Metabonomic Phenotyping Reveals an Embryotoxicity of Deca

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Metabonomic Phenotyping Reveals an Embryotoxicity of Deca-Brominated Diphenyl Ether in Mice Yi Chi,†,# Hongfei Xia,||,# Mingming Su,§,# Peipei Song,|| Xin Qi,† Yi Cui,|| Yu Cao,† Tianlu Chen,† Yunping Qiu,^ Aihua Zhao,† Xu Ma,*,|| Xiaoying Zheng,‡ and Wei Jia*,^ †

School of Pharmacy, Shanghai Jiao Tong University, Shanghai, P. R. China WHO Collaborating Center for Reproductive Health and Population Science/Institute of Population, Peking University, Beijing, P. R. China § David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, North Carolina, United States Reproductive and Genetic Center of National Research Institute for Family Planning, Beijing, P. R. China ^ Department of Nutrition, University of North Carolina at Greensboro, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina, United States

)

‡

bS Supporting Information ABSTRACT: Recent studies have demonstrated that polybrominated diphenyl ethers (PBDEs), a group of industrial chemicals, could disrupt thyroid hormone homeostasis and exhibit neurotoxicity, reproductive toxicity, and embryotoxicity. However, clear evidence of embryotoxicity and neurotoxicity of many of these congeners, such as deca-BDE, one of the least bioactive congeners of PBDEs, is still lacking. In the present study, we investigated deca-BDE embryotoxicity by quantitative analysis of two essential thyroid hormones (T4 and T3) and a variety of small-molecule metabolites in the serum of deca-BDE-dosed pregnant mice. Four groups of pregnant C57 mice were administrated with deca-BDE in 20% fat emulsion at a dose of 150, 750, 1 500, or 2 500 mg/kg body weight via gastric intubation on gestation days (g.d.s) 7 to 9, while a control group was given 20% fat emulsion. Maternal mice were euthanized on g.d. 16 and examined for external malformations of the fetus. Maternal serum samples were collected and analyzed by the enzyme linked immunosorbent assay (ELISA) and gas chromatography time-of-flight mass spectrometry (GC TOF MS). Using multivariate statistical analysis, we observed a significantly altered metabolic profile associated with deca-BDE embryotoxicity in maternal serum. Our results also demonstrated that deca-BDE at a dose of 2 500 mg/kg body weight induced significant disruption of thyroid hormone metabolism, the TCA cycle, and lipid metabolism in maternal mice, which subsequently led to a significant inhibition of fetal growth and development. We concluded that deca-BDE-induced embryotoxicity closely correlated with global metabolic disruption that can be characterized by thyroid hormone deficiency, disrupted lipid metabolism, and a depleted level of cholesterol in maternal mice.

’ INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are widely used as flame-retardant additives in a variety of consumer products including mattresses, carpets, furniture, electrical appliances, and textiles.1 As PBDEs are simply blended with, rather than covalently bound to these materials, they will gradually leach out from the products over time. Therefore, PBDEs are often detected in sediments, dust, air, fish, marine species, human breast milk, human blood, and adipose tissue samples.2 Many animal studies have demonstrated that PBDEs could induce the disruption of thyroid hormone homeostasis, developmental neurotoxicity, and reproductive toxicity.3 5 Although the underlying mechanism of metabolic disorders in humans exposed to PBDEs has not been fully explored, emerging evidence suggests that the increased concentration of PBDEs detected in human biological samples is associated closely with many developmental disorders.6 8 For instance, prenatal exposure to PBDEs substantially correlated r 2011 American Chemical Society

with low birth weight9 and adverse neurodevelopmental effects in offspring.10 Among all of the BDE congeners that are being used in household products, deca-BDE is among the most poorly absorbed, rapidly eliminated, and the least bioactive congeners,11,12 thus, the embryonic and developmental toxicity of deca-BDE has not been well studied. However, recent studies indicate that deca-BDE may convert to lower congeners under certain circumstances.13,14 Some BDE congeners were suspected to disrupt thyroid hormone homeostasis through an increased elimination of thyroxin (T4) that could be caused by activating uridine diphosphoglucuronosyl transferase (UDPGT),15 competitively binding to transthyretin,16 or directly binding to thyroid hormone receptors.17 The direct impact of deca-BDE on the thyroid hormone was Received: July 20, 2011 Published: October 13, 2011 1976

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Chemical Research in Toxicology revealed by an increased incidence of thyroid hyperplasia18 and a decreased thyroid hormone level in offspring.19,20 In addition to thyroid hormone disruption, deca-BDE could also lead to neurobehavioral disorders and cancers.13,21 23 Some countries suspended the use of PBDEs in products as early as the mid1980s; however, restriction of deca-BDE was not fully implemented in the European Union and many US states until 2008.1 Because these chemicals are still widely present in products manufactured before PBDEs were banned, its exposure will continue to expand and impact future generations. For this reason, deca-BDE continues to pose a serious public health threat. As an integral part of systems biology, metabonomics/ metabolomics has been applied in nutrition, toxicology, and clinical diagnosis.24 27 In this study, we investigated the embryotoxicity of deca-BDE using a metabonomic approach employing gas chromatography time-of-flight mass spectrometry (GC TOF MS). We also quantitatively measured T4 and T3 in serum samples of maternal mice to assess the impact of deca-BDE on thyroid hormone homeostasis due to the substantial influence of thyroid hormone on fetal development.28 The goal of our work was to gain mechanistic insights into the embryotoxicity of decaBDE through measuring metabolic end points of the maternal mice under deca-BDE exposure.

’ EXPERIMENTAL PROCEDURES Animal Handling and Sampling. The animal study was approved by the Ethics Committee of National Research Institute for Family Planning (Permit Number: SYXK (JING) 2009-0033; Beijing, P. R. China). Sexually mature, healthy female C57 mice were purchased from and raised in the Laboratory Animal Center, the Academy of Military Medical Sciences, Chinese Academy of Sciences (Beijing, P. R. China). The mice were housed under a controlled condition of 12 h light/12 h dark cycle at approximately 22 24 C with a relative humidity of 60 70% and acclimatized to the environment for two weeks prior to commencing the experiment. The mice had free access to chow and water ad libitum. The standard mouse chow was provided by Laboratory Animal Center, The Academy of Military Medical Sciences, Chinese Academy of Sciences (Beijing, P. R. China), and the composition of the chow included protein (22.70%), fat (5.54%), lysine (1.25%), methionine (0.40%), calcium (1.24%), phosphorus (1.07%), water (9.00%), fiber (4.02%), and nitrogen-free extract (NFE, 51.50%), and had calories (352 kcal). Each female mouse was mated with a sex ratio of 3:1 (female:male), and gestation day (g.d.) 0 was defined by the presence of a vaginal plug or a sperm positive vaginal smear. Deca-BDE was prepared by mixing different quantities of deca-BDE (1.5, 7.5, 15, and 25 mg) in a 20% fat emulsion vehicle, where the vehicle is a mixture of egg lecithin (Sigma-Aldrich, St. Louis, MO) and olive oil (Sigma, St. Louis, MO) (1:10, w/w). The pregnant female mice were randomly divided into five groups: four model groups received deca-BDE in 20% fat emulsion vehicle with a daily dose of 150, 750, 1 500, and 2 500 mg/kg body weight (n = 10 per group) by gastric intubation on g.d. 7 9, and a control group (n = 10) received 20% fat emulsion vehicle. The dose volume was about 100 μL per day per mouse on g.d.s. 7 9. All of the mice were euthanized by an intraperitoneal injection of pentobarbital (50 mg/kg body weight) at the 16th day of gestation. The blood samples were collected by enucleation of the eyeball, and the resulting serum samples were stored at 80 C until analysis. Placenta weight, number of implantation sites per litter, number of fetal resorptions per litter, and number of live and dead fetuses per litter were recorded. The percentage of postimplantation loss per litter, live and dead fetuses per litter, and fetal resorptions per litter were calculated accordingly. Live fetuses and their brains, hearts, and livers were also weighed. All of the organs of live fetuses were examined

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for external abnormalities. The number of serum samples used for the thyroid hormone assay and metabolite analysis was eight per group except for 150 mg/kg group (n = 7) due to the limited volume of serum available. Thyroid Hormone Assay. The determination of thyroid hormone in maternal sera was conducted using the enzyme linked immunosorbent assay (ELISA). Total T4 and T3 were measured by commercial mouse ELISA kits purchased from R&D Systems China Co. Ltd. (Shanghai, P. R. China). The measurement was carried out following the vendor’s standard instructions (Supporting Information). The linear concentration ranges were 0 40 ng/mL for T4 and 0 240 ng/mL for T3, respectively. Optical density (OD) values of each well were obtained using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific Inc., San Jose, CA). Serum Metabolite Extraction and Derivatization. Metabolites were extracted with organic solvent and derivatized following our published procedures with minor modifications.29 Briefly, each 100 μL of serum sample was spiked with two standard solutions (10 μL of L-2chlorophenylalanine and heptadecanoic acid). The two internal standards were used to monitor the analytical variations during sample handling and instrument analysis. The relative standard deviation (R.S.D.) was set less than 15% in this study.30 A mixture (300 μL) of chloroform/methanol = 1:3 (v/v) was added for protein precipitation. After storing for 10 min at 20 C, the mixture was centrifuged at 13 000 rpm for 10 min. An aliquot of a 300-μL supernatant was diverted into a 2-mL sample vial and dried under vacuum. The residue was dissolved in 80 μL of methoxylamine hydrochloride (15 mg/mL in pyridine) for 90 min at 30 C. The resulting products were silylated with 80 μL of N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA) (containing 1% trimethylchlorosilane) (Sigma-Aldrich, St. Louis, MO) for 1 h at 70 C. GC TOF MS Analysis. A 1-μL aliquot of derivatized mixture was injected in splitless mode through an Agilent 7683B Series autosampler (Agilent, Atlanta, GA) into an Agilent 6890N GC system coupled with a Pegasus HT TOFMS (Leco, St. Joseph, MI). A DB-5MS capillary column coated with 5% diphenyl cross-linked 95% dimethylpolysiloxane (30 m  250 μm i.d., 0.25-μm film thickness; Agilent J&W Scientific, Folsom, CA) was used. The injector, transfer line, and ion source temperatures were set to 270, 260, and 200 C, respectively. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The initial oven temperature was set at 80 C for 2 min, followed by 10 C/min, and the oven temperature was ramped to 180 C, 6 C/min to 230 C, and 40 C/min to 295 C, and finally held at 295 C for 7 min. The measurement was made with electron impact ionization (70 eV) in a full scan mode (m/z 30 600), with an acquisition rate of 20 spectra/s. The samples were ordered at a full-crossed matter, i.e., group 1, rat 1 f group 2, rat 1 f group 3, rat 1 f group 4, rat 1 f group 5, rat 1 f group 1, rat 2 f group 5 rat 8. The conditioning samples were a subset of five study samples, and the solvent blank samples were run every 10 study samples to eliminate carryover. Data Analysis. The observational data obtained from animal experiments and ELISA were subjected to one-way analysis of variance (one-way ANOVA) with an LSD test in the SPSS 18 statistical software package (The SPSS Inc., Chicago, IL). A threshold of p value less than 0.05 was considered statistically significant in this study. The acquired data files from GC TOF MS analysis were exported as NetCDF format by ChromaTOF software (version 4.41, Leco Co., St. Joseph, MI). The data pretreatment including baseline correction, peak deconvolution, and alignment, etc. and was conducted by a published procedure.31 Briefly, the ratio of signal to noise was set to 30. The minimum number of samples that contained the analyte was set as 10 (20% of total number of samples), and the minimum percent of samples in a class that contained the analyte was set as 50%. The resulting data set included sample information, retention times, and peak intensities. Subsequent to data extraction, the data set was mean-centered and autoscaled (scaling 1977

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Table 1. Gestation Parametersa dose (mg deca-BDE/kg body weight/day) 0

150

750

1 500

2 500

number treated

10

10

10

10

10

number of implantation sites per litter

7.60 ( 0.37

7.00 ( 0.47

7.20 ( 0.44

7.50 ( 0.31

8.00 ( 0.52

resorptions per litter

0.30 ( 0.21

0.30 ( 0.15

0.30 ( 0.21

0.50 ( 0.27

1.00 ( 0.30

dead fetus per litter

0.00 ( 0.00

0.00 ( 0.00

0.20 ( 0.13

0.00 ( 0.00

0.10 ( 0.10

number of live fetuses per litter

7.30 ( 0.37

6.70 ( 0.47

6.7 ( 0.42

7.00 ( 0.45

6.90 ( 0.60

% postimplantation loss per litter

3.95%

4.29%

6.95%b

6.67%b

13.75%c

% dead fetus per litter

0.00%

0.00%

2.78%

0.00%

1.25%

% resorptions per litter % live fetuses per litter

3.95% 96.05%

4.29% 95.71%

4.17% 93.05%

6.67%b 93.33%

12.50%c 86.25%b

a Data are presented as the means ( SD. b p < 0.05 as compared with the control group. c p < 0.01 (one-way ANOVA) as compared with the control group.

Table 2. Effect of deca-BDE on Fetal Developmenta dose (mg/kg/day) 0

150

750

1 500

2 500

no. of fetuses

72

62

67

69

70

fetal brain weight(g) fetal heart weight(g)

0.0448 ( 0.0021 0.0059 ( 0.0006

0.0454 ( 0.0023 0.0056 ( 0.0003

0.0433 ( 0.0033 0.0054 ( 0.0002

0.0410 ( 0.0016 0.0054 ( 0.0005

0.0362 ( 0.0010b 0.0043 ( 0.0002c

fetal liver weight(g)

0.0510 ( 0.0055

0.0457 ( 0.0021

0.0435 ( 0.0030

0.0447 ( 0.0028

fetal body weight(g)

0.4562 ( 0.018

0.4434 ( 0.0124

0.4086 ( 0.0093

0.4103 ( 0.0144

b

0.0400 ( 0.0017b b

0.3548 ( 0.0136c

fetal brain/body weight (%)

10.17 ( 0.18

10.24 ( 0.39

10.81 ( 0.81

10.21 ( 0.44

9.11 ( 0.50

fetal heart/body weight (%)

1.32 ( 0.09

1.31 ( 0.11

1.33 ( 0.04

1.36 ( 0.16

1.17 ( 0.08

fetal liver/body weight (%)

11.33 ( 0.82

10.63 ( 0.79

10.71 ( 0.65

10.97 ( 0.40

10.83 ( 0.57

placenta weight(g)

0.1190 ( 0.0043

0.1148 ( 0.0017

0.1122 ( 0.0019

0.1146 ( 0.0028

0.1097 ( 0.0024b

Data are presented as the means ( SD. p < 0.05 as compared with the control group. p < 0.01 (one-way ANOVA) as compared with the control group. a

b

to unit variance) before multivariate statistical analysis in the SIMCA-P+  Sweden). In this study, 12.0.1 software package (Umetrics, Umea, principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA), and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were employed. To evaluate the model’s validity and determine the number of principal components optimal for a valid model, a default cross-validation procedure with 7-fold (leave-1/7th samples-out) cross-validation was carried out,32 and two essential parameters that assess the model quality and reliability, i.e., R2Y and Q2Y were obtained accordingly. A model with R2Y and Q2Y approaching 1.0 indicates an ideal model with satisfactory predictive ability, while the PLS-DA model with Q2Y g 0.4 was acceptable in practical applications (www.umetrics.com). Differential metabolites between the dosed groups and the control group were selected by variable importance on projection (VIP) with a threshold of 1.0 in OPLS-DA models. A univariate Wilcoxon Mann Whitney (WMW) test was also conducted to cross-validate the significance of differential metabolites obtained from the OPLS-DA model at a univariate level. The critical p-value of the WMW test was set to 0.05 in this study. Compound Annotation. Initial compound annotation was performed by comparing the mass spectral data of each metabolite with those in the NIST/EPA/NIH (NIST 08) mass spectral library (National Institute of Standards and Technology, Gaithersburg, MD), and a match score greater than 700 was needed. Subsequently, identification of metabolites was further validated by our in-house GC-MS metabolite library (retention index and mass spectral data) that was established

c

using approximately 550 (endogenous metabolites) reference standards available in our laboratory.

’ RESULTS General Information of the Animal Experiment. The overall impact of deca-BDE on fetal development is described in Table 1. All of the deca-BDE-dosed group mice exhibited an increased rate of postimplantation and resorptions while also exhibiting a decreased rate of live fetuses per litter as compared with the controls. The disruption observed in the 2 500 mg/kg group was the most severe among the dosed groups, and the altered rates were statistically significant as compared to the control group. The percentage of live fetuses per litter decreased from 96.05% to 86.25% in the 2 500 mg/kg group. The rate of resorptions and postimplantation loss per litter increased from 3.95% to 12.5% and 3.95% to 13.75% in this group, respectively. The weight of the placenta, fetal body, and organs (brain, heart, and liver) was also decreased in the four dosed groups, of which the 2 500 mg/kg group exhibited a statistically significant decline (p < 0.05) (Table 2). In addition, there was apparent relative weight loss in the 2 500 mg/kg group as compared to the control group, but the change was not significant. None of the dosed group exhibited visible external abnormities as seen in fetal mice. Thyroid Hormone Measurement. As illustrated in Figure 1, total T4 and T3 concentrations in the serum significantly 1978

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Figure 1. Box-plots of the concentration of maternal serum T4 and T3. (A) Serum T4; (B) Serum T3. n = 8 for each group except for the 150 mg/kg group (n = 7). **p < 0.05 obtained from one-way ANOVA with the LSD test. The results indicated that serum T4 and T3 were significantly decreased in the 2 500 mg/kg group. T4 was also significantly reduced in the 1 500 mg/kg group.

Figure 2. Typical total ion current (TIC) chromatograms of maternal serum: (A) control group; (B) 2 500 mg/kg group. Labels are listed in Table 3.

decreased in the 2 500 mg/kg group in comparison with the control group (p < 0.01). In the 1 500 mg/kg group, T4 was significantly decreased, and T3 was decreased but not significantly. In both the 750 mg/kg and 150 mg/kg groups, no obvious changes were observed. However, a dose-dependent decrease of T3 and T4 was implicated across the four dosed groups (Figure 1). Metabonomic Phenotyping. Representative total ion current (TIC) chromatograms of maternal serum from a control mouse (Figure 2A) and a 2 500 mg/kg group mouse (Figure 2B) clearly presents many differences in peak intensities between the two rats from the two different groups. However, it is not feasible to compare chromatograms of all the subjects simultaneously and quantitatively. Therefore, an unsupervised PCA was initially utilized to visualize general clustering trends and supervise outliers in all of the samples. In the three-dimensional PCA scores plots (R2X = 0.588, Figure S1 (Supporting Information)), the separation trend was observed between the deca-BDE groups at high dose groups

versus the control group. However, PCA did not provide sufficient separation in low dose groups from the controls. Therefore, a supervised PLS-DA was established by maximizing metabolic profile variations among all the groups. The resulting three-component PLS-DA classification plot (Figure 3A, R2X = 0.460, R2Y = 0.444, Q2Y = 0.243) presents an apparent separation of metabolic profiles between the dosed groups versus the control group. As illustrated in Figure 3A, metabolic profiles of the 150 mg/kg and 750 mg/kg groups, as well as the 1 500 mg/kg and 2 500 mg/kg groups, tend to cluster together, suggesting a dose-dependent metabolic shift in mice upon deca-BDE administration. To attain the differential metabolites most associated with deca-BDE toxicity, a more sophisticated OPLS-DA model was employed accordingly. As shown in an OPLS-DA scores plot (Figure 3B), there is a distinct separation between the 2 500 mg/ kg group and the control group. On the basis of the combined criteria including VIP values (VIP g 1.0) of the OPLS-DA model 1979

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Table 3. Differential Metabolites Derived from the OPLS-DA Model with the Wilcoxon Mann Whitney Test Correlated with PBDE (2 500 mg/kg versus Control) Exposure 2 500 mg/kg vs control no.

metabolite

rt/min VIPb

1

glycerol

8.39

2.2

2

fumaratea

9.49

1.7

fold changec 2.8

pd 0.0008

2.1

0.0117

2.2

0.0087

3

malate

11.26

1.8

4

glycerol 3-phosphatea

14.71

2.5

2.8

0.0008

5 6

gluconatea palmitelaidic acida

18.2 18.46

1.4 1.7

2.0 2.0

0.0209 0.0157

7

palmitic acida

18.76

2.5

2.8

0.0008

8

linolenic acid

20.72

2.5

2.8

0.0008

9

linoleic acida

20.92

2.5

2.8

0.0007

10 oleic acida

20.98

2.4

2.8

0.0008

11 stearic acida

21.2

2.6

2.8

0.0007

12 arachidonic acida

21.97

2.5

2.8

0.0008

13 docosahexaenic acida 14 2-(9Z-octadecenoyl)-glycerol

22.91 23.72

2.4 2.4

2.8 2.8

0.0008 0.0008

15 1-(9Z-octadecenoyl)-glycerol

23.92

2.4

16 1-stearoyl-glycerola

24.06

2.2

17 cholesterola

27.22

2.6

2.8 2.8 2.8

0.0008 0.0008 0.0008

a

Figure 3. Score plots of the PLS-DA model (4 dose groups and a control group) and OPLS-DA model (2 500 mg/kg vs control) derived from the GC-TOFMS spectral data of maternal serum. (A) PLS-DA score plot; (B) OPLS-DA score plot. The PLS-DA score plot demonstrated the apparent separation of metabolic profiles in four dose groups from the control group and showed a similar metabolic pattern between 150 mg/kg and 750 mg/kg, as well as between 1 500 mg/kg and 2 500 mg/kg.

and p values (p < 0.05) of the Wilcoxon Mann Whitney test, a total of 64 differential peak signals were attained. Seventeen of these peak signals (Table 3) were annotated by comparing the mass spectral data with that from commercial databases and validated by our internal metabolite library. The majority of the altered metabolites were involved in lipid metabolism. In addition, the intermediates from the TCA cycle such as fumaric acid and malic acid were decreased. Metabolic profile differentiation between other dose groups versus the control group is provided in Figures S2 S4 (Supporting Information).

’ DISCUSSION In this study, the administration of deca-BDE at a dose of 2 500 mg/kg body weight to mice during pregnancy (g.d.s 7 9) resulted in significantly increased postimplantation and resorptions, and decreased live fetuses. The weight of the fetuses and their vital organs was also significantly decreased in this group. These findings demonstrate severe embryotoxicity of deca-BDE at a high dose, although no visible external abnormities in fetal mice was observed in any of the dosed group. This finding is consistent with a previous teratogenicity study on deca-BDE in which intrauterine exposure to commercial deca-BDE in doses of 10, 100, or 1 000 mg/kg body weight per day on g.d.s 6 15 did not induce external malformations in Sprague Dawley rats.33

Metabolites verified by the conference compound; others were identified using available commercial library databases. b Variable importance on the projection (VIP) was obtained from the OPLS-DA model. c Fold change (FC) with a positive value means that the concentration of a metabolite is relatively higher in the dose group, while a negative value suggests a relatively lower concentration as compared to the control group. d The p-value and FC are calculated from nonparametric Wilcoxon Mann Whitney test.

The administration of deca-BDE at a dose of 1 500 mg/kg or 2 500 mg/kg caused a decrease of serum total T4 and T3, and the decrease was also significant in the 2 500 mg/kg group. Blood T4 is the major product secreted by thyroid gland, and T3 is mainly formed from deiodination of T4 in the liver or other tissues.34 As thyroid hormones play a critical role in tissue accretion and differentiation,28 and maternal serum is the primary source for hormones that a fetus needs, the imbalance of thyroid hormones in dams caused by deca-BDE may impair normal fetal development. It has been reported that maternal hypothyroidism resulted in pronounced growth retardation.35 Therefore, fetal growth retardation in the 2 500 mg/kg group could correlate with the depletion of thyroid hormone of the maternal serum. By means of a GC TOF MS-based metabonomic approach, a total of 17 differential metabolites were identified. These significantly perturbed metabolites are mainly involved in lipid metabolism and the TCA cycle (Table 3 and Figure 4). For instance, fumaric acid and malic acid are intermediates in the TCA cycle36 and were significantly decreased in the 2 500 mg/kg group. This implicated an inhibitory effect of deca-BDE on aerobic energy metabolism. As a result, the production of lactic acid increased (FC = 1.6, p = 0.12) in the 2 500 mg/kg group as compared to the control group, but such an increase is not statistically significant. Together with the alterations in lipid metabolism including increased glycerol, glycerol 3-phosphate, free fatty acids, 1-stearoyl-glycerol, and decreased 2-(9Z-octadecenoyl)glycerol and 1-(9Z-octadecenoyl)-glycerol, we conceive that deca-BDE could induce an inhibition of energy metabolism in 1980

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synthesis of fatty acids. The depleted level of cholesterol observed in the study is derived from an inhibition of upstream cholesterol synthesis, which is reported to lead to teratogenic action during prenatal development. Dysregulated glucose and lipid metabolism is a result of or associated with cholesterol depletion and thyroid hormone deficiency, with an outcome of impaired fetal development during maternal pregnancy. The animal experiment demonstrated an inhibitory effect of decaBDE on fetal growth and development where no visible malformations were observed in fetus in any of the dosed groups.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional experimental methods (thyroid hormone assay methods) and PCA score plot of all the groups; OPLS-DA score plot of 150 mg/kg, 750 mg/kg, or 1 500 mg/kg versus the control group. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Figure 4. Disrupted metabolic pathways associated with deca-BDE administration. Metabolites in orange represent a higher concentration, while metabolites and hormone in green represent a lower concentration in 2 500 mg/kg group. Dotted arrows and red characters are proposed interactions.

Corresponding Author

*E-mail: [email protected] (X.M.; questions regarding animal experiments); ([email protected] (W.J.; questions regarding metabonomics/metabolomics work). Author Contributions

maternal mice because an increased metabolic rate of body fat is expected to provide energy for life activities of the fetus. Therefore, acceleration of lipolysis will lead to the increased levels of fatty acids, monoglyceride, and related metabolites in maternal serum. The declined levels of 2-(9Z-octadecenoyl)-glycerol and 1-(9Z-octadecenoyl)-glycerol remain to be interpreted. It has been demonstrated that thyroid hormones can remarkably influence carbohydrate and lipid metabolism.37 39 Thyroid hormone deficiency would suppress the TCA cycle and cause fatty acid oxidation.40 Thus, the disrupted T3 and T4 also support the observation of abnormal energy and lipid metabolism. In addition, the significantly decreased level of cholesterol was observed. The depleted level of cholesterol observed in the study suggests that there is significant inhibition of upstream cholesterol synthesis, which has teratogenic action during prenatal development.41 It has also been reported that cholesterol is essential to proper signaling in fetal development and that a lower level of cholesterol in maternal serum may increase the risk of fetal agenesis.42 Since glucose is the main energy substrate for fetal development,43 the deposition of maternal body fat is significantly increased in early gestation,44 and maternal lipolysis will be enhanced in late gestation45 to supply energy for mother and provide substrate for gluconeogenesis such that glucose is sufficient for fetus.46,47 However, excessive lipolysis will cause hyperlipidaemia that may induce oxidative stress,48 and maternal oxidative stress will obviously impair fetal development during maternal pregnancy.49,50 In summary, we investigated the embryotoxicity of deca-BDE in pregnant mice using a metabonomics profiling approach. Figure 4 is a schematic depiction of dysregulated metabolic pathways observed in experimental mice with deca-BDE-induced embryotoxicity. A depletion of thyroid hormone in the maternal serum is directly linked to the observation of fetal growth retardation in the 2 500 mg/kg group. Thyroid hormone deficiency impairs the TCA cycle, as evidenced by depleted levels of fumarate and malate, which are also involved in anabolic

#

These authors contributed equally to this work.

’ ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (also called the 973 Program. Grant numbers: 2007CB511905 and 2007CB511901). ’ ABBREVIATIONS PBDEs, polybrominated diphenyl ethers; deca-BDE, decabrominated diphenyl ether; g.d., gestation day; ELISA, enzyme linked immunosorbent assay; GC TOF MS, gas chromatography time-of-flight mass spectrometry; BSTFA, N,O-bis-trimethylsilyl-trifluoroacetamide; ANOVA, analysis of variance; PCA, principal component analysis; PLS-DA, partial least-squares-discriminant analysis; OPLS-DA, orthogonal partial least-squares-discriminant analysis; VIP, variable importance on projection ’ REFERENCES (1) Talsness, C. E. (2008) Overview of toxicological aspects of polybrominated diphenyl ethers: A flame-retardant additive in several consumer products. Environ. Res. 108, 158–167. (2) Costa, L. G., and Giordano, G. (2007) Developmental neurotoxicity of polybrominated diphenyl ether (PBDE) flame retardants. Neurotoxicology 28, 1047–1067. (3) Zhang, S., Bursian, S. J., Martin, P. A., Chan, H. M., Tomy, G., Palace, V. P., Mayne, G. J., and Martin, J. W. (2009) Reproductive and developmental toxicity of a pentabrominated diphenyl ether mixture, DE-71, to ranch mink (Mustela vison) and hazard assessment for wild mink in the great lakes region. Toxicol. Sci. 110, 107–116. (4) Yu, L. Q., Deng, J., Shi, X. J., Liu, C. S., Yu, K., and Zhou, B. S. (2010) Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae. Aquat. Toxicol. 97, 226–233. (5) Daubie, S., Bisson, J. F., Lalonde, R., Schroeder, H., and Rychen, G. (2011) Neurobehavioral and physiological effects of low doses of 1981

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