Bioaccumulation, Biotransformation, and Toxicity of BDE-47, 6-OH

Article Environmental Science & Bioaccumulation, Technology is published by the American Chemical biotransformation and Society. 1155 Sixteenth Street N.W., Washington, toxicity ofDC BDE-47, 20036 by RUTGERS Subscriber access provided Published by American UNIVERSITY Chemical Society. Copyright © American Chemical Society.

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PageEnvironmental 1 of 44 Science & Technology

ACS Paragon Plus Environment

Environmental Science & Technology

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Bioaccumulation, biotransformation and toxicity of BDE-47, 6-OH-BDE-47 and

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6-MeO-BDE-47 in early life-stages of zebrafish (Danio rerio)

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Hongling Liu1#*, Song Tang2#, Xinmei Zheng1, Yuting Zhu1, Zhiyuan Ma1, Chunsheng Liu1,

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Markus Hecker2,3, David M.V. Saunders3, John P. Giesy1,3,4,5, Xiaowei Zhang1, Hongxia Yu1*

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Nanjing University, Nanjing, Jiangsu 210023, China

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2

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N

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5B3, Canada

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3

Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada

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Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK

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S7N 5B3, Canada

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5

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Kong, SAR, China

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#

These authors contributed equally to this work.

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*

Correspondence to: Drs. Hongling Liu and Hongxia Yu, School of the Environment,

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Nanjing University, Nanjing, Jiangsu 210023, China

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Tel: +86-25-89680356; Fax: +86-25-89680356; Email: [email protected] (Dr. Hongling Liu)

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and [email protected] (Dr. Hongxia Yu)

Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong

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Key words: PBDEs, endocrine disruption, nuclear receptor, aryl hydrocarbon receptor,

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estrogen receptor, androgen receptor, thyroid hormone receptor

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2



26

Abstract

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2,2',4,4'-Tetrabromodiphenyl ether (BDE-47), 6-hydroxy-tetrabromodiphenyl ether

28

(6-OH-BDE-47), and 6-methoxy-tetrabromodiphenyl ether (6-MeO-BDE-47) are the most

29

detected congeners of polybrominated diphenyl ethers (PBDEs), OH-BDEs, and MeO-BDEs,

30

respectively, in aquatic organisms. Although it has been demonstrated that BDE-47 can

31

interfere with certain endocrine functions that are mediated through several nuclear hormone

32

receptors (NRs), most of these findings were from mammalian cell lines exposed in vitro. In

33

the present study, embryos and larvae of zebrafish were exposed to BDE-47, 6-OH-BDE-47,

34

and 6-MeO-BDE-47 to compare their accumulation, biotransformation, and bioconcentration

35

factors (BCF) from 4 to 120 hpf. In addition, effects on expression of genes associated with

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eight different pathways regulated by NRs were investigated at 120 hpf. 6-MeO-BDE-47 was

37

most bioaccumulated and 6-OH-BDE-47, which was the most potent BDE, was least

38

bioaccumulated. Moreover, the amount of 6-MeO-BDE-47, but not BDE-47, transformed to

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6-OH-BDE-47 increased in a time-dependent manner, approximately 0.01%, 0.04%, and 0.08%

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at 48, 96, and 120 hpf, respectively. Expression of genes regulated by the aryl hydrocarbon

41

receptor (AhR), estrogen receptor (ER), and mineralocorticoid receptor (MR) was affected in

42

larvae exposed to 6-OH-BDE-47, while genes regulated by AhR, ER, and the glucocorticoid

43

receptor (GR) were altered in larvae exposed to BDE-47. The greatest effect on expression of

44

genes was observed in larvae exposed to 6-MeO-BDE-47. Specifically, 6-MeO-BDE-47

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affected the expression of genes regulated by AhR, ER, AR, GR, and thyroid hormone

46

receptor alpha (TRα). These pathways were mostly down-regulated at 2.5 µM. Taken together,

47

these results demonstrate the importance of usage of an internal dose to assess the toxic

48

effects of PBDEs. BDE-47 and its analogs elicited distinct effects on expression of genes of

49

different hormone receptor-mediated pathways, which have expanded the knowledge of

50

different mechanisms of endocrine disrupting effects in aquatic vertebrates. Because some of 3


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these homologues are natural products assessments of risks of anthropogenic PBDE need to

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be made against the background of concentrations from naturally occurring products. Even

53

though PBDEs are being phased out as flame retardants, the natural products remain.

4



54

Introduction

55

Polybrominated diphenyl ethers (PBDEs) have been extensively employed as flame

56

retardants (FRs) in various consumer and commercial products for decades.1, 2 As a result of

57

the substantial production, long-term use, disposal, and recycling processes, these chemicals

58

are now frequently found in the environment.3 The persistence, bioaccumulation potential

59

and toxic potency (PBT criteria) of 2,2',4,4' -tetrabromodiphenyl ether (BDE-47), one of the

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primary PBDEs found in the environment, has raised concern about its potential adverse

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effects to ecosystems and human health.3-5 In addition to the synthetic BDE-47, its

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hydroxylated (OH-) or methoxylated (MeO-) forms, 6-OH-BDE-47 and 6-MeO-BDE-47,

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have been suggested to be natural products of marine organisms6 and have been detected in a

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wide variety of freshwater and marine organisms including mollusks, mussels, shellfish, clam,

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fish, seal, dolphin, and whale.4, 7-13 Moreover, it has been conclusively demonstrated that

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MeO-BDEs, and not PBDEs, are precursors of OH-BDEs.6, 17-19 In zebrafish (Danio rerio),

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6-MeO-BDE-47 can be transformed into 6-OH-BDE-47; however, BDE-47 cannot be

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transformed into 6-OH-BDE-47.20 In addition, inter-conversion between 6-MeO-BDE-47 and

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6-OH-BDE-47 has been observed during dietary exposure of Japanese medaka (Oryzias

70

latipes).17

71

To date, increasing evidence has shown that exposure to BDE-47 and its two natural

72

analogs, 6-OH-BDE-47 and 6-MeO-BDE-47, can elicit a number of adverse effects in

73

aquatic organisms including disruption of the endocrine system,21, 22 disruption of molting,23

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developmental defects,20, 24-26 and neurobehavioral toxicity.27-29 OH- and MeO-BDEs have

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been shown to exhibit greater toxic potencies than PBDEs for certain endpoints such as 5


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estrogenicity and androgenicity.21, 30 However, mechanisms of their toxicity are complex and

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have not been fully resolved.31 PBDEs as well as OH- and MeO-BDEs are structural analogs

78

to thyroid hormones, T3 and T4, as well as dioxin-like chemicals such as polychlorinated

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biphenyls (PCBs), dioxins (TCDD), and furans (PCDF).31 This raises the question of whether

80

the adverse biological outcomes resulting from exposure to PBDEs and OH- or MeO-BDEs

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are due to their ability to simulate thyroid hormones or whether they elicit effects similar to

82

those of the above dioxin-like chemicals.

83

Nuclear receptors (NRs) are a superfamily of ligand-activated, transcription factors that

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act globally to regulate a broad range of biological processes, including development,

85

reproduction, and metabolism.32, 33 NRs mediate signaling by ligands such as endogenous

86

hormones, lipids, and xenobiotics.34, 35 Upon binding of a ligand to the ligand binding domain

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of several kinds of NRs, a complex array of cellular responses is initiated. Recently, several

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in vivo and in vitro studies have investigated effects of BDE-47, OH-BDE-47 or

89

MeO-BDE-47 on certain NR mediated physiological pathways, in particular the pathways

90

involving the thyroid hormone receptor (TR), estrogen receptor (ER), androgen receptor

91

(AR), and aryl hydrocarbon receptor (AhR). For example, in adult fathead minnows

92

(Pimephales promelas), dietary exposure to BDE-47 induced transcription of TRα in the

93

brain of females, and decreased the transcription of TRβ in the brain of fish of both sexes.41

94

In porcine ovarian follicles, both BDE-47 and 6-OH-BDE-47 did not alter expression of AR

95

mRNA or associated protein, but decreased expression of ERβ mRNA and protein following

96

exposure to BDE-47 and increase both ERα and ERβ gene and protein expression following

97

exposure to 6-OH-BDE-47.42 In an AhR-responsive luciferase reporter assay, 6-OH-BDE-47 6



98

exhibited greater potency to induce AhR activity than that of 6-MeO-PBDEs and BDE-47.43

99

However, so far, most NR studies of BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47 were

100 101

completed by use of mammalian or cellular assays. The zebrafish represents an excellent vertebrate model organism in environmental

102

toxicology studies,44-46 especially in context with investigating effects of endocrine disrupting

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chemicals (EDCs) on reproductive and developmental systems.47-49 Moreover, developmental

104

profiling of zebrafish gene expression patterns has confirmed a high degree of conservation

105

in NR expression patterns between zebrafish and other vertebrate models.50 Therefore, in the

106

present study, zebrafish embryos and larvae were used to determine the time-course of

107

accumulation, biotransformation, and bioconcentration factors (BCFs) of BDE-47,

108

6-OH-BDE-47, and 6-MeO-BDE-47. Additionally, in order to gain a more comprehensive

109

understanding of the molecular mechanisms of the toxicity of BDE-47 and related OH- and

110

MeO-analogs on the endocrine system, their effects on expression of genes associated with

111

eight nuclear hormone receptor pathways, particularly ER, AR, AhR, TRα, peroxisome

112

proliferator-activated receptor alpha (PPARα), glucocorticoid receptor (GR),

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mineralocorticoid receptor (MR), and pregnane x receptor (PxR), were investigated and

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compared.

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Materials and methods

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Materials and reagents

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BDE-47 (98% purity) was purchased from Chem Service (West Chester, PA, USA).

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6-MeO-BDE-47 and 6-OH-BDE-47 were synthesized at City University of Hong Kong, and

119

purities were more than 98% as described previously.51 13C-PCB-178, 13C-2’-OH-BDE-99

120

and 13C-BDE-139 were purchased from Cambridge Isotope Laboratories (Andover, MA,

121

USA). BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47 were dissolved in dimethyl sulfoxide

122

(DMSO, Generay Biotech, Shanghai, China) to prepare stock solutions and then diluted with

123

embryonic rearing water (60 mg/L instant ocean salt in aerated distilled water) to the desired

124

test concentrations. Concentration of DMSO in final test solutions did not exceed 0.1%.

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RNAlater, RNA Stabilization Reagents, and RNeasy® Mini Kit were purchased from

126

QIAGEN (Hilden, Germany). Maxima® First Strand cDNA Synthesis Kits were purchased

127

from Fermentas (St Leon-Rot, Germany). SYBR® Real time PCR Master Mix Plus Kits were

128

purchased from Toyobo (Tokyo, Japan).

129

130 131

Animals and exposure experiment Adult (7-months old) AB strain zebrafish maintenance and culturing were performed as

132

previously described.20 The eggs were examined under a stereomicroscope and only normally

133

developed embryos were used for exposure experiments. Briefly, twenty embryos were

134

randomly distributed into a 25 mL glass beaker containing 20 mL of exposure solution. Fish

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were exposed until 120 hour post-fertilization (hpf), by which time they had developed into

136

free-swimming larvae and most organs had completed development.52 The control group

137

received 0.1% DMSO (v/v) only. 100% of the exposure solutions were replaced by fresh

138

exposure solution every 48 h. For 6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47 exposures, 8



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the experiments included two parts: First, zebrafish embryos were exposed to 6-OH-BDE-47

140

(0, 0.008, 0.02, 0.05, 0.1, 0.5 µM), 6-MeO-BDE-47 (0, 0.02, 0.1, 0.5, 2.5 µM) and BDE-47

141

(0, 0.02, 0.1, 0.5, 2.5 µM) from 4 to 120 hpf to study the morphologic toxicity of compounds

142

as previously described.20 Secondly, based on the results of acute toxicity test, three

143

comparable exposure concentrations for each compound were chosen: 6-OH-BDE-47 at

144

0.008, 0.02, and 0.05 µM, and at 0.1, 0.5, and 2.5 µM for both 6-MeO-BDE-47 and BDE-47

145

from 4 to 120 hpf to study the effects on expression of 63 genes involved in eight

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receptor-mediated pathways by q-RT-PCR. After exposure for 120 hpf larvae were

147

anesthetized with ethyl 3-aminobenzoate methanesulfonate (MS-222, Suzhou Xin Yong

148

Biological Medicine Technology Co Lt, Jiangsu, China), and were preserved in RNAlater

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RNA Stabilization Reagents until total RNA isolation.

150 151

Bioavailability analysis and QA/QC

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This experiment was designed to analyze bioaccumulation of the three chemicals in early

153

life-stages of zebrafish. In each treatment, 600 zebrafish embryos were exposed to

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6-OH-BDE-47, 6-MeO-BDE-47 or BDE-47 at 300 µg/L (0.6, 0.58, and 0.62 µM,

155

respectively) from 4 to 120 hpf in glass beakers. Variation among concentrations of the three

156

compounds in both the exposure medium and the embryos/larvae was determined. 80

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embryos/larvae and corresponding exposure solutions were collected at 12, 24, 48, 72, 96,

158

and 120 hpf. Detailed protocols for extraction, clean up, and quantification, and quality

159

assurance and quality control (QA/QC) are provided in previous studies20,

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Supporting Information Methods and Table S1

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and

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Quantitative RT-PCR Total RNA was isolated from zebrafish larvae using RNeasy® Mini Kit. The

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concentration and quality of total RNA were determined in accordance with the procedures

165

described in a previous study.45 First-strand cDNA synthesis and quantitative RT-PCR were

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performed using Maxima® First Strand cDNA Synthesis and SYBR® Realtime PCR Master

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Mix Plus Kits.20 Quantitative RT-PCR was performed by an Applied Biosystems Stepone

168

Plus Real-time PCR System (Foster City, California, USA). The primers were either mined

169

from previous literature55 or designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/).

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Primer sequences were listed in Supporting Information Table S2. The housekeeping gene18S

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small subunit ribosomal RNA (18S rRNA) was used as an internal control.56 The thermal

172

cycle was set at 95 oC for 2 min, followed by 40 cycles of 95 oC for 15 s and 60 oC for 1 min.

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Melting curves were derived during RT-PCR to validate that all cDNA samples amplified

174

only a single product. Levels of expression of genes were normalized to 18S rRNA mRNA

175

contents using the 2-∆∆Ct method. Each concentration was measured in triplicate or

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quadruplicate in a composite sample containing 20 larvae.

177 178 179

Nuclear receptor pathway analysis For genes relating to AhR and ER pathways, the Agilent Literature Search application

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was used to construct a biological interaction network within the Cytoscape software v3.1.1

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(Cytoscape consortium, San Diego, CA, USA).57-59 The gene networks of the other six NR

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pathways were retrieved by either WikiPathways (http://www.wikipathways.org)60 or

183

SABioscience Gene Network Central 10



184

(http://www.sabiosciences.com/genenetwork/genenetworkcentral.php), and integrated with

185

AhR and ER pathways as “associations” and visualized as one network by Cytoscape. Only

186

genes of interest were shown in this pathway network. The resulting network genes (nodes)

187

were colored by the Enhanced Graphics application within Cytoscape according to the

188

significant fold changes of gene expressions in the respective treatments.

189 190 191

Statistical analysis SPSS 12.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A

192

Kolmogorov-Smirnov test was used to verify the normality of the data, and the homogeneity

193

of variances was analyzed by Levene’s test as previously described.20 If the data failed the

194

Kolmogorov-Smirnov test, logarithmic transformation was performed and data was checked

195

again for homogeneity of variances. A one-way analysis of variance (ANOVA) followed by

196

LSD test was used to evaluate differences between the control and exposure groups. A value

197

of p < 0.05 was considered statistically significant. To capture the likely nonlinearity in

198

concentrations in exposure water or in zebrafish embryos-larvae across different time-points,

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generalized additive models (GAMs) were used by the ‘mgcv’ package in R software version

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3.10 (R Core Team, Vienna, Austria). Hierarchical cluster analysis for the gene expression

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was performed by the "complete" method in R. A heatmap of gene expression results was

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implemented by ‘pheatmap’ package version 0.7.7 in R.

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Results

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Morphologic effects of 6-OH-BDE-47, 6-MeO-BDE-47 and BDE-47

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Among the three compounds, 6-OH-BDE-47 was the most potent to zebrafish

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embryos/larvae (Supporting Information Figures S1. B, C, D, and F and Table S3). Exposure

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to all concentrations caused delayed development of embryos for up to 6-8 h at 24 hpf. In

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embryos exposed to 0.5 µM 6-OH-BDE-47, mortality significantly increased to 22.5 ± 4.08%,

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at 48 hpf. At 48 hpf, in groups exposed to concentrations greater than 0.1 µM, the embryos

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developed hypopigmentation. At 72 hpf, development was arrested in all embryos exposed to

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0.5 µM 6-OH-BDE-47 (Figures S1. B and C) while development of embryos at 12-18 hpf

212

was not altered. Larvae exposed to 0.1 µM 6-OH-BDE-47 exhibited spinal curvatures (Figure

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S1. D), decreased heartbeats, and reduced body lengths (3516 ± 250 µm in 0.1µM group and

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4040 ± 55 µm in control). The LC50 values of 6-OH-BDE-47 for teratogenic effects were 0.28

215

µM (0.21-0.38) at 72 hpf, 0.13 µM (0.11-0.16) at 96 hpf, and 0.09 µM (0.04-0.10) at 120 hpf.

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The most sensitive toxicological endpoint was spinal curvature (Figure S1. F), which was

217

manifested in a concentration-dependent manner with maximal effects resulting from

218

exposure to 0.08 µM (0.07-0.09) 6-OH-BDE-47 at 120 hpf.

219

There were no significant differences in developmental alterations in individuals exposed

220

to 6-MeO-BDE-47 and the control group until 96 hpf. The most sensitive toxicological

221

endpoints were concomitant spinal curvature and pericardial edema at 120 hpf at 2.5 µM

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(Figure S1. G). Although no statistically significant differences were observed within 96 hpf

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following exposure to BDE-47 up to 2.5 µM, concomitant spinal curvature and pericardial

224

edema occurred in embryos at 120 hpf. The NOEC of spinal curvature and pericardial edema

225

was 0.5 µM of BDE-47, although there were significant differences at 2.5 µM and the

226

proportion of affected larvae was 27.6 ± 18.3% (Figure S1 H). In addition, at 120 hpf

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following exposure to 2.5 µM BDE-47the body lengths of larvae (3751 ± 152 µm) were 12



228

significantly reduced compared to those in the control group (4029 ± 201µm).

229

230 231

Accumulation by zebrafish Concentrations of BDE-47, 6-OH-BDE-47, or 6-MeO-BDE-47 were below their

232

analytical method detection limits in the control group (Supporting Information Table S4). At

233

120 hpf, 100% mortality occurred following exposure to 300 µg/L 6-OH-BDE-47, therefore,

234

no data is available for this time point. GC/MS results indicated that the chemical

235

concentrations in exposure solutions decreased and the doses in embryos and larvae increased

236

in a time-dependent manner (Figure 1). Moreover, the concentrations of BDE-47 and

237

6-MeO-BDE-47 were approximately 10- to 100-fold greater than concentrations of

238

6-OH-BDE-47 in zebrafish embryos and larvae that were exposed to the same concentrations

239

of the three compounds (Figure 1). The three BDE congeners ranked as follows regarding

240

their in vivo accumulation (values from greater to lesser potential): 6-MeO-BDE-47 >

241

BDE-47 > 6-OH-BDE-47.

242

Calculated BCF values for 6-OH-BDE-47 were 4.07, 9.88, 21.7, 26.9, and 23.3 at 12 hpf,

243

24 hpf, 48 hpf, 72 hpf, and 96 hpf, respectively (Figure 2A). For 6-MeO-BDE-47, the BCF

244

values were 17.2, 42.6, 89.7, 390, 1207, and 935 at 12 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, and

245

120 hpf, respectively. For BDE-47, bioconcentration factor (BCF) values were 25.7, 68.0,

246

489, 750, 2489, and 2430 at 12 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, 120 hpf, respectively.

247

These results indicated that trends of BCF values after various durations of exposure were

248

similar for 6-MeO-BDE-47 and BDE-47, reaching a maximum at 96 hpf, and showing a

249

slight decrease at later time points.

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Biotransformation in zebrafish Exposure of zebrafish to 300 µg/L 6-MeO-BDE-47 resulted in increasing tissue

253

concentrations of 6-OH-BDE-47 in their tissues were quantified in a time-dependent manner,

254

with 0.02, 0.11, 0.12, and 0.25 µg/g, wm (wet mass) at 48, 72, 96, and 120 hpf, respectively

255

(Figure 2B). However, no transformation of BDE-47 into 6-OH-BDE-47 or 6-MeO-BDE

256

occurred. Also, 6-OH-BDE-47 was not transformed into either BDE-47 or 6-MeO- BDE-47.

257

Based on these transformation ratios, the amounts of biotransformed 6-OH-BDE-47 were

258

expected to be 0.04, 0.21, and 1.07 µg/g, wm after 120 hpf exposure to 0.1, 0.5, or 2.5 µM

259

6-MeO-BDE-47, respectively (Table 1). Amounts of biotransformed 6-OH-BDE-47 in larvae

260

exposed to 0.5 and 2.5 µM of 6-MeO-BDE-47 were greater than the accumulated

261

concentration of 6-OH-BDE-47 (0.21 and 1.07 µg/g, wm vs. 0.15 µg/g, wm) in larvae

262

exposed to 0.05 µM of 6-OH-BDE-47 (Table 1).

263 264

265

Transcriptional responses of NR pathways to 6-OH-BDE-47 A hierarchical cluster analysis of gene expression data showed a dendrogram which

266

highlighted five principal clusters (Figure 3A). Exposures to 2.5 µM 6-MeO-BDE-47 resulted

267

in a unique clustering of gene expression data that revealed a significantly different gene

268

expression profile from the other exposures (Figure 3A). Exposures to the same compound

269

but at different concentrations generally clustered in the same group, especially for exposures

270

to 0.008, 0.1, and 0.5 µM 6-OH-BDE-47 (Figure 3A).

271

Zebrafish embryos exposed to 6-OH-BDE-47 had significant alterations in the

272

expression of genes associated with several NR pathways. The most significant effects

273

occurred along the AhR pathway (Figures 3 and 4 and Supporting Information Table S5) with 14



274

exposure to 0.008 µM 6-OH-BDE-47 causing a significant up-regulation in the expression of

275

ahr1a, ahr1b, ahr2, and arnt2 by 2.32-, 1.71-, 1.62-, and 1.62-fold, respectively (p < 0.05),

276

and a significant down-regulation of ahrra and cyp19b expression by 2.27- and 1.81-fold,

277

respectively (p < 0.05). Exposure to 0.02 µM 6-OH-BDE-47 significantly induced expression

278

of ahr1b and ahr2 by 1.63- and 1.67-fold, respectively, and reduced the expression of ahrra

279

and cyp1a1 by 1.96- and 1.75-fold (p < 0.05). Exposure to 0.05 µM 6-OH-BDE-47

280

significantly reduced the expression of cyp1a1, cyp1b1, arnt1la, ahrra, and cyp19b by 1.92-,

281

2.63-, 1.25-, 1.96-, and 2.23-fold, respectively (p < 0.05). In addition to the ahr receptor,

282

following exposure to 0.008 and 0.05 µM 6-OH-BDE-47 the expression of mr and er2b were

283

significantly up-regulated by 1.51- and 1.83-fold, respectively (p < 0.05).

284 285

Transcriptional responses of NR pathways to 6-MeO-BDE-47

286

Some NR-mediated pathways such as AhR, ER, AR, TR, and GR were affected by

287

exposure to 6-MeO-BDE-47 with the greatest effects occurring at the greatest concentration

288

tested, 2.5 µM (Figures 3 and 4). Exposure to lesser concentrations of 6-MeO-BDE-47, 0.1

289

µM, also induced the expression of several genes in these pathways. Specifically, arnt2, dut,

290

ugtlal, ctnnb1, pa2g4a, dap3, and rela were significantly induced by 2.08-, 1.67-, 2.23-, 1.56-,

291

1.78-, and 1.51-fold, respectively (p < 0.05). Exposure to 0.5 µM 6-MeO-BDE-47 did not

292

significantly alter the expressions of most genes, except for the down-regulation cyp1a1 and

293

er2a by 9.09- and 1.56-fold, respectively (p < 0.05). However, exposure to a greater

294

concentration of 6-MeO-BDE-47, 2.5 µM, reduced the expression of most altered genes.

295

Along the AhR pathway, ahr2 was down-regulated by 1.92-fold and associated genes such as

296

cyp1a1, cyp1b1, cyp365a, and sp1 were also down-regulated by 50-, 100-, 3.03-, and

297

1.89-fold, respectively (p < 0.05). However, both ahrra and ahrrb were significantly induced 15


Page 16 of 44

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298

by 3.16- and 5.71-fold, respectively (p < 0.05). In the ER pathway, er2a and ccnd1 were

299

down-regulated by 1.43- and 1.85-fold, respectively, while er2b was up-regulated by

300

1.44-fold following exposure to 2.5 µM 6-MeO-BDE-47 (p < 0.05). In the AR pathway, ar,

301

ctnnb1, pa2g4b, and ncoa1 were down-regulated by 1.85-, 1.89-, 1.47-, and 1.67-fold,

302

respectively (p < 0.05). Exposure to 2.5 µM 6-MeO-BDE-47 also decreased the expression of

303

thra by 2.17-fold and TR associated genes such as ncor and fus were significantly

304

down-regulated by 2.08- and 1.85-fold (p < 0.05). Also, following exposure to 2.5 µM

305

6-MeO-BDE-47, the expression of gr and tgfb1 were down-regulated by 2.04- and 1.72-fold,

306

respectively (p < 0.05).

307 308 309

Transcriptional responses of NR pathways to BDE-47 Exposure to BDE-47 significantly affected the expression of receptors in the AhR, ER,

310

and GR pathways (Figures 3 and 4). The number of altered genes increased in a concentration

311

dependent manner. The expression of gr was significantly down-regulated by 1.28-fold

312

following exposure to 0.5 µM BDE-47 (p < 0.05). When exposed to 2.5 µM, expressions of

313

ahr1b, er2a, and er2b were significantly greater by 1.64-, 1.35-, and 1.40-fold, respectively

314

(p < 0.05) than those of the respective genes controls. Expressions of genes along AhR and

315

ER pathways, such as cyp1a1, cyp19a, cyp3a65, and ccnd1, were significantly less by factors

316

of 5.88-, 2.88-, 2.04-, and 1.52-fold, respectively, relative to that of the controls (p < 0.05).

16



317 318

Discussion Hazard assessment of contaminants is typically based on the exposure of aquatic

319

organisms to chemical solutions for a defined exposure time and the adverse outcomes

320

observed are then correlated with the concentrations of the compounds in the ambient media.

321

Since chemicals need to be accumulated into organisms and distributed to target sites for the

322

induction of toxic effects, usage of target site effect concentrations are postulated to best

323

represent the hazards of a compound in vivo.61, 62 However, for small-bodied organisms such

324

as the zebrafish effect concentrations at the target site are difficult to determine, particularly

325

at earlier stages of development. Average body concentrations of contaminants in zebrafish

326

embryos and larvae are subject to competitive dynamic processes, which include the ability

327

of compounds to penetrate the chorion, in vivo biotransformation, distribution, and excretion.

328

Hence, internal effect concentrations need to be determined. Moreover, the ratio of the

329

internal concentration in a fish to the surrounding concentration at a steady state represents

330

the compound’s BCF, which is an important metric for regulatory assessment of chemicals.63

331

In the present study accumulation, biotransformation, varied among BCF of BDE-47,

332

6-OH-BDE-47, and 6-MeO-BDE-47 during multiple developmental stages of zebrafish. The

333

time points during which accumulation of BDE-47 and 6-MeO-BDE-47 increased

334

substantially coincide with the hatching period of zebrafish embryos (48 hpf). In the late

335

developmental periods of the larvae (after 96 hpf), the bioaccumulation of BDE-47 and

336

6-MeO-BDE-47 reached a plateau, which might be due to an increase in metabolism and/or

337

excretion activities as well as the rapid growth of larvae at this time that might dilute their

338

body concentrations over this time period. In this study, the BCF of 6-OH-BDE-47 at 96 hpf 17


Page 18 of 44

Page 19 of 44


339

was 23.3, which is similar to previously reported results.64 in which BCF values were

340

calculated in liver of zebrafish after 96 h exposure to 100 nM 6-OH-BDE-47. At all six

341

durations of exposure, 6-OH-BDE-47 was the least accumulated into the body, though it had

342

the greatest toxic potency. It is known that a compound with greater logKow generally has

343

greater bioaccumulation potential. Values of logKow were 6.76, 7.17, and 6.59 for BDE-47,

344

6-MeO-BDE-47, and 6-OH-BDE-47, respectively.65 Thus, differences in lipophilicity were

345

considered to be the most important parameters for the different accumulation properties of

346

the test chemicals. For compounds such as 6-OH-BDE-47, the hydroxyl group by making the

347

compound more polar, result in greater excretion, and might also be an important parameter

348

for the lesser in vivo concentration. Hence, our results confirmed that in aquatic exposure

349

tests, it is not sufficient to evaluate the ecotoxicological risk of a compound based solely on

350

the exposure concentration. In addition to accumulation in the body, the results of this study

351

indicated that the amounts of 6-MeO-BDE-47, but not BDE-47, that were transformed to

352

6-OH-BDE-47 increased in a time-dependent manner, (approximately 0.01%, 0.04%, and

353

0.08% at 48, 96, and 120 hpf, respectively), which is indicative of an increasing metabolic

354

capability of zebrafish embryos/larvae with increasing age.

355

Photomicrographs demonstrated that exposures to 6-OH-BDE-47, 6-MeO-BDE-47, and

356

BDE-47 resulted in developmental abnormalities in zebrafish embryos and larvae. The

357

embryo-toxic effects of BDE47, 6-OH-BDE47 and 6-MeO-BDE47 have been investigated in

358

a previous study with zebrafish embryos exposed from 3 to 72 hpf.64 Those authors showed

359

that 6-OH-BDE-47 was the most toxic BDE-47 congener inducing a range of developmental

360

defects including pericardial edema, yolk sac deformations, lesser pigmentation, lessened 18



361

heart rate, and delayed development at concentrations of 25-50 nM, which is consistent with

362

the morphology findings of this study (Figures S1 B, C and D).64 Furthermore, the lack of

363

toxic effects of BDE47 or 6-MeO-BDE47 at 2.5 µM until 120 hpf (Figures S1 G and H) is

364

consistent with previous findings that showed that no toxicity were observed for both BDE47

365

and 6-MeO-BDE47 in zebarfish at 72 hpf.64

366

Since disruptions of cellular molecules or processes are thought to precede adverse

367

outcomes, changes to normal molecular processes might function as sensitive biomarkers to

368

predict adverse biological outcomes.66 Moreover, alteration of NR mediated pathways has

369

been shown to be associated with adverse endocrine and developmental effects that were

370

linked with morphological deformities.20, 55 For example, previous studies have demonstrated

371

that BDE-47 can alter thyroid status and thyroid hormone-regulated gene transcription in the

372

pituitary and brain of adult fathead minnows,67 and both 6-OH-BDE-47 and 6-MeO-BDE-47

373

were shown to affect expression of TRα and TRβ genes in the TR pathway, which can result

374

in teratogenic effects such as pericardial edema, developmental retardation, and curved spine

375

in zebrafish embryos.20 Also, all BDE-47, TBBPA and BPA have been demonstrated to alter

376

expression of genes along the hypothalamus-pituitary-thyroid (HPT) axis of zebrafish larvae

377

as well as induce acute toxicity.68 Additionally, zebrafish has been used to investigate effects

378

of EDCs on the expression of genes in six NR mediated pathways.55 In this study, two

379

additional receptor pathways-AR and PxR were added, and the interactions of sixty-three

380

genes involved in eight zebrafish receptor pathways were integrated. This pathway network

381

might represent a novel tool for the examination of the molecular function of each individual

382

receptor as well as for the study of their combinatorial regulatory network within NRs. 19


Page 20 of 44

Page 21 of 44

383


The structures of the three compounds tested are similar. The cluster dendrogram

384

showed that expression of genes in individuals exposed to various concentrations of

385

6-OH-BDE-47 clustered together. Nevertheless, clustering is a function of concentration for

386

6-MeO-BDE-47 and BDE-47. Patterns of expression of genes following exposures to 2.5 µM

387

BDE-47 and 0.5 µM 6-MeO-BDE-47 were grouped into the same cluster, which indicates

388

BDE-47 likely has fewer effects on zebrafish NR-mediated pathways than 6-MeO-BDE-47 at

389

similar waterborne exposure concentrations. Exposure to 2.5 µM of the more

390

bioaccumulative 6-MeO-BDE-47 resulted in a unique gene expression profile compared to

391

BDE-47 and 6-OH-BDE-47. In addition, 1.07 µg/g, wm of biotransformed 6-OH-BDE-47

392

were detected in larvae exposed to 2.5 µM of 6-MeO-BDE-47, which was much greater than

393

the detected amount of 6-OH-BDE-47, 0.15 µg/g, which resulted from exposure to 0.05 µM

394

6-OH-BDE-47. The greater body burden of 6-OH-BDE-47 resulting from exposure to 2.5 µM

395

of 6-MeO-BDE-47 might explain the significant and great effect on gene transcription of NR

396

pathways observed in this exposure group. The effects that occurred in the greatest exposure

397

group of 6-MeO-BDE-47, therefore, were attributed to the combined effects of

398

biotransformed 6-OH-BDE-47 as well as 6-MeO-BDE-47. The clustering of the three

399

compounds correlated well with their respective accumulation potency, indicating the great

400

importance of the usage of internal dose to assess the dose-response relationship for studies

401

of PBDEs, especially MeO-PBDEs.

402

Further analyses of endocrine pathways indicated general disruption of receptor

403

pathways by all three BDEs congeners, which correlated well with the observed teratogenic

404

effects in zebrafish. Specifically, 6-OH-BDE-47 altered the expression of AhR, ER, and MR 20



405

receptor-mediated pathways, while AhR, ER, and GR were the primary pathways altered by

406

BDE-47. Yet, exposure to the more bioaccumulative 6-MeO-BDE-47 affected AhR, ER, AR,

407

GR, and TRα pathways. Molecular structures of OH-BDEs closely resemble those of thyroid

408

hormones (THs) and 6-OH-BDE-47 can disrupt normal thyroid homeostasis and functions as

409

either an agonist or an antagonist.31, 69 For example, expression of genes along the

410

hypothalamus-pituitary-thyroid (HPT) axis that is responsible for regulation of metabolism

411

and early life-stage development was affected by BDE-47 and its OH- or MeO- forms in

412

zebrafish embryos-larvae.20, 68 However, in the present experiment, expression of thra was

413

not significantly altered following exposure to 6-OH-BDE-47, which contradicted previous

414

findings showing thra was reduced in zebrafish exposed to 200 µg/L 6-OH-BDE-47.20 This

415

difference might be due to the almost 10 times greater concentrations used in the previous

416

study. However, exposure to 2.5 µM 6-MeO-BDE-47 significantly decreased the expression

417

of thra, nocr and fus in TRα pathway. Since significant quantities of biotransformed

418

6-OH-BDE-47 were found in vivo, 6-MeO-BDE-47 may exert adverse effects indirectly via

419

transformation into 6-OH-BDE-47, which then can bind directly to TH targeted genes by

420

mimicking THs.

421

Apart from the TRα pathway, recent studies have also focused on disruption of the AhR

422

and ER pathways by PBDEs. Cross talk between ER- and AhR-signaling pathways in fish

423

has been hypothesized previously.72, 73 The pathway analyses conducted in this study also

424

suggest interactions between these pathways as visualized in the constructed networks. All

425

three compounds altered AhR and ER pathways in zebrafish. 6-OH-BDE-47 significantly

426

increased expression of er2b in zebrafish, which is consistent with previous in vitro findings 21


Page 22 of 44

Page 23 of 44


427

that both ERα and ERβ gene and protein expression were induced by 6-OH-BDE-47.42

428

Exposure to 2.5 µM 6-MeO-BDE-47 significantly reduced expression of er2a but induced

429

er2b indicating the compound might cause endocrine disrupting effects through interfering

430

with the ER signaling pathway.21 In addition, 6-OH-BDE-47 increased the expression of

431

several AhR receptors including ahr1a, ahr1b, and ahr2 in vivo, while 6-MeO-BDE-47 and

432

BDE-47 only affected ahr2 and ahr1a transcription, respectively. These results have

433

confirmed that OH-BDEs can induce greater dioxin-like activity than corresponding

434

MeO-BDEs and parent PBDEs in vitro.43, 74 AR and PxR pathways have been previously shown to be affected by PBDEs in vitro.21,

435 436

75-77

437

exhibited potent antiandrogenicity, with potencies ranking as follows: 6-OH-BDE-47 (IC50 =

438

0.34 µM) > BDE47 (IC50 = 3.83 µM) > 6-MeO-BDE-47 (IC50 = 41.8 µM).21, 76 However, in

439

zebrafish, both BDE-47 and 6-OH-BDE-47 did not significantly alter AR expression, which

440

is consistent with a study in porcine ovarian follicular cells, showing BDE-47 and its OH-

441

metabolites had no effect on the expression of AR mRNA and protein expression.42 The PxR,

442

a steroid and xenobiotic nuclear receptor (SXR), can be activated by BDE-47 in mice.75

443

Nevertheless, in zebrafish, BDE-47 significantly down-regulated PxR associated genes of

444

cyp3a65, hnf4a and ugtlal, but increased pou1f1. The incongruities between these results

445

could be due to differences between species and lesser concentrations used in our

446

experiments. Furthermore, PxR associated genes cyp24a1 and hnf4a were significantly

447

up-regulated by 6-OH-BDE-47 exposure, indicating 6-OH-BDE-47 might be an agonist of

448

zebrafish PxR.

In the MDA-kb2 human cell line AR receptor binding assay, all three compounds

22



449

Reports on the effect of PBDEs on PPARα, MR, and GR are limited. PPARα plays an

450

important role in lipid homeostasis, inflammation, adipogenesis, reproduction, and

451

carcinogenesis.78 In this study, none of three compounds significantly affected the expression

452

of PPARα in zebrafish. However, treatment with a PBDE mixture, BDE-71 and BDE-47

453

caused increases in PPARγ transcript levels at day 8th in 3T3-L1 mouse embryo fibroblast

454

cells.79 GR and MR are essential for regulation of multiple physiological functions, such as

455

glucose metabolism, mineral balance, and behavior.80 In this study, exposure to 2.5 µM

456

6-MeO-BDE-47 or 0.5 µM BDE-47 caused down-regulation of GR, while 0.008 µM

457

6-OH-BDE-47 increased MR expression, which suggests that GR or MR signaling pathways

458

might be involved in the endocrine disrupting effects induced by PBDEs.

459

Altogether, the results of present study, which compared the toxicities of BDE-47

460

with its OH- and MeO- analogs in zebrafish via multiple quantitative approaches, ranging

461

from in vivo toxicity tests, bioaccumulation and biotransformation, to the molecular analysis

462

of response patterns of genes along NR pathways, highlight the importance of the usage of

463

internal dose to evaluate the toxic effects for PBDEs, and the use of early life-stages of

464

zebrafish as an efficient and reliable vertebrate model to assess toxicological effects of

465

endocrine disruptors. Our data also elucidated several molecular aspects of BDE-47,

466

6-OH-BDE-47, and 6-MeO-BDE-47 induced toxicities. Specifically, the data provided

467

valuable insights into the early interaction of these compounds with steroid hormone receptor

468

pathways, which provided novel clues for their in vivo mechanisms of subsequent endocrine

469

disruption and developmental toxicities.

470 23


Page 24 of 44

Page 25 of 44

471 472


Acknowledgements We thank Dr. Richard A. Erickson (Upper Midwest Environmental Sciences Center, U.S.

473

Geological Survey) for providing the expertise in statistical analyses. This work was funded

474

by National Natural Science Foundation (No. 21377053 and 20977047) and Major National

475

Science and Technology Projects (No. 2012ZX07506-001 and 2012ZX07501-003-02) of

476

China. J. P. G. and M. H. were supported by the Canada Research Chair Program. J. P. G. was

477

supported by the Program of 2012 "Great Level Foreign Experts" (#GDW20123200120)

478

funded by the State Administration of Foreign Experts Affairs, China to Nanjing University,

479

and the Einstein Professor Program of the Chinese Academy of Sciences. He was also

480

supported by a Visiting Distinguished Professorship in the Department of Biology and

481

Chemistry and State Key Laboratory in Marine Pollution at City University of Hong Kong.

482 483 484

Supporting Information Further details on the analytical methods and additional tables and figures as noted in the

485

text are available in Supporting Information. This information is available free of charge via

486

the Internet at http://pubs.acs.org/.

24



487

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36



Page 38 of 44

Table 1 Estimated internal doses of BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47 exposures in zebrafish larvae at 120 hpf. The calculation of internal dose for each compound was based on the ratio of the exposure concentration at 300 µg/L and the measured concentration in larvae at 120 hpf. Chemical

BDE-47

6-MeO-BDE-47

6-OH-BDE-47

Nominal Concentration (µM)

0.1

0.5

2.5

0.1

0.5

2.5

0.008

0.02

0.05

BDE-47 (µg/g)

9.05

45.27

226.33

—

—

—

—

—

—

6-MeO-BDE-47 (µg/g)

—

—

—

18.17

90.87

454.37

—

—

—

6-OH-BDE-47 (µg/g)

—

—

—

—

—

—

0.024

0.061

0.15

Biotransformed 6-OH-BDE-47 (µg/g)

—

—

—

0.04

0.21

1.07

—

—

—

37


Page 39 of 44


Figure 1 Measured concentrations in exposure medium (µg/L) and in zebrafish embryos-larvae (mg/g, wm) after exposure to 300 µg/L of BDE-47 (0.62 µM), 6-MeO-BDE-47 (0.58 µM), or 6-OH-BDE-47 (0.6 µM) across time-points (hpf). Generalized additive model (GAM) plots between concentrations (after log transformation) in exposure water or in zebrafish embryos-larvae and time (hpf) are given. Shaded areas are the 95% confidence intervals for each GAM. The F-statistics, p-values, and adjusted R2 for the specific GAMs are given in each plot, while D shows the deviance explained.

µ 00 10

0µ 10

Concentration

µ 10

1

g/L

g/L

g/L

F=87.29, p=3.89e-05, R-sq.(adj)=0.935, D=94.6%

6-MeO-BDE-47-Zfish

F=53.18, p=0.0151, R-sq.(adj)=0.973, D=98.9%

6-OH-BDE-47-Water

F=169.8, p=0.00265, R-sq.(adj)=0.992, D=99.7%

6-OH-BDE-47-Zfish

/g mg r o /L µg

0.1

6-MeO-BDE-47-Water

BDE-47-Water

F=10.13, p=0.124, R-sq.(adj)=0.873, D=94.8% F=107.7, p=7.98e-06, R-sq.(adj)=0.982, D=98.9%

BDE-47-Zfish

F=40.68, p=0.00562, R-sq.(adj)=0.953, D=97.3%

/g mg

/g mg 1 0.0

g/g m 01 0.0

0

12

24

48

72

96

Time (hpf) 38


120


Page 40 of 44

Figure 2 A). Bioconcentration factors (BCF) calculated after exposure to 300 µg/L of BDE-47 (0.62 µM), 6-MeO-BDE-47 (0.58 µM), and 6-OH-BDE-47 (0.6 µM) across time-points (hpf). BCF was calculated based on the ratio of measured concentrations in zebrafish embryos-larvae (µg/kg, wm) and measured concentrations in exposure medium (µg/L) at a specific time (hpf). B). Measured concentrations of biotransformed 6-OH-BDE-47 in zebrafish embryos-larvae (µg/g, wm) after exposure to 300 µg 6-MeO-BDE-47/L (0.58 µM) across several time-points (hpf). A linear regression between concentrations in zebrafish embryos-larvae and time (hpf) is given (R2=0.911; p < 0.05).

A.

B. 2500

0.3

R2 = 0.911 y = -0.120 + 0.003

x

0.2 1500 6-MeO-BDE-47 6-OH-BDE-47 BDE-47

1000

Concentration (µg/g)

Bioconcentration Factors (BCFs)

2000

0.1

500

0.0

0

12

24

48

72

96

120

48

72

96

Time (hpf)

Time (hpf)

39


120

Page 41 of 44


Figure 3 Dendrogram displaying similarities of chemicals and doses based on effects on genes in nuclear receptor pathways for BDE-47, 6-MeO-BDE-47, and 6-OH-BDE-47 in zebrafish larvae at 120 hpf. A). The dendrogram of hierarchical cluster analysis was calculated using the average gene expression values (63 genes in total) of the three or four biological replicates per exposure. Samples names are composed by the name of exposure compound followed by the exposure concentration (µM). Different colors in the dendrogram denoted five clustering groups; B). The heatmap of gene expression profiles was generated using the average gene expression values of the three or four biological replicates per exposure. The fold-changes of gene expression are given in the respective cells and genes involved in different receptor pathways are given different colors (see legend). A.

100

60 40

40


6 - OH - 0.02

6 - OH - 0.05 6 - OH - 0.008

6 - MeO - 0 BDE - 47 - 0

6 - OH - 0

6 - MeO - 0.1 BDE - 47 - 0.1 BDE - 47 - 0.5

0

6 - MeO - 0.5 BDE - 47 - 2.5

20 6 - MeO - 2.5

Distance

80


B.

41


Page 42 of 44

Page 43 of 44


Figure 4 Interaction network of selected genes in nuclear steroid receptor pathways of zebrafish. Nodes represent single genes, edges either protein-protein or protein-DNA interactions. Statistically significant changes (p < 0.05) in gene expression following different concentrations of treatment of BDE-47, 6-MeO-BDE-47, and 6-OH-BDE-47 at 120 hpf are given in the respective boxes (see legend).

42



43


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Bioaccumulation, Biotransformation, and Toxicity of BDE-47, 6-OH

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