μm).

ACCEPTED MANUSCRIPT Liquid chromatography system was connected to a Waters XevoTM TQ-S triple-quadrupole mass spectrometer (Waters Corp., Milford, MA) operating with a negative electrospray ionization source. Multiple reaction monitoring mode was used to quantitatively measure the analytes. The instrumental parameters were set as following: capillary voltage = 1.0 kV; source temperature = 135ºC; desolvation

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temperature = 500ºC; desolvation gas (N2) = 1,000 L/h; and collision cell pressure = 4.95 × 10−4 mbar.

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Quality control

With every batch of samples analyzed, procedural blanks (n = 3) were included to confirm sample

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preparation and investigate instrumental system contamination. Approximately 0.005 μg/L of TCC in

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procedural sample blanks was observed, which was below the detection limit of the method and may be explained by the ubiquity and high sensitivity of TCC. Mean TCC and TCS concentrations measured in

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procedural blanks for each analysis batch were subtracted from concentrations detected in samples. Moreover, a calibration standard and solvent blank were injected after every tenth sample to monitor

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background TCC and TCS. Mean recoveries of both analytes were validated in six replicates by spiking

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at three fortification levels between 93.7–103.6% for samples. The intra-day variability was less than 20%, as represented by the relative standard deviation percent (RSD%) at each fortification level for

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each compound. The limit of detection (LOD) and limit of quantification (LOQ) were defined by determining signal-to-noise (S/N) ratios of 3:1 and 10:1, respectively. The LODs (LOQs) were 0.005

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μg/L (0.01 μg/L) and 0.002 μg/L (0.006 μg/L) for TCS and TCC, respectively. For TCC, a 7-point calibration standard curve was obtained at concentrations between 0.005 and 100 μg/L (TCS was 10 times greater than TCC). Furthermore, concentrations measured in the samples were corrected for recoveries of internal standards (13C12-TCS and 13C6-TCC).

Data analyses All statistical analyses were performed using SPSS 17.0 (IBM, Chicago, IL). Concentrations below the

ACCEPTED MANUSCRIPT LOD were assigned a value of half the LOD [15]. Detection rate, mean ± standard deviation (SD), geometric mean (GM), median, and range were used to describe the TCS and TCC in samples. Detection rate was analyzed using Chi-squared test. TCS and TCC data were assessed with rank sum tests due to the skewed distribution. Differences and correlations were tested with non-parametric tests: Mann-Whitney U-test, Wilcoxon signed rank test and Spearman rank correlation. Significance level was

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set at 0.05.

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Results

This study covers 39 cases in the fetal-defect group (40 samples; one case was twins), and 52 cases in

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the control group (Table 1). There was no significant difference in mothers’ age and gestational age between the two groups. Fetal defects of the study subjects were classified according to ICD-10

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guidelines. The distribution of defects is shown in Table 2. Congenital malformations of the circulatory system, eye, ear, face, neck, urinary system and musculoskeletal system were among the most frequent

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abnormalities.

Descriptive statistics for TCS and TCC are summarized in Table 3. The detection rate of TCS in

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maternal sera in fetal anomaly group was significantly higher than that in the control group (80.00% vs 53.80%, p=0.009). Detection rates of TCS in cord serum were not significantly different in the fetal

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anomaly and control groups (p=0.188). No significance was found in the detection rates of TCC in either maternal sera (p=0.157) on in cord sera (p=0.173).

Data analysis indicated that there was no significant difference in TCS (p=0.058) and TCC (p=0.528) in maternal sera from fetal anomaly and control groups. Similarly, no significant difference was found in cord serum TCS (p=0.344) and TCC (p=0.934) between the two groups. The cases are stratified according to malformations into 7 groups and further analyzed for groups with more than 5 cases.

ACCEPTED MANUSCRIPT Average TCS and TCC levels of each group are compared to those of the control group. No significant difference was found by further stratification.

Linear regression analysis indicated that TCS levels in maternal sera were significantly correlated positively with TCS levels in cord sera (r=0.649; P<0.01, Figure 1). Similar results are obtained for

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TCC (r=0.683; P<0.01, Figure 2). However, neither the TCS levels in maternal (r=0.005, P=0.96,

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Figure 3) nor in cord sera (r=0.029, P=0.79, Figure 4) were significantly correlated with the

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corresponding TCC levels. (Figure 3 and 4)

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Discussion

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Results from animal studies on TCS and TCC have confirmed the connection between fetal development and exposure to these compounds [16]. In an early study in 2006, TCS was found to alter

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thyroid hormone-associated gene expression and to disrupt the postembryonic anuran development [17].

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Recent studies demonstrated that low doses of oral TCS exposure (1mg/kg) by the pregnant and lactating rats alters the appetite regulatory system on offspring and predispose them to metabolic

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abnormalities in the adulthood. Adult offspring were found to have a heavier body weight. Not surprisingly, food intake was higher and these rats were found to have higher serum cholesterol and

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glucose levels when compared with the control group [18]. Influences on human fetal development by environmental pollution of TCS have been investigated, especially in areas that known to be heavily polluted such as Beijing. As a type of phenols, TCS is suspected to have a weak inverse association with pubic hair development, but further analysis showed it is not an independent risk factor [19]. Benny et al reported human fetal exposure to TCS and TCC in an urban population from Brooklyn, New York. TCS and TCC were detected in 8 (22.9%) and 16 (45.7%),respectively,out of 35 human umbilical cord blood samples [11]. A limitation to the study, however, is that the newborn’s outcomes were not

ACCEPTED MANUSCRIPT investigated in that study.

Our data demonstrates that TCS and TCC can be detected in cord sera, and the maternal serum levels of these compounds are closely related to their cord serum levels, suggesting a maternal-to-fetal transmission. In this context, Benny et al. reported that the maternal urinary concentrations of TCS and

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TCC in the third trimester could not well predict fetal exposure as measured via cord blood at birth [11].

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It is unclear if this discrepancy was caused by maternal metabolism of these compounds or by renal

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barrier function. Regardless of the cause of this difference, our finding raised a serious concern on maternal-to-fetal transmission because recent work suggested that these compounds may interfere with

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fetal development. High doses of TCS can disrupt intrauterine blastocyst implantation in mice [14], and TCS may have estrogenic function [20]. On the other hand, TCS may inhibit estrogen sulfotransferase in

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sheep placenta [13], resulting in reduced total placental estrogen secretion and activity in target tissues. Uterine blood flow is regulated to an extent by estrogen, and it has been shown that placental estrogen

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biosynthesis is required for fetal development. Moreover, a series of studies in rats indicated that TCS

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could lower maternal thyroxine (T4) levels [12,21]. Thyroxine is a critical hormone for fetal development, especially for brain maturity. It was reported that a slight decrease in maternal T4 level

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could cause adverse effects on childhood cognition and motor function [22,23]. Although the mechanisms underlying the TCS effects on human endocrine system are not fully understood, it is clear

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that through these effects, intrauterine exposure of TCS could imposes a threat to the growth and differentiation of fetal cells/tissues, leading to various fetal malformations. The complex nature of the relationship between teratogen exposure and fetal malformation makes it difficult to identify the true culprit out of all the potential factors, and we could not exclude the contribution by other factors to the malformation observed in this study.

We found that the detection rate of TCS is significantly higher in the fetal anomaly group than normal

ACCEPTED MANUSCRIPT controls. However, the quantitative analysis demonstrates no significant difference between the levels of TCS in the two groups. The divergent results of the two compounds could be caused by their differential absorption, stability, and/or metabolism in vivo. Although the median concentrations of TCS in maternal (0.215 ng/ml) and cord sera (0.07 ng/m) in the fetal anomaly group were several folds higher than those of controls (0.055 versus 0.025 ng/ml), the difference did not reach a statistically significant level. Data

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shows that median TCC concentrations in maternal (0.048 ng/ml) and cord sera (0.03 ng/ml) of the fetal

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anomaly group was lower than in controls (0.065 versus 0.03 ng/ml). Further studies using larger sample

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size are required to accurately assess the risk for fetal malformation by these chemicals.

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We combined the data from the anomaly group and the control group and asked whether the concentrations of TCS and TCC in maternal blood could be associated with levels in fetal blood. Further

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analysis of the data showed a significant positive correlation in TCS (r=0.649, P<0.01) and TCC (r=0.683, P<0.01) levels of maternal and cord sera. Thus, the levels of compounds in maternal sera may

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predict those in cord sera, and consequently fetal exposure. These observations indicated that although

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the levels of these compounds are not necessarily correlated with the malformation, monitoring the maternal blood levels of these compounds could be used as a screening tool for fetal exposure to

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TCC/TSC in pregnancy.

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In summary, TCS and TCC can disrupt intrauterine blastocyst implantation, inhibit estrogen sulfotransferase and reduce the thyroxine (T4) level. Our suggested a possible association of TCS and TCC levels in maternal and cord sera with fetal anomalies. Future research efforts in this area are required to confirm this observation. Additionally, further investigation is needed to determine the dose/time effects of TCS and TCC on fetal anomalies and to investigate the underlying molecular mechanisms by which TCC/TCS exposure may lead to fetal anomalies.

ACCEPTED MANUSCRIPT Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments This study was funded by the National Natural Science Foundation of China (No. 21177014), Beijing

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Natural Science Foundation (Grant No. 7142026), Beijing Municipal Science & Technology

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Commission (No. Z141107002514006) and the Beijing Municipal Administration of Hospitals’Ascent

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Plan, Code:DFL20151302.

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milk from Swedish nursing mothers and their exposure via personal care products. Sci Total Environ 2006;372:87–93. 9.

Schebb NH, Inceoglu B, Ahn KC, et al. Investigation of human exposure to triclocarban after showering, and preliminary evaluation of its biological effects. Environ Sci Technol 2011;45: 3109–15.

10. Philippat C, Wolff MS, Calafat AM, et al. Prenatal exposure to environmental phenols: concentrations in amniotic fluid and variability in urinary concentrations during pregnancy. Environ

ACCEPTED MANUSCRIPT Health Perspect 2013;121:1225–1231. 11. Pycke BF, Geer LA, Dalloul M, et al. Human fetal exposure to triclosan and triclocarban in an urban population from Brooklyn, New York. Environ Sci Technol 2014;48:8831–38. 12. Zorrilla LM, Gibson EK, Jeffay SC, et al. The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicol Sci 2009;107:56–64.

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13. James MO, Li W, Summerlot DP, et al. Triclosan is a potent inhibitor of estradiol and estrone

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sulfonation in sheep placenta. Environ Int 2010;36:942–49.

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14. Crawford BR, DeCatanzaro D. Disruption of blastocyst implantation by triclosan in mice: impacts of repeated and acute doses and combination with bisphenol-A. Reprod Toxicol 2012;34: 607–13.

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15. Hornung RW, Reed LD. Estimation of average concentration in the presence of non-detectable values. Appl Occup Environ Hyg 1990;5:46–51.

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16. Zorrilla LM, Gibson EK, Jeffay SC, et al. The effects of triclosan on puberty and thyroid hormones in male wistar rats.Toxicol Sci 2009;107:56–64.

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17. Veldhoen N, Skirrow RC, Osachoff H, et al. The bactericidal agent triclosan modulates thyroid

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hormone-associated gene expression and disrupts postembryonic anuran development. Aquat Toxicol 2006;80: 217–27.

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18. Rabaglino MB, Moreira-Espinoza MJ, Lopez JP, et al. Maternal Triclosan consumption alters the appetite regulatory network on Wistar rat offspring and predispose to metabolic syndrome in the

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adulthood. Endocr J 2016 Aug 26. [Epub ahead of print] 19. Wolff MS, Teitelbaum SL, Pinney SM, et al. Investigation of relationships between urinary biomarkers of phytoestrogens, phthalates, and phenols and pubertal stages in girls. Environ Health Perspect 2010;118 :1039-46. 20. Jung EM, An BS, Choi KC, Jeung EB. Potential estrogenic activity of triclosan in the uterus of immature rats and rat pituitary GH3 cells. Toxicol Lett 2012;208:142–48. 21. Axelstad M, Boberg J, Vinggaard AM, et al. Triclosan exposure reduces thyroxine levels in

ACCEPTED MANUSCRIPT pregnant and lactating rat dams and in directly exposed offspring. Food Chem Toxicol 2013;59:534–40 . 22. Ghassabian A, Bongers-Schokking JJ, Henrichs J, et al. Maternal thyroid function during pregnancy and behavioral problems in the offspring: the generation R study. Pediatr Res 2011;69:454–59. 23. Henrichs J, Bongers-Schokking JJ, Schenk JJ, et al. Maternal thyroid function during early

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pregnancy and cognitive functioning in early childhood: the generation R study. J Clin Endocrinol

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Metab 2010;95:4227–34.

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Figure legends

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Figure 1. Correlation of TCS concentrations (ng/mL) in maternal and cord sera (r=0.649; P<0.01). r:

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Spearman correlation coefficient.

Figure 2. Correlation of TCC concentrations (ng/mL) in maternal and cord sera in this study (r=0.683;

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P<0.01). r: Spearman correlation coefficient.

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r: Spearman correlation coefficient.

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Figure 3. Correlation between TCS and TCC concentrations (ng/mL) in maternal sera. (r=0.005, P=0.96).

Figure 4. Correlation between the TCS and TCC concentrations (ng/mL) in cord sera. (r=0.029, P=0.79).

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r: Spearman correlation coefficient.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4

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Table 1 Detailed sample characteristics in this study. Control group

40

52

Average age (years)

32.2

29.5

Average gestation week

25.4

39.2

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Number

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Fetal defect group

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Characteristics

ACCEPTED MANUSCRIPT Table 2 Detailed classification of birth defect group by ICD-10.

Congenital malformations of the eye, ear, face and neck

8

Congenital malformations of the urinary system

7

Congenital malformations of the musculoskeletal system

7

Multiple deformity

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Congenital malformations of the nervous system

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Sacrococcygeal teratoma

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9

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Congenital malformations of the circulatory system

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Number

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ACCEPTED MANUSCRIPT Table 3 Descriptive statistics of triclosan (TCS) and triclocarban (TCC) in the maternal and umbilical cord serum. TCS (ng/mL)

TCC (ng/mL)

Maternal

Maternal

Statistics

Cord serum

80.00%*

60.00%

Mean ± SDb

0.549±1.355

0.160±0.293 0.190±0.365

Median

0.215

GMc

0.191

Range

ND-8.54d

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0.059

0.035

ND-1.75

ND-2.00

ND-0.82

53.80%*

46.20%

86.50%

71.20%

0.649±1.562

0.245±0.478 0.292±0.586

0.106±0.171

Median

0.055

0.025

0.065

0.03

GMc

0.11

0.073

0.081

0.042

ND-8.00

ND-2.28

ND-2.75

ND-0.53

Detection (%)

65.20%

52.20%

81.50%

65.20%

Mean ± SD

0.606±1.468

0.208±0.408 0.248±0.502

0.105±0.178

Median

0.115

0.060

0.055

0.030

GM

0.140

0.075

0.071

0.039

Mean ± SDb

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(n=52)

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Range

a: values above the limit of detection SD: standard deviation

0.103±0.187 0.03

Detection (%)a

b

57.50%

0.048

Control group

Total

75.00%

0.07

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(n=40)

Detection (%)a

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Fetal anomaly group

Cord serum serum

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serum

ACCEPTED MANUSCRIPT c

GM: geometric mean

d

ND: not detected

* The detection rates of TCS in maternal sera were significantly different between the two groups (P<0.01). The concentrations of the detectable samples below the limit of quantification (LOD) were assigned a

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value of half the LOD.

ACCEPTED MANUSCRIPT Highlights

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1.Significantly increased levels of TCS were detected in maternal sera from mothers of fetus with malformations. 2.These is a significant correlation between TCS/TCC concentrations in maternal and umbilical cord sera from both anomaly and control group. 3.Maternal blood test could be a useful assay for detecting fetal exposure to TCS and TCC. 4.High exposure to TCS may be potentially associated with increased risk for fetal malformations.