Article
pubs.acs.org/est
Accumulation and Biotransformation of BDE-47 by Zebrafish Larvae
and Teratogenicity and Expression of Genes along the
Hypothalamus−Pituitary−Thyroid Axis
Xinmei Zheng,† Yuting Zhu,† Chunsheng Liu,† Hongling Liu,*,† John P. Giesy,†,‡,§ Markus Hecker,⊥
Michael H. W. Lam,§ and Hongxia Yu*,†
†
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023,
China
‡
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada
§
Department of Biology and Chemistry and State Key Laboratory for Marine Pollution, City University of Hong Kong, Kowloon,
Hong Kong, SAR, China
⊥
School of Environment and Sustainability and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
S Supporting Information
*
ABSTRACT: Accumulation and effects of BDE-47 and two analogues, 6-OH-BDE47 and 6-MeO-BDE-47, on ontogeny and profiles of transcription of genes along the
hypothalamus−pituitary−thyroid (HPT) axis of zebrafish (Danio rerio) embryos
exposed from 4 h post fertilization (hpf) to 120 hpf were investigated. The 96 h-LC50
of the most toxic compound, based on teratogenicity, was 330 μg of 6-OH-BDE-47/
L. 6-OH-BDE-47 significantly down-regulated expression of mRNA of thyroid
stimulating hormone receptor (TSHR), thyroid hormone receptors (TRs, including
TRα and TRβ), sodium/iodide symporter (NIS), and transthyretin (TTR) while upregulating expression of thyroglobulin (TG) and thyrotropin-releasing hormone
(TRH). Spontaneous movement was affected by 1 mg of 6-OH-BDE-47/L or 5 mg
of 6-MeO-BDE-47/L. BDE-47 did not alter activity of larvae at any concentration
tested. 6-MeO-BDE-47 significantly up-regulated expression of mRNA of TRH,
TRα, TRβ and NIS. Both 6-OH-BDE-47 and 6-MeO-BDE-47 affected the thyroid
hormone pathway. BDE-47 and 6-MeO-BDE-47 were accumulated more than 6-OH-BDE-47. 6-MeO-BDE-47 was transformed
into 6-OH-BDE-47, but BDE-47 was not transformed into it. In summary, the synthetic brominated flame retardant, BDE-47, did
not elicit the adverse effects caused by the other two analogues and appeared to have less toxicological relevance than the two
natural product analogues 6-OH- and 6-MeO-BDE-47.
■
dietary intake of fish was positively correlated with concentrations of PBDEs in human milk. BDE-47 was the
predominant congener observed among fishes, with a maximum
concentration of 1000 pg/g wet weight (dw).5 Two structural
analogues of BDE-47, 6-OH-BDE-47 and 6-MeO-BDE-47,
were also detected in aquatic biota such as blood and tissues of
fish and marine mammals and algae and have been shown to be
accumulated through the food chain into top predators.6−10
6-OH-BDE-47 and 6-MeO-BDE-47 were originally thought
to be biotransformation products or byproducts during
synthesis of PBDEs.11 However, recent studies have confirmed
that 6-OH-BDE-47 and 6-MeO-BDE-47 are likely naturally
occurring compounds, with interconversion between 6-MeO-
INTRODUCTION
As one of the predominant polybrominated diphenyl ethers
(PBDEs), 2,2′,4,4′-tetrabromodiphenylether (BDE-47) is a
contaminant of concern due to its ubiquity and potential to
bioaccumulate, especially in aquatic systems.1,2 Concentrations
of BDE-47 in water and sediments in south China were 21 pg/
L and greater than 100 ng/g dw, respectively.3 BDE-47 was
detected in electrical and electronic equipment waste (e-waste)
recycling sites at concentrations of 0.83 ng/L in the dissolved
phase and 4.3 ng/L in the particulate phase. 4 Mean
concentrations of BDE-47 in the Little River sewage treatment
plant (LRSTP) in Windsor, Ontario, Canada were 102 ± 83
ng/L in the influent to the primary sedimentation tanks, 36 ±
29 ng/L in primary sedimentation tank effluent, 14 ± 4 ng/L in
final effluent, 586 ± 207 ng/g dry weight (dw) in primary
sludge, and 963 ± 415 ng/g dw in waste activated sludge.
Concentrations of BDEs in fish, vegetables, meat, and human
milk in Japan were determined, and it was concluded that
© 2012 American Chemical Society
Received:
Revised:
Accepted:
Published:
12943
August 13, 2012
October 25, 2012
October 30, 2012
October 30, 2012
dx.doi.org/10.1021/es303289n | Environ. Sci. Technol. 2012, 46, 12943−12951
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MA, USA). MeO-PBDEs and OH-PBDEs (12 MeO-PBDEs: 6MeO-BDE-47, 6′-MeO-BDE-17, 2′-MeO-BDE-28, 5-MeOBDE-47, 4′-MeO-BDE-49, 2′-MeO-BDE-68, 6-MeO-BDE-85,
6-MeO-BDE-90, 4-MeO-BDE-90, 3-MeO-BDE-100, 6′-MeOBDE-123, 6-MeO-BDE-137; 12 OH-PBDEs: 6-OH-BDE-47,
2′-OH-BDE-7, 3′-OH-BDE-7, 2′-OH-BDE-17, 2′-OH-BDE-25,
2-OH-BDE-28, 4′-OH-BDE-49, 2′-OH-BDE-66, 2′-OH-BDE68, 6-OH-BDE-85, 6-OH-BDE-90, 6-OH-BDE-137) were
synthesized in the Department of Biology and Chemistry at
City University of Hong Kong, and purities of greater than 98%
have been confrmed.30 Stock solutions of chemicals (6-OHBDE-47, BDE-47, and 6-MeO-BDE-47) were prepared in
dimethyl sulfoxide (DMSO, Generay Biotech, Shanghai, China)
and diluted with embryonic rearing water (60 mg/L instant
ocean salt in aerated distilled water) to the final concentrations
immediately before use. The final concentration of solvent
(DMSO) in the test solution did not exceed 0.1%. RNAlater
RNA Stabilization Reagents, RNeasy Mini Kit, and Omniscript
RT Kit were purchased from Qiagen (Hilden, Germany). SYBR
Real time PCR Master Mix Plus Kits were obtained from
Toyobo (Tokyo, Japan).
Maintenance of Zebrafish and Exposure. Adult zebrafish (4-month old) were obtained from the Institute of
Hydrobiology, Chinese Academy of Sciences (Wuhan,
China), and maintained in a semiautomatic system with treated
tap water (no residual ammonia, chlorine, chloramines, and
disinfected with UV light) under 14/10 h light/dark photoperiod. Fish were fed frozen blood worms or dry food twice a
day. Nylon nets were used at the bottom of each tank to isolate
eggs and adult fish. Spawning was induced in the morning when
the light was turned on. Collected embryos were rinsed with
embryonic rearing water and examined under an inverted
stereomicroscope. The majority of embryos developed
normally at the early cleavage stage with cytoplasm streams
toward animal pole to form the blastodisc as determined by
means of a stereomicroscope at magnification of 50×. 6-well
cell culture plates (Corning Inc. Steuben, New York, USA)
were used in the experiments. Twenty normally shaped
fertilized embryos were randomly assigned to each well
including 10 mL of exposure or vehicle control (DMSO)
solution and a medium control each with three replicates at 4 h
post fertilization (hpf). The experiment was terminated at 120
hpf. In order to directly compare potencies of the three test
chemicals (6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47),
fertilized embryos were exposed to the same nominal
concentrations of each compound (0, 8, 40, 200, 1000, or
5000 μg/L). Wells were covered with foil to avoid evaporation
of test solutions. Development of the embryos was not affected
by the covering of foil on the wells during the exposure since
mortality of embryos was less than 20%. Embryos were
examined under a multipurpose zoom microscope (Nikon AZ
100) at different developmental stages (4, 8, 12, 24, 48, 72, 96,
and 120 hpf). Coagulated embryos before hatching are opaque,
milky white, and appear dark under the microscope.
Toxicological endpoints included whether embryos were clear
or opaque, edema at 48, 72, or 96 hpf, structural malformations
at 72 or 96 hpf, and body lengths measured after hatching until
120 hpf. Malformations of the crooked spine were defined as
scoliosis and curvature of the tail. Mortalities included
coagulated embryos before hatching and dead larvae after
hatching until 120 hpf. Each exposure experiment was
replicated three times. The mRNA expression studies were
conducted under the same conditions described above with
BDE47 and 6-OH-BDE47 observed in Japanese medaka
(Oryzias latipes).12,13
Several studies of effects of BDE-47 on aquatic organisms
have been conducted. Inflated swim bladder and dorsal
curvature of zebrafish (Danio rerio) larva were observed when
zebrafish embryos were exposed to 25 mg BDE-47/L.14 25−50
nM (12−24 μg/L) of 6-OH-BDE-47 caused a range of
developmental defects such as pericardial edema, yolk sac
deformations, lesser pigmentation, lessened heart rate, and
delayed development.15 PBDEs, OH-PBDEs, and MeO-PBDEs
are structural analogues to chlorinated biphenyls (PCBs),
dioxins (PCDD), and furans (PCDF), some of which have
structure similarities to thyroid hormones (THs). However,
PBDEs are larger and contain a flexible ether linkage such that
they are not agonists of the aryl hydrocarbon receptor (AhR).16
Therefore, PBDEs were hypothesized to potentially affect
thyroid hormone homeostasis. Concentrations of BDE-47 were
negatively correlated with circulating concentrations of free T4
(FT4) in blood plasma of white whales (Delphinapterus leucas)
from Svalbard.17 BDE-47 has been reported to alter thyroid
status and thyroid hormone-regulated gene transcription in
pituitary and brain of adult fathead minnows. 18 The
hypothalamus−pituitary−thyroid (HPT) axis, also known as
thyroid homeostasis or thyrotropic feedback control, is part of
the endocrine system that is responsible for regulation of
metabolism and early life-stage development.19,20 In addition to
the gonads, the HPT axis is the target of endocrine-disrupting
chemicals (EDCs).21 Altered function of the HPT axis is
associated with endocrine and developmental effects.22
Regulation of expression of genes was found to be affected in
embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 h
post fertilization (hpf) due to potential disruption of the
cholinergic system and thyroid hormone homeostasis.23
Mostly, in vitro studies have shown that PBDEs can have
potential endocrine disrupting properties, a number of which
can be attributed to the hydroxylated metabolites.24 OHPBDEs can bind competitively to transthyretin (TTR), the
thyroid hormone transport protein, and also can cause
estrogenic effects through interaction with the estrogen
receptor.25,26 However, to our knowledge, there has been no
published information on effects of 6-MeO-BDE-47 on thyroid
function in fishes. Comparison of the developmental toxicity of
these three compounds in fish is also limited.
Zebrafish embryos/larvae previously have been shown to be
a good model to study effects of chemicals on the HPT axis,
and polymerase chain reaction (PCR) methods have been used
to measure expression of mRNA of genes in the thyroid
system.27−29 In the study upon which we report here, an HPTPCR array for zebrafish larvae was used to investigate effects of
6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47 on the HPT axis
and on pathways of thyroid synthesis and to compare these
effects with developmental and behavioral effects. Furthermore,
accumulation and biotransformation of 6-OH-BDE-47, BDE47, and 6-MeO-BDE-47 by zebrafish larvae were assessed by
liquid−liquid extraction coupled with GC/MS.
■
MATERIALS AND METHODS
Chemicals. BDE-47 (98% purity) was purchased from
Chem Service, Inc. (West Chester, PA, USA). All other PBDEs
(11 PBDEs: BDE-17, BDE-28, BDE-71, BDE-66, BDE-100,
BDE-99, BDE-85, BDE-154, BDE-138, BDE-183, BDE-190),
13
C-PCB-178, 13C-2′-OH-BDE-99, and 13C-BDE-139 were
purchased from Cambridge Isotope Laboratories (Andover,
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Figure 1. Photomicrographs demonstrating changes in morphology at several stages of development after zebrafish embryos were exposed to 6-OHBDE-47 and 6-MeO-BDE-47. (A) Normal developed embryo (36 hpf). (B) Delayed development embryo exposed to 1000 μg of 6-OH-BDE-47/L
(36 hpf). (C) Normal hatched larva (72 hpf). (D) Abnormal embryo exposed to 1000 μg of 6-OH-BDE-47/L (72 hpf). (E) Normal hatched larva
(72 hpf). (F) Abnormal larva with pericardial edema after exposure to 5000 μg of 6-MeO-BDE-47/L (72 hpf). (G) Normal hatched larva (96 hpf).
(H) Abnormal larva with edema and malformed spine after exposure to 5000 μg of 6-MeO-BDE-47/L (96 hpf).
exposure concentrations of 0, 8, 40, or 200 μg/L for each of the
three chemicals. Duration of exposure was chosen on the basis
of the fact that most genes expressed along the HPT axis can be
detected before 96 hpf and that thyroid follicles continue to
grow until 120 hpf in zebrafish.31,32 At 120 hpf, larvae were
randomly sampled and stored in RNAlater solution at −20 °C
for subsequent gene assays. A subset of larvae was analyzed for
bioaccumulation of BDEs and presence of metabolites.
Isolation of RNA, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction (RT-PCR).
Effects of 6-OH-BDE-47, BDE-47, and 6-MeO-BDE-47 on
relative transcription of RNA of key genes involved in HPT axis
were determined by RT-PCR as described previously.33 Whole
bodies of larvae were used as the samples because they were
only 3−4 mm long. Isolation of total RNA was performed using
the RNeasy Mini Kit (Hilden, Germany), and quantification
and verification were performed by use of a previously reported
protocol.33 The Omniscript RT Kit was used to synthesize
cDNA following the manufacturers’ instructions. Quantitative
real-time PCR was performed using the SYBR Green PCR kit
under Applied Biosystems Stepone Plus Real-time PCR System
(Applied biosystems inc. Foster city, CA, USA). The online
Primer 3 program (http://frodo.wi.mit.edu) was used to design
the primer sequences for the selected genes (Table S1 of
Supporting Information). Conditions of the RT-PCR reaction
were as follows: initial denaturation step at 95 °C for 2 min,
followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
Melt curves were derived during RT-PCR to validate that all
cDNA samples amplified only a single product. The expression
of mRNA for each target gene was normalized to the mRNA
content of housekeeping gene (rpl8), and the change in the
mRNA expression of the relevant genes was analyzed by the
2−ΔΔCt method.29 Each concentration was measured in
triplicate in a composite sample containing 15 larvae.
Instrumental Analysis. Zebrafish larvae were collected at
120 hpf, rinsed with Milli-Q water, and gently dried. Larvae
from each exposure were composited, weighed, and stored
separately at −80 °C until analysis. Detailed protocols for
extraction, clean up, identification, and quantification and
quality assurance and quality control (QA/QC) are provided in
Wen et al.34 Internal dose and potential products of
transformation of the three chemicals were determined as
follows: Briefly, concentrations of individual OH-PBDEs, MeOPBDEs, and PBDEs were quantified by liquid−liquid extraction
combined with gas chromatography and mass spectrometry
(GC/MS). Approximately 0.1 g of larvae sample was
homogenized and transferred into amber serum bottles. The
sample was then spiked with the surrogate recovery standard,
and 2 mL of Milli-Q water, 50 μL of hydrochloric acid (HCl, 2
M), and 2 mL of 2-propanol were added. The thus prepared
sample was then extracted three times with 10 mL of n-hexane/
methyl tert-butyl ether (MTBE) (1:1, v/v). Extracts were
concentrated by rotary evaporation and dried under nitrogen.
The derivation of PBDEs, MeO-PBDEs, and OH-PBDEs and
their purification were conducted in accordance with the
method of Wen et al.34 Concentrations of 12 PBDEs, 12 OHPBDEs, and 12 MeO-PBDEs were determined by use of a TSQ
Quantum GC/MS (Thermo Scientific, USA) coupled with an
Agilent DB-XLB column (15 m × 0.25 mm × 0.25 μm, J&W
Scientific, USA) in 3 separate runs. Identification of specific
PBDEs, OH-PBDEs, and MeO-PBDEs was performed by
comparing relative retention times versus internal standard and
product ions in SRM mode with the standard chemicals.
QA/QC and Statistical Analysis. Quality assurance and
quality control were performed by regular analysis of
procedural blanks (RSD < 22.6% for 6 replicates). Rates of
recovery for standard compounds ranged between 93−146%,
67−113%, and 103−133% for PBDEs, OH-PBDEs, and MeOPBDEs, respectively. The limit of detection (LOD) for each
compound was defined as three times the SD of the laboratory
blanks. For congeners not detected in the blanks, the LOD was
set to the instrumental limit of quantification (LOQ). Method
LODs ranged between 9.2 × 10−2 and 2.9 × 101 ng/g wet
weight (ww) for individual PBDEs, OH-PBDEs, and MeOPBDEs congeners. Concentrations less than the method LOD
were assumed to be not detectable. PBDEs, OH-PBDEs, and
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hormone (TRH) was significantly greater (1.77-fold) in larvae
exposed to 40 μg of 6-OH-BDE-47/L and up-regulated by
1.60- and 1.72-fold in larvae exposed to 40 or 200 μg of 6MeO-BDE-47/L, respectively, while no significant alterations
were observed in larvae exposed to BDE-47 (Figure 2A).
Transcription of thyrotropin-releasing hormone 1 (TRHR1)
and thyroid stimulating hormone β (TSHβ) genes was not
affected by any of the three chemicals at the selected
concentrations (Figure 2B,C). Expression of mRNA of thyroid
hormone receptors (TRs, including TRα and TRβ) showed
contrary effects in larvae exposed to 6-OH-BDE-47 and 6MeO-BDE-47 (Figure 2D,E). Exposure of 200 μg of 6-OHBDE-47/L larvae resulted in lesser expression of TRα and TRβ
mRNA by 2.24- and 3.14-fold, respectively, while 6-MeO-BDE47 resulted in up-regulation of expression of TRα and TRβ
mRNA by 1.62- and 2.01-fold, respectively. mRNA expression
of TRα and TRβ after exposure to BDE-47 decreased slightly
but the difference was not statistically significant.
Transcriptional profiles of Na+/I− symporter (NIS) and
transthyretin (TTR) genes were both significantly affected by
exposure to 6-OH-BDE-47 and 6-MeO-BDE-47 (Figure 3).
The effects of 6-OH-BDE-47 and 6-MeO-BDE-47 on the
expression of the NIS genes were concentration dependent
(Figure 3A). Expression of the NIS gene was down-regulated
by 1.43- and 3.33-fold in larvae exposed to 40 or 200 μg of 6OH-BDE-47/L, respectively. However, expression of the NIS
gene was 3.05-fold greater in larvae exposed to 200 μg of 6MeO-BDE-47/L. 6-OH-BDE-47 significantly affected expression of the TTR gene (Figure 3B). Exposure to 40 or 200 μg of
6-OH-BDE-47/L resulted in 1.48- and 1.50-fold downregulation of mRNA of TTR, respectively, while 6-MeOBDE-47 did not significantly affect expression of TTR. No
significant effect on expression of mRNA of either the NIS or
the TTR gene was observed after exposure to BDE-47.
Transcription of the thyroid stimulating hormone receptor
(TSHR) gene was significantly down-regulated by 7.88-fold in
larvae exposed to 200 μg of 6-OH-BDE-47/L. However, there
was no significant effect of 6-MeO-BDE-47 or BDE-47 (Figure
4A). Expression of thyroglobulin (TG) mRNA was significantly
affected by only 6-OH-BDE-47 (Figure 4B), expression of
which was up-regulated by 1.52- and 1.71-fold in larvae exposed
to 8 or 40 μg/L, respectively. Transcriptions of Dio1 and Dio2
were not significantly affected by any of the three chemicals
(Figure 4C,D).
Accumulation and Biotransformation. A concentrationdependent bioconcentration of 6-OH-BDE-47, BDE-47, and 6MeO-BDE-47 was observed in larvae after 5 days of exposure
(Figure 5). Larvae exposed to 200 μg of 6-MeO-BDE-47/L,
accumulated 117 ng of 6-OH-BDE-47/g ww (Figure 5). There
were also small percentages of other isomers detected in
zebrafish exposed to each compound. These included 2’-OHBDE-28, BDE-28, BDE-85 and BDE-99 in zebrafish larvae
exposed to BDE-47; 2’-OH-BDE-28, 6-OH-BDE-85, 6-MeOBDE-47 and BDE-47 observed in zebrafish larvae exposed to 6OH-BDE-47; and 2’-OH-BDE-28, 2’-MeO-BDE-28 and BDE47 observed in zebrafish larvae exposed to 6-MeO-BDE-47.
The percentages were all very small and due exclusively to
impurities in test compounds. In larvae exposed to 200 μg of 6MeO-BDE-47/L, 117 ng of 6-OH-BDE-47/g ww were
observed (Figure 5). No 6-OH-BDE-47 or 6-MeO-BDE-47
was observed in zebrafish exposed to BDE-47, and no 6-MeOBDE-47 occurred in individuals exposed to 6-OH-BDE-47. In
MeO-PBDEs were all quantified in samples extracts relative to
13
C-PCB-178. Recoveries of surrogate standards 13C-BDE-139
and 13C-2-OH-BDE-99 averaged 67.3−118.5% and 77.7−
121%, respectively.
Statistical analyses were performed with the SPSS 12.0 for
Windows. The Kolmogorov−Smirnov test was used to verify
the normality of the data, and homogeneity of variances was
analyzed by the Levene’s test. If the data failed the
Kolmogorov−Smirnov test, logarithmic transformation was
performed and then checked again for homogeneity of
variances. Once the data satisfied the assumptions of
homogeneity of variances, one-way analysis of variance
(ANOVA) followed by LSD test was used to evaluate the
differences between the variables. A value of p < 0.05 was
considered as statistically significant. The probit model was
used to calculate the LC50 for the endpoints used to
characterize teratogenic effects at the different developmental
stages of zebrafish embryos. All values were expressed as mean
± standard error (SEM).
■
RESULTS
Developmental Toxicity. (Raw data of morphological
effects are shown in Tables S2−S4, Supporting Information.)
Zebrafish embryos grew well in culture medium with or without
carrier solvent (0.1% DMSO). On the basis of nominal
concentrations, 6-OH-BDE-47 was more toxic to zebrafish
embryos than was 6-MeO-BDE-47 or BDE-47. Development of
embryos exposed to 1000 μg of 6-OH-BDE-47/L was slower,
and embryos had significantly less melanin at 36 hpf compared
with controls (Figure 1). Development of these larvae was
arrested at 72 hpf. By 96 hpf, all embryos exposed to 1000 or
5000 μg/L had died. LC50 values were 1550 and 330 μg 6-OHBDE-47/L after 48 and 96 h, respectively (Table 1).
Table 1. Lethal Concentrations (LC50; μg/L) of 6-OH-BDE47 to Developing Zebrafish Embryos (μg/L)
duration of development (hpf)
LC50 (μg/L)
confidence interval (95%)
24
36
48
54
60
72
96
4620
1690
1550
1500
1190
540
330
2810−13760
820−5720
870−3710
880−3270
890−1720
340−940
160−990
Concentrations less than 200 μg of 6-OH-BDE-47/L did not
cause significantly mortality compared with the controls after
120 hpf. Pericardial edema was observed after 96 hpf in
embryos exposed to 5000 μg of 6-MeO-BDE-47/L. None of
the tested concentrations of 6-MeO-BDE-47 or BDE47 affected
mortality or body lengths in 96 hpf larvae. However, at 120 hpf,
edema and curved spine occurred in embryos exposed to 5000
μg of 6-MeO-BDE-47/L (Figure 1). Lengths of embryos were
significantly less when exposed to 5000 μg of 6-MeO-BDE-47/
L (3568 ± 167 μm) compared with controls (4241 ± 76 μm).
The three target BDE congeners ranked as follows regarding
their toxic potencies (values from greater to lesser potency): 6OH-BDE-47 > 6-MeO-BDE-47 > BDE-47.
Transcriptional Responses. Exposure to 6-OH-BDE-47
or 6-MeO-BDE-47 but not BDE-47 resulted in significant
changes in gene expression profiles of genes along the HPT axis
(Figures 2−4). Expression of mRNA of thyrotropin-releasing
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Figure 2. Expressions of (A) TRH, (B) TRHR1, (C) TSHβ, (D) TRα, and (E) TRβ, determined by real-time PCR in zebrafish larvae after exposure
to 8, 40, or 200 μg/L of individual chemicals. Results are expressed as means ± SEM of three replicates. *P < 0.05 and **P < 0.01 indicate significant
difference between exposure groups and the control.
Figure 3. Expression of (A) NIS and (B) TTR in zebrafish larvae determined by real-time PCR after exposure to 8, 40, or 200 μg/L individual
chemicals. Results are means ± SEM of three replicate samples. *P < 0.05 and **P < 0.01 indicate significant difference between exposure groups
and the control.
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Figure 4. Expression of (A) TSHR, (B) TG, (C) Dieo1, and (D) Dieo2 in zebrafish larvae determined by real-time PCR after exposure to 8, 40, or
200 μg/L of individual chemicals. Results expressed as means ± SEM of three replicate samples. *P < 0.05 and **P < 0.01 indicate significant
difference between exposure groups and the control.
nominal concentrations of 8, 40, and 200 μg 6-OH-BDE-47/L,
respectively. Estimated BCFs were 13, 4, and 22, when larvae
were exposed to 8, 40, or 200 μg BDE-47/L, respectively, and
7, 13, and 70 when exposed to 8, 40, or 200 μg 6-MeO-BDE47/L, respectively.
■
DISCUSSION
On the basis of the small percentage of each compound present
as an impurity in BDE-47, the concentrations of 2′-OH-BDE28, BDE-28, BDE-85, and BDE-99 observed in zebrafish larvae
exposed to BDE-47 probably resulted from impurities in the
original BDE-47, which indicates that zebrafish might not
transform BDE-47 into OH-PBDEs or MeO-PBDEs. This
result is consistent with that of a previous study in which BDE47 was not converted to 6-OH-BDE-47 or 6-MeO-BDE-47.36
In larvae exposed to 6-OH-BDE-47, 2′-OH-BDE-28, 6-OHBDE-85, 6-MeO-BDE-47, and BDE-47 were observed, which
might also come from impurities in the parent compounds. In
zebrafish larvae exposed to 6-MeO-BDE-47, in addition to a
large amount of the precursor, small amounts of 2′-OH-BDE28, 2′-MeO-BDE-28, and BDE-47 were also observed because
of the impurities in the precursor material. In addition, 117 ng
of 6-OH-BDE-47/g ww was determined in embryos exposed to
200 μg/L, so it indicated that 6-OH-BDE-47 could be
metabolized from 6-MeO-BDE-47.
This study, for the first time, demonstrated that both 6-OHBDE-47 and 6-MeO-BDE-47 can affect expression of specific
genes along the HPT axis as well as resulted in teratogenic
effects in zebrafish embryos, while the man-made BDE-47 did
not result in molecular or pathological effects at exposure
concentrations up to 200 or 5000 μg of BDE-47/L,
respectively. Previous studies demonstrated that changes
Figure 5. Bioconcentration and metabolism of 6-OH-BDE-47, BDE47, and 6-MeO-BDE-47 measured in zebrafish larvae after exposure to
nominal concentrations of the three chemicals (0, 8, 40, or 200 μg/L)
in water with DMSO at 0.1% (v/v) for 5 days.
the control group, neither BDE-47 nor any of the analogues or
products of biotransformation were detected (Figure 5).
Concentrations of BDE-47 and 6-MeO-BDE-47 were
approximately 10- to 100-fold greater than concentrations of
6-OH-BDE-47 in larvae that were exposed to similar
concentrations of the three compounds at a nominal
concentration of 200 μg/L. The log Kow for 6-OH-BDE-47 is
known to be less than that of the other compounds,35 which
might make its excretion more likely to potentially contribute
to its lower concentration in zebrafish larvae. The estimated
bioconcentration factors (BCF) for 6-OH-BDE-47 based on
measured concentrations in larvae divided by the nominal
concentration in water were 0, 3, and 3 with treatment of
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of the thyroid gland. Its primary function is to serve as a
macromolecular substrate for coupling of iodide to its tyrosine
residues during synthesis of TH.46 THs bind to and activate
nuclear TRs, via 5′ deiodination of T4 in several organs,
including brain, pituitary, brown adipose tissue (BAT), skin,
placenta, human heart, muscle, and thyroid.47 Modulation of
transcription of TRH and TSH genes is also influenced by
concentrations of THs via negative feedback.29 In this study,
most genes including TSHR, TRα, TRβ, NIS, and TTR were
down-regulated after exposure to 6-OH-BDE-47. This is in
accordance with the significant impact of greater concentrations
of this congener on larval development and is indicative of the
involvement of the disruptions along the thyroid axis in the
pathologies observed. Further studies are required, however, to
elucidate the specific mechanism by which 6-OH-BDE-47
causes developmental effects through the thyroid axis in fish
and to help establish specific adverse outcome pathways for this
chemical.
As members of the nuclear receptor superfamily, thyroid
hormone receptors (TRs) have been shown to be associated
with postnatal development of birds, metamorphosis of
amphibians, and smoltification of fish.48 TRs including TRα
and TRβ can bind to specific DNA sequences (thyroid
hormone response elements) on promoters to regulate target
genes, which are involved in resistance to thyroid hormone
(TRH).49 The TRα isoform is predominantly expressed in
adipocytes and mediates actions of thyroid hormone in these
cells.50 Thus, up-regulation of TRs could reduce concentrations
of THs. TSH enhances the ability of the thyroid gland to trap
iodide. So when TSH is decreased, expression of NIS is downregulated. TTR is a carrier protein for TH in blood and
regulates the supply of the TH to various target tissues.51
Concentrations of THs in blood were directly proportional to
expression of mRNA of TTR, and lesser expression of TTR was
accompanied by lesser THs in blood plasma.51 The significant
decrease in TTR gene expression observed in this study
indicates that concentrations of THs in zebrafish were likely
decreased by 6-OH-BDE-47. In contrast, the significant
increase in the expression of TRH mRNA could also have
resulted in an increase of THs in larvae exposed to 6-OH-BDE47. Considering the negative impacts exposure to 6-OH-BDE47 had on larval development, however, it is hypothesized that
the increase in TRH mRNA is more likely a compensatory
effect in an attempt to offset the negative impacts along the
HPT axis.
Accumulation and toxic potencies varied among the three
compounds tested. While 6-OH-BDE-47 was the least
accumulated into larvae, it exhibited the greatest toxicity. 6MeO-BDE-47 was accumulated more than the other two
chemicals, and significant amounts of 6-MeO-BDE-47 were
transformed to 6-OH-BDE-47, even though the biotransformation capability of larvae was limited (approximately 0.1% after
120 h). This study revealed significant conversion of 6-MeOBDE-47 but not BDE-47 to 6-OH-BDE-47, which is in
accordance with other studies on different fish species that
hypothesized that natural MeO-BDEs and not the man-made
BDEs are the main source of the more toxic OH-congeners.13
Considering the greater concentration of 6-MeO-BDE-47 in
marine environments and its greater potential for bioaccumulation and biotransformation of 6-MeO-BDE-47, it is the likely
source of 6-OH-BDE-47 observed in fishes.
In conclusion, this study demonstrated that 6-OH-BDE-47,
and to a lesser extent 6-MeO-BDE-47, had significant effects at
along the HPT axis were often related to specific pathological
phenomena. For instance, when zebrafish embryos were
exposed to 500 μg/L microcystin-LR, changes in gene
expression along the HPT axis could be correlated with a
significant decrease of body length.37 All BDE-47, TBBPA and
BPA were shown to induce alteration of genes along the HPT
axis of zebrafish larvae as well as cause acute toxicity.38 In this
study, 6-OH-BDE-47 was acutely toxic with a 96 h-LC50 of 330
μg/L while reduced body length and teratogenic effects were
observed at 5000 μg/L at 120 hpf. These results further suggest
a relationship between early molecular-level effects and
subsequent morphological changes. The observed impact on
development of zebrafish is in accordance with the critical role
of the HPT axis in early life-stage development of
vertebrates.19,20
In female C57BL/6 mice, BDE-47 had previously been
reported to decrease total serum T4 concentrations by 43% at
100 mg/kg/day treatment, activate the nuclear receptor, CAR,
and decrease Mdr1a mRNA expression at 3 mg/kg/day.39 An
earlier study indicated that 96 h-EC50 values of BDE-47 for
zebrafish embryos based on hatching success were 20.30 mg/L
between 2.03 and 15.23 mg/L, significantly affecting expression
of some genes along the HPT.38 In this study, BDE-47 did not
show developmental or molecular toxicity at exposure
concentrations up to 5000 or 200 μg/L, respectively. Perhaps,
because the concentrations used in those studies were greater
than concentrations tested in our experiments. It could also
have been due to differences in exposures and different
organisms. The environmental relevance of such great
exposures, however, would be negligible. In contrast, 6-OHBDE-47, and to a lesser extent 6-MeO-BDE-47, had significant
effects at the molecular level on the HPT axis in larval zebrafish
that were indicative of developmental effects in this species.
The fact that most genes along the HPT axis, such as TSHR,
TRα, TRβ, NIS, and TTR, were down-regulated and only TRH
and TG were up-regulated by 6-OH-BDE-47, while exposed to
6-MeO-BDE-47, resulted in up-regulation of the expression of
TRH, TRα, TRβ, and NIS, indicating that these two chemicals
affect the HPT axis of zebrafish through different toxic
pathways (Figure S1 of Supporting Information). These results
are consistent with the findings of a previous study that
reported that cytotoxicity of 6-OH-BDE-47 to E. coli was via a
mechanism that was different from that of either BDE-47 or 6MeO-BDE-47.40 In mammals, the hypothalamic tripeptide
TRH stimulates thyroid stimulating hormone (TSH) synthesis
and release from the anterior pituitary to bind to TSHR in the
thyrocytes.41 TRH increases locomotor activity and intake of
food, both of which are associated with increases in the
hypothalamic expressions of OX and CART in goldfish.42 TSH
is a member of a glycoprotein hormone family which is
composed of common α- and specific β-subunits. TSHR is a
key protein in the control of thyroid function, by stimulating
TH synthesis after binding its ligand, the thyrotropin. This
pathway is involved with both growth and functional
characteristics, by acting via the cAMP pathway.43 TSHR
plays a critical role in certain thyroid diseases, including Graves’
disease (GD), multinodular thyroid goiter (MTG), and
Hashimoto’s thyroiditis (HT).44 When TSH was transferred
to thyroid, T4 was produced with the assistant of NIS and TG.
As an integral plasma membrane glycoprotein, NIS is localized
in the baso-lateral membrane of thyrocytes. It is necessary for
the transport of iodide for the biosynthesis of TH.45 TG is a
large glycoprotein produced and stored in the follicular lumen
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pollutants resulted from e-wastes recycling and bioaccumulation of
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Japanese medaka. Environ. Sci. Technol. 2010, 44 (22), 8729−8735.
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1694−1699.
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the molecular level on the HPT axis in larval zebrafish that were
indicative of developmental effects in this species. In contrast,
exposure to the man-made BDE-47 did not result in any effects
either at the molecular or the pathological level. Furthermore,
this study revealed significant conversion of 6-MeO-BDE-47
but not BDE-47 to 6-OH-BDE-47, which is in accordance with
other studies on different fish species that hypothesized that
natural MeO-BDEs and not the man-made BDEs are the main
source of the more toxic OH-congeners. Although 6-OH-BDE47 was less bioaccumulative than BDE-47 and 6-MeO-BDE-47,
the significant transformation of the highly bioaccumulative
MeO-BDE is likely to represent a continuous internal source
for the toxic OH-BDE.
■
ASSOCIATED CONTENT
S Supporting Information
*
Figure S1, pathway of hypothalamic-pituitary-thyroid (HPT)
axis in zebrafish; Table S1, primer sequences for the
quantitative reverse transcription-polymerase chain reaction
(q-PCR); Table S2, raw data of morphological effect of 6-OHBDE-47; Table S3, raw data of morphological effect of 6-MeOBDE-47; Table S4, raw data of morphological effect of BDE-47.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: 86-25-89680356. Fax: 86-25-89680356. E-mail: hlliu@
nju.edu.cn (H.L.); yuhx@nju.edu.cn (H.Y.).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was jointly funded by the National Natural Science
Foundation of China (Nos. 20977047, 20737001), Major
National Science and Technology Projects (Nos.
2012ZX07506-001, 2012ZX07529-003-02), and the Environmental Monitoring Research Foundation of Jiangsu Province
(No. 1114). The research was supported by a Discovery Grant
from the Natural Science and Engineering Research Council of
Canada (Project # 326415-07). J.P.G. and M.H. were
supported by the Canada Research Chair program. Furthermore, J.P.G. was supported by an at large Chair Professorship
at the Department of Biology and Chemistry and State Key
Laboratory in Marine Pollution, City University of Hong Kong,
and the Einstein Professor Program of the Chinese Academy.
■
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Accumulation and biotransformation of BDE-47 by zebrafish larvae and
teratogenicity and expression of genes along the hypothalamus-pituitary-thyroid
axis
Xinmei Zheng1, Yuting Zhu1, Chunsheng Liu1, Hongling Liu1*, John P. Giesy1,2,3,
Markus Hecker4, Michael H. W. Lam3, Hongxia Yu1*
1 State Key Laboratory of Pollution Control and Resource Reuse, School of the
Environment, Nanjing University, Nanjing 210046, China
2 Department of Veterinary Biomedical Sciences and Toxicology Centre, University
of Saskatchewan, Saskatoon, Saskatchewan, Canada
3 Department of Biology & Chemistry and State Key Laboratory for Marine
Pollution,, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
4 School of Environment and Sustainability and Toxicology Centre, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada
Authors for correspondence:
School of the Environment
Nanjing University
Nanjing 210046, China
Tel: 86-25-89680356
Fax: 86-25-89680356
E-mail: hlliu@nju.edu.cn (Hongling Liu)
yuhx@nju.edu.cn (Hongxia Yu)
Figure
S1.
Schematic
of
endocrine
hypothalamic-pituitary-thyroid (HPT) axis in zebrafish.
pathways
along
the
Table S1. Primer sequences for quantitative reverse transcription-polymerase chain
reaction (q-PCR).
Gene
Sense primer (5’-3’)
name
Antisense
primer Gene
(5’-3’)
accession no.
bank
rpl8
ttgttggtgttgttgctggt
ggatgctcaacaggggttcat
NM_200713
TSHβ
gcagatcctcacttcacctacc
gcacaggtttggagcatctca
AY135147
TSHR
gctccttgatgtgtccgaat
cgggcagtcaggttacaaat
NM_001145763
TG
ccagccgaaaggatagagttg
atgctgccgtggaatagga
XM_001335283
Dio1
gttcaaacagcttgtcaaggact
agcaagcctctcctccaagtt
BC076008
Dio2
gcataggcagtcgctcattt
tgtggtctctcatccaacca
NM_212789
TTR
cgggtggagtttgacacttt
gctcagaaggagagccagta
BC081488
TRα
ctatgaacagcacatccgacaagag cacaccacacacggctcatc
NM_131396
TRβ
tgggagatgatacgggttgt
ataggtgccgatccaatgtc
NM_131340
NIS
ggtggcatgaaggctgtaat
gcctgattggctccatacat
NM_001089391
TRH
cacacagatggaggagcaga
agcagcatcaggtagcgttt
NM_001012365
TRHR1
ctggtggtggtcaactcctt
gctttccaccgttgatgttt
NM_001114688
Table S2. Raw data for morphological effects of 6-OH-BDE-47.
Repeat
1
Repeat
2
Repeat
3
Concentrati
on (µg/L)
Total
embryos
24 hpf 36 hpf 48 hpf 72 hpf 72 hpf
96/120 hpf
coagu coagulat coagulat coagula hatching
coagulation
lation ion
ion
tion
rate
0
20
4
4
4
4
0.88
4
8
20
4
4
4
4
1
4
40
20
5
5
5
5
1
5
200
20
3
3
3
3
1
3
1000
20
4
4
6
15
0
20
5000
20
8
20
20
20
0
20
Concentrati
on (µg/L)
Total
embryos
24 hpf 36 hpf 48 hpf 72 hpf 72 hpf
96/120 hpf
coagu coagulat coagulat coagula hatching
coagulation
lation ion
ion
tion
rate
0
20
4
4
4
4
1
4
8
20
6
6
6
6
1
6
40
20
2
2
2
2
0.89
2
200
20
2
2
2
2
1
2
1000
20
2
2
3
14
0
20
5000
20
11
20
20
20
0
20
Concentrati
on (µg/L)
Total
embryos
24 hpf 36 hpf 48 hpf 72 hpf 72 hpf
96/120 hpf
coagu coagulat coagulat coagula hatching
coagulation
lation ion
ion
tion
rate
0
20
4
4
4
4
1
4
8
20
7
7
7
7
0.92
7
40
20
4
4
4
4
0.94
4
200
20
5
5
5
5
1
5
1000
20
3
3
5
19
0
20
5000
20
14
20
20
20
0
20
Table S3/ Raw data of morphological effect of 6-MeO-BDE-47.
Repeat
1
Repeat
2
Repeat
3
Concentration Total
(µg/L)
embryos
72 hpf
120 hpf
24
hpf
96
hpf
hatching
spinal
coagulation
coagulation
rate
curvature
0
20
3
1
3
0
8
20
4
0.88
4
0
40
20
4
0.88
4
0
200
20
1
1
1
0
1000
20
7
1
7
0
5000
20
7
0.92
7
12
Concentration Total
(µg/L)
embryos
72 hpf
120 hpf
24
hpf
96
hpf
hatching
spinal
coagulation
coagulation
rate
curvature
0
20
3
1
3
0
8
20
4
1
4
0
40
20
3
1
3
0
200
20
1
1
1
0
1000
20
4
1
4
0
5000
20
1
1
1
19
Concentration Total
(µg/L)
embryos
72 hpf
120 hpf
24
hpf
96
hpf
hatching
spinal
coagulation
coagulation
rate
curvature
0
20
2
0.94
2
0
8
20
3
0.94
3
0
40
20
2
1
2
0
200
20
4
1
4
0
1000
20
4
1
4
0
5000
20
1
0.95
1
2
Table S4. Raw data of morphological effect of BDE-47.
Repeat 1
Repeat 2
Repeat 3
Concentration
(µg/L)
Total embryos
72
hpf
24
hpf
120
hpf
hatching
coagulation
coagulation
rate
0
20
4
1
4
8
20
4
1
4
40
20
4
1
4
200
20
3
1
3
1000
20
4
1
4
5000
20
3
1
3
Concentration
(µg/L)
Total embryos
72
hpf
120
hpf
24
hpf
hatching
coagulation
coagulation
rate
0
20
4
1
4
8
20
5
1
5
40
20
2
1
2
200
20
2
1
2
1000
20
4
1
4
5000
20
3
1
3
Concentration
(µg/L)
Total embryos
72
hpf
24
hpf
120
hpf
hatching
coagulation
coagulation
rate
0
20
4
1
4
8
20
4
1
4
40
20
3
1
3
200
20
2
1
2
1000
20
5
1
5
5000
20
4
1
4