ARTICLE pubs.acs.org/est
Parental Transfer of Polybrominated Diphenyl Ethers (PBDEs) and Thyroid Endocrine Disruption in Zebrafish Liqin Yu,†,|| James C. W. Lam,§,|| Yongyong Guo,† Rudolf S. S. Wu,‡ Paul K. S. Lam,*,§ and Bingsheng Zhou*,† †
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China ‡ School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China § State Key Laboratory in Marine Pollution; Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, China
bS Supporting Information ABSTRACT: Polybrominated diphenyl ethers (PBDEs) have the potential to disrupt the thyroid endocrine system. The objective of the present study was to characterize the disrupting effects of longterm exposure on the thyroid endocrine system in adult fish and their progeny following parental exposure to PBDEs. Zebrafish (Danio rerio) embryos were exposed to environmentally relevant concentrations (1, 3, and 10 μg/L) of the PBDE mixture DE-71 for 5 months until sexual maturation. In the F0 generation, exposure to DE-71 significantly increased plasma thyroxine (T4) but not 3,5,30 -triiodothyronine (T3) in females. This increased T4 was accompanied by decreased mRNA levels of corticotropinreleasing hormone (CRH) and thyrotropin β-subunit (TSHβ) in the brain. The F1 generation was further examined with or without continued DE-71 treatment conditions. Exposure to DE-71 in the F0 fish caused significant increases in T4 and T3 levels in the F1 larvae and modified gene expressions in the hypothalamic pituitarythyroid axis (HPT axis) under both conditions. Decreased hatching and inhibition of growth in the F1 offspring were observed in the condition without DE-71 treatment. Continued DE-71 treatment in the F1 embryos/larvae resulted in further decreased hatching, and increased malformation rates compared with those without DE-71 exposure. Analysis of F1 eggs indicated that parental exposure to DE-71 could result in a transfer of PBDEs and thyroid hormones (THs) to their offspring. For the first time, we demonstrated that parental exposure to low concentrations of PBDEs could affect THs in the offspring and the transgenerational PBDE-induced toxicity in subsequent nonexposed generations.
’ INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are additives in flame retardants. They are hydrophobic and lipophilic, and some congeners of PBDEs share many characteristics with classical persistent organic pollutants (POPs). Tetra-, penta-, hexa-, and hepta-BDE have recently been added to the POPs list under the Stockholm Convention1 due to great concerns that have arisen concerning the potential environmental and human health risks associated with PBDE exposure. Because of their structural similarity to thyroid hormones (THs), PBDEs are thyroid endocrine disruptors. Decreased concentrations of circulating total thyroxine (T4) have been observed in rats and mice following short- to long-term exposure to various PBDEs.27 In fish, studies have also shown that PBDEs perturb TH homeostasis. Plasma levels of T4 are lower in PBDEexposed juvenile lake trout (Salvelinus namaycush) compared with control fish after dietary PBDE exposure.8 Reduced plasma T4 levels have been observed in fathead minnows (Pimephales promelas) r 2011 American Chemical Society
administered BDE-47.9 Exposure of the PBDE mixture DE-71 to zebrafish embryos/larvae has also been shown to result in a reduction of T4 in the larvae.10 Like other lipophilic organic compounds, maternal transfer of several PBDEs has previously been observed in various species, such as frogs,11 birds,12,13 rats,14 and humans.15 Recently, maternal transfer of PBDEs to the offspring following parental exposure via feed was observed in zebrafish in laboratory experiments16 and in the field.17 This vertical transfer provides an opportunity for PBDEs to interfere with developmental effects on the progeny. Many fish species are known to be most sensitive to organic contaminants in their early life stages. In fish, PBDEs may be transferred maternally in the lipid stores of oocytes, and the Received: August 28, 2010 Accepted: October 31, 2011 Revised: September 28, 2011 Published: October 31, 2011 10652
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Table 1. Effects of DE-71 on Zebrafish Developmental Parametersa hatching rates (%) fish F0
F1(DE-71)
F1(+DE-71)
DE-71 (μg/L)
malformation rates (%)
3d
5d
survival (%) 5d
10d
body weight (mg) 5d
10d
0
78.1 ( 2.1
1.67 ( 0.7
80.3 ( 1.8
78.3 ( 1.3
0.34 ( 0.01
0.38 ( 0.02
1
79.8 ( 2.7
2.33 ( 0.3
81.6 ( 1.6
70.5 ( 2.5
0.35 ( 0.02
0.38 ( 0.02
3
72.6 ( 1.5
2.00 ( 0.6
76.2 ( 1.7
73.7 ( 1.2
0.31 ( 0.01
0.35 ( 0.01
10
71.2 ( 2.7
2.67 ( 0.3
75.7 ( 0.6
69.3 ( 5.8
0.31 ( 0.01
0.33 ( 0.03
0
82.4 ( 2.7
1.17 ( 0.2
80.0 ( 2.0
74.8 ( 3.6
0.33 ( 0.01
0.38 ( 0.02
1
86.0 ( 0.8
1.25 ( 0.3
81.4 ( 1.4
71.1 ( 1.1
0.31 ( 0.03
0.36 ( 0.01
3
74.8 ( 1.5*
1.64 ( 0.5
76.0 ( 2.7
72.0 ( 4.4
0.26 ( 0.02*
0.28 ( 0.00*
10 0
73.2 ( 1.5* 82.4 ( 2.7
1.31 ( 0.3 1.17 ( 0.2
79.8 ( 5.0 80.0 ( 2.0
76.1 ( 4.3 74.8 ( 3.6
0.28 ( 0.01* 0.33 ( 0.01
0.31 ( 0.01* 0.38 ( 0.02
1
69.8 ( 2.5*a
1.36 ( 0.3
76.8 ( 4.2
74.9 ( 4.2
0.26 ( 0.01*
0.28 ( 0.01*
3
62.2 ( 2.6*b
2.89 ( 0.8*b
74.8 ( 2.8
68.9 ( 2.8
0.28 ( 0.01*
0.29 ( 0.03*
10
67.3 ( 4.9*c
2.59 ( 0.3*c
76.5 ( 2.7
64.5 ( 4.2
0.28 ( 0.02*
0.31 ( 0.03*
a
Hatching, malformation, survival and growth in the F0 embryos/larvae exposed to DE-71 and F1 embryos/larvae with (+DE-71) or without (DE-71) continued DE-71 treatments were evaluated. Asterisk indicates significantly different between exposure groups and their corresponding control (P < 0.05) (One way ANOVA, followed by Tukey’s test). In the F1 generation, the differences between groups treated with or without continued DE-71 treatment were also compared (Student’s t-test). a,b,cRepresents significant differences between continued (+DE-71) treatment at 1, 3, and 10 μg/L and without (DE-71) treatment, respectively. Results are given as mean values of three replicates of 50 embryos or larvae for each exposure condition at 5 and 10 dpf. All data are expressed as means ( SEM.
offspring can be exposed to these compounds during the earliest stages of embryogenesis. Due to limited reports regarding the thyroid endocrine effects of PBDEs in fish, and because little information is known about transgenerational toxicity in particular, the purpose of our study was to evaluate the endocrine disrupting activities of PBDE exposure in the thyroid system and the impact on offspring. We selected the DE-71 commercial mixture since it contains tetraand penta-brominated congeners that are prevalent in environmental and biological samples (BDE-47, BDE-99, BDE-100, BDE-153, and BDE-154).18,19 This study focused on parental exposure to DE-71 and the transfer of these compounds to the offspring, and investigated the effects on the thyroid endocrine system in both generations. Specifically, thyroid hormone levels, gene expression patterns in the hypothalamicpituitarythyroid axis, and developmental toxicity were examined.
’ MATERIALS AND METHODS Chemicals. The commercial PBDE mixture DE-71 (purity >99.9%) was obtained from Wellington Laboratory, Inc. (Ontario, Canada). The TRIzol reagent and SYBR Green PCR kit were purchased from Invitrogen (New Jersey) and Toyobo (Osaka, Japan), respectively. All other chemicals used in the present study were of analytical grade. Zebrafish Maintenance and Experimental Design. Adult zebrafish (AB strain) were maintained and the embryos were exposed to DE-71 as described previously.10 Briefly, the embryos that had developed normally and reached the blastula stage (2 h postfertilization, hpf) were selected for the experiments. The embryos were randomly distributed into glass beakers containing 500 mL of DE-71 exposure solution (0, 1, 3, and 10 μg/L). There were three replicates for each exposure concentration, and each beaker contained 100 embryos. The selected exposure concentrations were based on our previous study.10 At 10 days post fertilization (dpf), the larvae were transferred into 20-L tanks. At 40 dpf, the fry were transferred into 30-L tanks. After 150 days of
exposure, the survival and growth (length and weight) were determined, from which a condition factor was obtained. The fish were paired (18 males and 18 females), and eggs were immediately collected for DE-71 and TH assays. The embryos were divided into two groups: one group received continued treatment with the same DE-71 concentrations as did their parents, and the other group received no further DE-71 treatment. In the no treatment group, the embryos were washed with freshwater five times and placed in glass dishes in freshwater without DE-71 to evaluate the parental transfer of PBDEs and transgenerational toxicity. During all the experimental period, 50% of the exposure solution was renewed daily, and the appropriate concentration of PBDEs was maintained. The control and treated groups received 0.003% (v/v) dimethyl sulfoxide (DMSO). The hatching, malformation, survival, and growth were determined in both generations. The F1 larvae were randomly sampled at 5 and 10 dpf, immediately frozen in liquid nitrogen, and stored at 80 °C for subsequent gene expression analysis and TH assays. TH Assays. After 150 days of exposure, the adult fish were anesthetized in 0.03% MS-222, and blood was collected from the caudal vein of each fish. The blood samples from four fish of the same sex were pooled as one replicate (about 40 μL). The plasma was stored at 80 °C until analysis. The methods for extraction of whole body THs content in eggs and larvae were from a previous method in fathead minnow with a small modification (Text S1, Supporting Information). For THs levels in plasma of adult zebrafish and in the F1 eggs and larvae, the total T4 and T3 levels were measured using the commercial enzyme-linked immunosorbent assay (ELISA) test kits purchased from Wuhan EIAab Science Co. Ltd. (Wuhan, China) following the manufacturer’s instructions. Quantitative Real-Time PCR Assay. The liver and brain (including hypothalamus and pituitary) were collected and preserved in TRIzol reagent at 80 °C. Extraction, purification, and quantification of total RNA and first-strand cDNA synthesis were performed as described previously10 (Text S2, Supporting Information). The primer sequences of the selected genes were 10653
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Table 2. Total T4 and T3 Levels in F0 Adult Zebrafish after Exposure to DE-71 and in the F1 Eggs and Larvaea DE-71 (μg/L) fish F0
TH levels T4 T3
eggs F1(-DE-71)
F1(+DE-71)
sex
0
1
3
10
female
20.50 ( 1.08
22.18 ( 1.40
21.06 ( 0.17
27.52 ( 1.52*
male
22.59 ( 1.85
22.50 ( 1.81
20.80 ( 1.60
26.11 ( 4.61
female
2.58 ( 0.30
3.36 ( 0.61
3.63 ( 0.46
4.03 ( 1.30
male
2.67 ( 0.23
3.13 ( 0.59
3.32 ( 0.44
3.41 ( 0.44 11.00 ( 1.27*
T4
7.52 ( 0.18
6.54 ( 1.17
7.80 ( 0.38
T3
0.75 ( 0.02
0.80 ( 0.11
1.11 ( 0.10*
1.28 ( 0.08**
T4
5d
15.30 ( 0.37
14.58 ( 1.05
20.22 ( 2.36
23.97 ( 3.49*
T3
10d 5d
16.53 ( 0.46 0.94 ( 0.11
17.55 ( 3.76 1.12 ( 0.09
21.06 ( 1.88 1.54 ( 0.04*
23.13 ( 1.39*a 1.61 ( 0.16*
10d
1.80 ( 0.24
1.98 ( 0.13
2.26 ( 0.08
2.17 ( 0.16
T4
5d
15.30 ( 0.37
17.68 ( 3.54
19.38 ( 2.65
26.39 ( 2.05**
10d
16.53 ( 0.46
18.48 ( 2.74
19.39 ( 1.13
29.23 ( 1.84**b
T3
5d
0.94 ( 0.11
1.42 ( 0.36
1.21 ( 0.24
1.75 ( 0.14*
10d
1.80 ( 0.24
2.09 ( 0.25
2.09 ( 0.28
2.15 ( 0.26
a For the adult zebrafish, plasma samples from four individual fish were pooled and tested with three replicates. For the F1 eggs and larvae, 200 eggs or 300 larvae were measured also with three replicates. The eggs were collected immediately after spawning, while the TH contents were measured at 5 and 10 dpf with or without continued DE-71 treatment. The TH levels in the F0 zebrafish are expressed as ng/mL and ng/g wet weight in the F1 eggs and larvae, respectively. All data are expressed as means ( SEM.*P < 0.05 and **P < 0.01 indicate significant differences between exposure groups and the corresponding control group. a,bRepresents significant differences between continued (+DE-71) treatment at 10 μg/L and without (DE-71) treatment (Student’s t-test, P < 0.05).
obtained by using the online Primer 3 program (http://frodo.wi. mit.edu/) (Table S1, Supporting Information). Quantification of PBDEs in F0 Zebrafish and F1 Eggs. Concentrations of PBDEs were determined in the adult fish (F0) and the eggs (F1). The detailed protocols for extraction, clean up, analysis, and quality assurance and quality control (QA/QC) are provided in the Supporting Information (Text S3). Statistical Analysis. All data are expressed as means ( standard error (SEM). The normality of the data was verified using the KolmogorovSmirnov test. The homogeneity of variances was analyzed by Levene’s test. The differences between the control and each exposure group were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test by using SPSS 13.0 software (SPSS, Chicago, IL). A P value 0.05) (Table 2). The plasma total T3 level also showed a trend toward an increase in exposure groups in both the females and males, but it was again not statistically significant relative to the control (P > 0.05) (Table 2). Whole body levels of THs were measured in the F1 eggs and F1 larvae at 5 and 10 dpf with or without continued DE-71 treatment. In the F1 eggs, the T4 levels were significantly increased in the 10 μg/L group (P < 0.05), and T3 levels were increased in the 3 and 10 μg/L groups with parental exposure of 3 and 10 μg/L groups (P < 0.05) (Table 2). At 5 dpf, in the F1 larvae without DE-71 treatment, the total T4 levels were significantly increased by 32.2% and 56.5% with parental groups exposed to 3 and 10 μg/L, respectively, compared with those in the control (P < 0.05) (Table 2). For the respective F1 larvae groups given continued treatment with 1, 3, and 10 μg/L of DE-71, concentration-dependent increases of T4 (15.6%, 26.7%, and 72.5%) were detected at 5dpf (P < 0.05) (Table 2); at 10 dpf, increases in T4 were also found in the F1 larvae with (11.8%, 17.3%, 76.8%; P < 0.05) or without (6.2%, 27.4%, 39.9%; P < 0.05) continued DE-71 treatment (Table 2). A significant increase in the total T4 levels was observed in the F1 generation with continued DE-71 treatment (+DE-71, 10 μg/L) compared with those without treatment (-DE-71) at 10 dpf (Student’s t-test, P < 0.05) (Table 2). At 5 dpf, in the group without DE-71 treatment, the T3 levels were significantly increased (63.8%, 71.3%) with parental groups exposed to 3 and 10 μg/L, respectively (P < 0.05). In the group where DE-71 treatment was continued, a significantly increased T3 level (86.2%) was observed with parental exposure to 10 μg/L (P < 0.01) (Table 2). At 10 dpf, a trend of increased T3 levels was 10654
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Environmental Science & Technology detected in the groups with (16.1%, 16.1%, 19.4%) and without (10.0%, 25.6%, 20.6%) continued DE-71 treatment, but the differences were not significantly significant (P > 0.05) (Table 2). There was no significant difference in the total T3 levels between the F1 generation with continued DE-71 treatment (+DE-71) and the F1 generation without treatment (DE-71) at both 5 and 10 dpf (Student’s t-test, P > 0.05) (Table 2). Gene Expression in F0 and F1 Generations. In the F0 fish, several genes involved in regulation, transport, binding, and metabolism of THs were examined. In the brain of the females, both corticotropin-releasing hormone (CRH) and thyroid-stimulating hormone (TSHβ) gene expressions were significantly down-regulated in the 10 μg/L exposure group compared with those in the control (P < 0.01). Likewise, in the males, a small but significant down-regulation of CRH gene expression was observed in the 10 μg/L exposure group (P < 0.05), and a concentration-dependent down-regulation of TSHβ gene expression was observed in the 1, 3, and 10 μg/L exposure groups (P < 0.05) (Table S3, Supporting Information). In the liver, transthyretin (TTR) gene expression was significantly down-regulated in the 10 μg/L exposure group in the females (P < 0.05), while hepatic uridine diphosphoglucuronosyl transferase (UGT1) was increased in the 10 μg/L exposure group (P < 0.01). Expressions of the deiodinases (Dio1 and Dio2) were significantly down-regulated in the 10 μg/L treated group compared with those in the control group (P < 0.05). In the liver of the males, down-regulation of TTR, and up-regulation of UGT1 were observed upon exposure to 10 μg/L (P < 0.01). The gene expressions of Dio1 and Dio2 were also down-regulated in the 10 μg/L exposure group (P < 0.05) (Table S3, Supporting Information). In zebrafish larvae (F1), CRH and TSHβ, marker genes involved in thyroid gland development and growth (e.g., hhex and nkx2.1), THs synthesis (e.g., thyroglobulin, TG) and binding (TTR) were examined at 5 and 10 dpf with or without continued DE-71 treatment. The gene expression levels were similar in the control group of both treatments. At 5 dpf, TSHβ gene expression was down-regulated in the groups without DE-71 exposure (P < 0.01). Hhex, nkx2.1, and TG (P < 0.05) were up-regulated in the groups under both conditions. Significant up-regulation of Dio1 (P < 0.01) and down-regulation of Dio2 (P < 0.05) were observed in the groups without DE-71 exposure. The down regulation of Dio1 was observed in the larvae subjected to continued treatment with DE-71 in the 3 and 10 μg/L (P < 0.05) and a small but significant upregulation of Dio2 was observed in the 10 μg/L group (P < 0.05). However, UGT1 gene expression was downregulated in the group without DE-71 treatment (P < 0.01). The TTR gene expression was not changed in the group without DE-71 exposure, but its up-regulation was found in the group with continued DE-71 treatment (P < 0.01) (Table S4, Supporting Information). At 10 dpf, the CRH gene expressions were significantly downregulated in the groups subjected to continued treatment with DE-71 (P < 0.01). The gene expressions of TSHβ were significantly down-regulated (P < 0.05) under both conditions, while the hhex and nkx2.1 gene expressions were up-regulated (P < 0.05). Decreases and increases in the TG gene expression were observed in the groups with and without continued DE-71 treatment (P < 0.05), respectively. Significant down- and upregulations of Dio1 were measured in the groups with (P < 0.01) and without continued DE-71 exposure (P < 0.01), respectively. Meanwhile, Dio2 gene expression was significantly inhibited
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(P < 0.01) under both treatment conditions. The gene expressions of UGT1 (P < 0.05) and TTR (P < 0.01) were significantly increased in the group without DE-71 exposure, while those in the DE-71 continued treatment group were down-regulated (P < 0.01) (Table S4, Supporting Information). PBDE Content in F0 Adult Fish and F1 Eggs. Seven congeners were detected in exposed F0 zebrafish, where BDE47 contributed to most of the total PBDE body burden, followed by BDE-100 and 99, in both females and males (Figure 1A). The contents of individual congeners and total PBDEs showed clear dose-dependent relationships between body burdens and their water exposure concentrations. The total body burdens of PBDEs were higher in males than in females in all the exposure groups. The detected total contents of PBDEs were 2.03 ( 0.88 ng/ g wet weight in the control females and 7706 ( 578, 19 954 ( 224, 55 029 ( 4444 in the 1, 3, and 10 μg/L exposure groups, respectively. In the males, the detected total PBDE contents were 2.58 ( 0.62 ng/g in the control and 11 816 ( 1314, 40 821 ( 2646, 118 419 ( 9087 ng/g in the 1, 3, and 10 μg/L exposure groups, respectively. The estimated bioaccumulation factors (detected concentrations in fish/nominated concentrations in water) are 7700, 6650, and 5530 with treatment of 1, 3, and 10 μg/L in the females and 11 820, 13 600, and 11 400 in the males, respectively. In the F1 eggs, seven congeners were detected; BDE-47 was the predominant congener, followed by BDE-100 and BDE-99 (Figure 1B). The total body burden also showed a dose-dependent relationship between parental exposure concentrations of 1, 3, and 10 μg/L with the values of 1689 ( 289, 2569 ( 528, and 13 701 ( 420 ng/g wet weight, respectively. The detected total PBDE content in the control eggs was 0.85 ( 0.34 ng/g.
’ DISCUSSION The aim of the study reported here was to investigate a possible transgenerational effect of DE-71 following parental exposure as this is potentially relevant for environmental risk assessments. The present study demonstrated that parental exposure to low concentrations of DE-71 significantly disturbed THs in both generations. Parental exposure to DE-71 did not cause any changes in hatching and maternal growth. However, offspring following DE71 parental exposure demonstrated reduced hatching and growth retardation compared to controls. This observation is consistent with recent reports on parental exposure of DE-71 to ranch minks (Mustela vison)20 and rats.14 Our results indicated that this alteration was transferred to the F1 generation, and developmental effects in offspring were more sensitive than those in their respective parents. A significant increase in the T4 level was observed in the adult females. In contrast, most studies have found reduced T4 levels with treatment at higher doses of PBDEs in lake trout (Salvelinus namaycush),8 fathead minnows (Pimephales promelas),9 flounder (Platichthys flesus),21 and mammalian models.27 A previous study also reported that total T4 plasma levels were elevated at 3 and 6 days in adult rats following a single exposure to PBDE99 (8.2 mg/kg), but they returned to normal levels after 12 days.22 In zebrafish, waterborne exposure to DE-71 (0, 5, 16, 50, 160, and 500 μg/L) for 30 days revealed a trend of concentrationdependent increase of plasma T4.21 In our previous study, zebrafish embryos were exposed to DE-71 (1, 3, and 10 μg/L) for 14 days, but T4 was significantly reduced at 10 μg/L in the 10655
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Figure 1. PBDE content in (A) F0 adult zebrafish after exposure to 0, 1, 3, or 10 μg/L DE-71 for 150 days; (B) PBDEs in the F1 eggs. For adult fish, the values represent means ( standard error (SEM) of three individual replicate fish. For eggs, PBDEs were measured in 100 eggs, with three replicate samples. a,b,c,dRepresent the eggs derived from parental exposure of 0, 1, 3, and 10 μg/L DE-71, respectively.
larvae.10 Therefore, the levels of THs may vary due to different exposure regimes, and given the evidence that in the environment where mostly low concentrations of PBDEs are detected, testing of long-term lower dose exposures would be better for assessing environmental risks. Decreased serum T3 upon exposure to PBDEs has not generally been observed in previous rodent studies, but a recent study did show a decrease in T3 concentrations from relatively low doses of DE-71 (0.5 mg/L) in ranch mink.20 In our study, an increasing trend of T3 was also observed in the adult zebrafish, and this result is consistent with a previous report, which demonstrated a concentration-dependent increase in T3 levels in zebrafish exposed to DE-71.21 In fish, THs are present in high quantities in eggs and are presumably of maternal origin.23 Thus, the TH content in eggs probably reflects that of the maternal plasma. In the F1 eggs,
significant increases in both T4 and T3 contents were measured, indicating maternal transfer of increased THs to the offspring. Thus, the significant increases in THs in offspring could also be due to maternal hyperthyroidism caused by DE-71 exposure. This may have a direct effect on the development of thyroid function in the young larvae. However, previous analysis of THs during embryo development in fathead minnow also showed a significant rise in both T4 and T3 during the prehatch period, indicating embryonic production of both thyroid hormones and significantly increased synthesis of THs in an early developmental stage after hatching in fish.24,25 In the present study, TH levels were increased in the larvae and the expressions of genes related to thyroid development and growth (e.g., hhex, nkx2.1) and TH synthesis (e.g., TG) were all significantly up-regulated as well, indicating that the increased THs in the larvae could also be 10656
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Environmental Science & Technology from enhanced synthesis of THs in the larvae. Increased T4 levels have also been observed in ranch mink juveniles after the adult had been treated with lower doses of DE-71 (0.5 mg/L)20 and in female rat offspring at postnatal day 60 following DE-71 exposure (1.7 and 10.2 mg/kg/day).14 Consistent with these previous reports, our study showed that parental exposure to PBDEs could result in increased TH levels and thus thyroid endocrine disruption in the developing offspring. In fish, CRH and TSH secretions function as common regulators of the thyroidal axis as feedback mechanisms are triggered by changes in the concentrations of circulating THs.26,27 Therefore, the down-regulation of both CRH and TSHβ genes observed in the present study could be explained as a negative feedback response of increased levels of T4. On the other hand, a low dietary dose of BDE-47 was shown to elevate expression of TSHβ in the pituitary gland, associated with reduced plasma levels of T4 in adult fathead minnows.9 In this regard, evaluation of CRH and TSHβ gene expression can be used to determine whether environmental chemicals cause thyroid dysfunction. Although the mechanisms of disturbing THs by PBDE exposure are not well-understood, three general explanations have been proposed, including direct interference at the thyroid gland by changes in thyroid gland histology and morphology,3 interference with TH metabolic enzymes, and interference with the plasma transport of THs.6,28 In the present study, TTR gene expression in adult exposed zebrafish was down-regulated, which may have reduced the amount of TTR proteins available to bind and transport free T4 to target organs. UGTs play a role in decreasing circulating THs, and up-regulation of UGT gene expression or enzyme activities have generally been observed in PBDE-treated animal models.3,29 In our study, the increased UGT1 gene expression could possibly be explained as an autoregulatory response to increased T4 levels, by increased biliary elimination of the conjugated hormone within the thyroid axis. Hepatic deiodinases are important regulators of circulating and peripheral TH levels in vertebrates. In fish, it has been demonstrated that Dio2 plays a pivotal role in producing active T3, allowing an adequate availability of local and systemic T3.30 Dio1 is mainly expressed in the kidney and is thought to play a minimal role in plasma TH homeostasis, while it has a considerable influence on iodine recovery and TH degradation.30,31 In our study, both Dio1 and Dio2 were significantly down-regulated in the adult fish liver. Our results are consistent with previous reports showing that hyperthyroidism suppresses Dio1 and Dio2 activities and expression of their mRNA, while hypothyroidism increases them in fish.30,31 The down-regulation of gene expression of the deiodinases may indicate a regulatory role in response to increased THs, indicating that the observed effects have functional consequences and indicate a true hyperthyroid state. In the F1 larvae, the gene expressions were examined with or without continued DE-71 treatment. In the F1 larvae without continued DE-71 treatment, Dio2 gene expression was downregulated, while Dio1 was up-regulated. The effects of THs on deiodinases in developing zebrafish embryos/larvae have been studied previously. Treatment with 5 nM T3 was shown to downregulate Dio2 mRNA expression in zebrafish larvae, without affecting Dio1 expression.25 Knockdown of Dio1 alone does not affect developmental progression, while Dio2 knockdown results in a clear developmental delay,32 suggesting that Dio2 is the major contributor to TH activation in developing zebrafish embryos/larvae. These results indicate that zebrafish larvae are dependent on T4 to T3 conversion for normal development, and
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down-regulation of Dio2 transcription may affect zebrafish development. The increased T4 levels were measured in F1 larvae without DE-71 treatment, while the gene expressions of CRH and TSHβ were significantly down-regulated in the F1 larvae. This response thus can indicate a negative response to the hyperthyroid levels. In addition, it is also well-known that expression levels of hepatic deiodinases respond to both hypo- and hyperthyroidism. Our results are thus consistent with a previous report, which showed that hyperthyroidism suppresses Dio2 mRNA in fish.30 It is also worth noting that different responses of Dio1 and Dio2 were observed in the present study. The upregulation of Dio1 gene expressions could possibly be explained as increased TH degradation and downregulation of Dio2 in response to hyperthyroidism. However, previous reports have also shown mixed results with Dio1 and Dio2 gene expressions and demonstrated that deiodinase genes are expressed in tissue and developmental stage specific patterns,23 especially during early development in zebrafish.25 Therefore the role of deiodinase enzymes as mediators of TH action in fish remains to be investigated. In our study, different UGT1 and TTR gene expressions were also observed at 5 and 10 dpf. The up-regulation of UGT1 at 10 dpf is consistent with those in adult fish toward increasing T4 clearance and glucuronidation. The temporally differential response of this gene expression is not clear. However, several previous studies did not find a strong correlation between serum T4 levels and T4-UGT activity following PBDE exposure.3,33 In addition, albumin and thyroxine-binding globulin (TBG) can also bind to THs in fish plasma.9 Therefore, other mechanisms may need to be examined in order to understand the role of TH-binding proteins. In the present study, we also investigated developmental toxicity, TH levels, and related gene expressions with continued DE-71 treatment of the F1 larvae. This experiment allowed the discrimination between a situation where PBDEs could have a direct effect and that where only the parental effects could play a role. Our results showed similar impacts on TH levels, suggesting that these effects could be due to a transgenerational effect on thyroid endocrine disruption. However, continued treatment until 5 and 10 dpf seemed to have contributed a further increase in T4, while an increasing trend of T3 was also observed. PBDEs have been shown to alter TH levels and clearance and can compete for the thyroid hormone binding protein. Our data suggest that there is a decreased binding or reduced clearance of THs in F1 upon further exposure to low doses of DE-71, as marked downregulation of UGT gene and TTR gene expressions was observed in the group with continued DE-71 treatment. It should also be noted that the malformation rate was further increased in the F1 larvae under continued DE-71 treatment compared with those without DE-71 exposure, indicating an increased sensitivity to toxic effects of PBDEs compared with their parental embryos/larvae (F0). THs have an important role in the regulation of early fish development. Both beneficial34 and harmful35 effects of increased levels of maternal T3 on subsequent larval development and survival have been reported in fish species. The reasons for the conflicting results are unexplained but may be associated with differences in the dose and mode of administration of the THs used in the experiments. Exogenous TH induces premature differentiation of the zebrafish pectoral fins; in particular, they inhibit the development of scales and pigment pattern, and impair the growth of both pectoral and pelvic fins.36,37 In 10657
dx.doi.org/10.1021/es2026592 |Environ. Sci. Technol. 2011, 45, 10652–10659
Environmental Science & Technology zebrafish, excessive exogenous T4 (30 nM) is toxic and causes severe developmental defects.38 A significant growth inhibition was also observed in F1 larvae in our study, which was consistent with the results of previous report indicating that PBDEs disruption of TH production in rat F1 offspring causes growth impairment.39 In our study, significant levels of DE-71 were detected in the eggs, indicating that DE-71 was transferred from exposed adult fish to their offspring. Furthermore, these PBDEs can also directly disrupt thyroid function and development in the larvae. Although the maternal transfer is complex, and the present study design did not allow for any detailed analysis of the transfer processes, it clearly indicates that hydrophobic compounds are more efficiently transferred than less hydrophobic compounds,16 resulting in increased prevalence of adverse health signs in the offspring. Taken together, our results highlighted the thyroid hormone disruption in the adults, as well as transgenerational thyroid disruption following parental exposure to PBDEs.
’ ASSOCIATED CONTENT
bS
Supporting Information. Text S1 outlines thyroid hormone extraction and assay; Text S2 is a description of gene expression method; Text S3 is a description of PBDE analysis and QA/QC procedures; Table S1 shows primer sequence; Table S2 contains F0 growth and condition factor; Table S3 shows gene expression in adult fish; Table S4 shows gene expression in F1. This information is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel: 852-2788 7681; fax: 852- 2788 7406; e-mail: bhpksl@cityu. edu.hk (P. K. S. L.). Tel: 86-27-68780042; fax: 86-27-68780123; e-mail:
[email protected] (B. Z.). )
Notes
Co-first authors.
’ ACKNOWLEDGMENT This work was supported by grants from the NSFC (20890113), the Chinese Academy of Sciences (KCZCX2-YW-Q02-05), and FEBL (2008FBZ10). ’ REFERENCES (1) International Institute for Sustainable development (IISD). Summary of the Fourth Conference of the Parties to the Stockholm Convention on Persistent Organic Pollutants; Geneva, Switzerland, 48 May 2009; http://www.unido.org/fileadmin/user_ media/Services/Environmental_ Management/Stockholm_Convention/POPs/ SummaryRepotrofCOP4_01.pdf. (2) Zhou, T.; Ross, D. G.; DeVito, M. J.; Crofton, K. M. Effects of short-term in vivo exposure to polybrominated diphenyl ethers on thyroid hormones and hepatic enzyme activities in weanling rats. Toxicol. Sci. 2001, 61, 76–82. (3) Zhou, T.; Taylor, M. M.; DeVito, M. J.; Crofton, K. M. Developmental exposure to brominated diphenyl ethers results in thyroid hormone disruption. Toxicol. Sci. 2002, 66, 105–116. (4) Stoker, T. E.; Laws, S. C.; Crofton, K. M.; Hedge, J. M.; Ferrell, J. M.; Cooper, R. L. Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols. Toxicol. Sci. 2004, 78, 144–155. (5) Ellis-Hutchings, R. G.; Cherr, G. N.; Hanna, L. A.; Keen, C. L. Polybrominated diphenyl ether (PBDE)-induced alterations in vitamin
ARTICLE
A and thyroid hormone concentrations in the rat during lactation and early postnatal development. Toxicol. Appl. Pharmacol. 2006, 215, 135–145. (6) Richardson, V. M.; Staskal, D. F.; Ross, D. G.; Diliberto, J. J.; DeVito, M. J.; Birnbaum, L. S. Possible mechanisms of thyroid hormone disruption in mice by BDE 47, a major polybrominated diphenyl ether congener. Toxicol. Appl. Pharmacol. 2008, 226, 244–250. (7) Szabo, D. T.; Richardson, V. M.; Ross, D. G.; Diliberto, J. J.; Kodavanti, P. R.; Birnbaum, L. S. Effects of perinatal PBDE exposure on hepatic phase I, phase II, phase III, and deiodinase 1 gene expression involved in thyroid hormone metabolism in male rat pups. Toxicol. Sci. 2009, 107, 27–39. (8) Tomy, G. T.; Palace, V. P.; Halldorson, T.; Braekevelt, E.; Danell, R.; Wautier, K.; Evans, B.; Brinkworth, L.; Fisk, A. T. Bioaccumulation, biotransformation, and biochemical effects of brominated diphenyl ethers in juvenile lake trout (Salvelinus namaycush). Environ. Sci. Technol. 2004, 38, 1496–1504. (9) Lema, S. C.; Dickey, J. T.; Schultz, I. R.; Swanson, P. Dietary exposure to 2,20 ,4,40 -tetrabromodiphenyl ether (PBDE-47) alters thyroid status and thyroid hormone-regulated gene transcription in the pituitary and brain. Environ. Health Perspect. 2008, 116, 1694–1699. (10) Yu, L.; Deng, J.; Shi, X.; Liu, C.; Yu, K.; Zhou, B. Exposure to DE-71 alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis of zebrafish larvae. Aquat. Toxicol. 2010, 97, 226–233. (11) Wu, J. P.; Luo, X. J.; Zhang, Y.; Luo, Y.; Chen, S. J.; Mai, B. X.; Yang, Z. Y. Bioaccumulation of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in wild aquatic species from an electronic waste (e-waste) recycling site in South China. Environ. Int. 2008, 34, 1109–1113. (12) Lundstedt-Enkel, K.; Asplund, L.; Nylund, K.; Bignert, A.; Tysklind, M.; Olsson, M.; Orberg, J. Multivariate data analysis of organochlorines and brominated flame retardants in Baltic Sea guillemot (Uria aalge) egg and muscle. Chemosphere 2006, 65, 1591–1599. (13) Verreault, J.; Villa, R. A.; Gabrielsen, G. W.; Skaare, J. U.; Letcher, R. J. Maternal transfer of organohalogen contaminants and metabolites to eggs of Arctic-breeding glaucous gulls. Environ. Pollut. 2006, 144, 1053–1060. (14) Kodavanti, P. R.; Coburn, C. G.; Moser, V. C.; MacPhail, R. C.; Fenton, S. E.; Stoker, T. E.; Rayner, J. L.; Kannan, K.; Birnbaum, L. S. Developmental exposure to a commercial PBDE mixture, DE-71: neurobehavioral, hormonal, and reproductive effects. Toxicol. Sci. 2010, 116, 297–312. (15) Schecter, A.; Johnson-Welch, S.; Tung, K. C.; Harris, T. R.; P€apke, O.; Rosen, R. Polybrominated diphenyl ether (PBDE) levels in livers of U.S. human fetuses and newborns. J. Toxicol. Environ. Health A 2007, 70, 1–6. (16) Nyholm, J. R.; Norman, A.; Norrgren, L.; Haglund, P.; Andersson, P. L. Maternal transfer of brominated flame retardants in zebrafish (Danio rerio). Chemosphere 2008, 73, 203–208. (17) Ostrach, D. J.; Low-Marchelli, J. M.; Eder, K. J.; Whiteman, S. J.; Zinkl, J. G. Maternal transfer of xenobiotics and effects on larval striped bass in the San Francisco Estuary. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19354–19359. (18) Mazdai, A.; Dodder, N. G.; Abernathy, M. P.; Hites, R. A.; Bigsby, R. M. Polybrominated diphenyl ethers in maternal and fetal blood samples. Environ. Health Perspect. 2003, 111, 1249–1252. (19) Hites, R. A. Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. Environ. Sci. Technol. 2004, 38, 945–956. (20) Zhang, S.; Bursian, S. J.; Martin, P. A.; Chan, H. M.; Tomy, G.; Palace, V. P.; Mayne, G. J.; Martin, J. W. 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. 2009, 110, 107–116. (21) Kuiper, R. V.; Vethaak, A. D.; Canton, R. F.; Anselmo, H.; Dubbeldam, M.; van den Brandhof, E. J.; Leonards, P. E.; Wester, P. W.; van den Berg, M. Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys 10658
dx.doi.org/10.1021/es2026592 |Environ. Sci. Technol. 2011, 45, 10652–10659
Environmental Science & Technology
ARTICLE
flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio). Chemosphere 2008, 73, 195–202. (22) Hakk, H.; Larsen, G.; Klasson-Wehler, E. Tissue disposition, excretion and metabolism of 2,20 ,4,40 ,5-pentabromodiphenyl ether (BDE99) in the male Sprague-Dawley rat. Xenobiotica 2002, 32, 369–382. (23) Power, D. M.; Llewellyn, L.; Faustino, M.; Nowell, M. A.; Bjornsson, B. T.; Einarsdottir, I. E.; Canario, A. V.; Sweeney, G. E. Thyroid hormones in growth and development of fish. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2001, 130, 447–459. (24) Crane, H. M.; Pickford, D. B.; Hutchinson, T. H.; Brown, J. A. Developmental changes of thyroid hormones in the fathead minnow, Pimephales promelas. Gen. Comp. Endrocrinol. 2004, 139, 55–60. (25) Walpita, C. N.; Van der Geyten, S.; Rurangwa, E.; Darras, V. M. The effect of 3,5,30 -triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen. Comp. Endrocrinol. 2007, 152, 206–214. (26) De Groef, B.; Van der Geyten, S.; Darras, V. M.; K€uhn, E. R. Role of corticotropin-releasing hormone as a thyrotropin-releasing factor in non-mammalian vertebrates. Gen. Comp. Endrocrinol. 2006, 146, 62–68. (27) Geven, E. J. W.; Flik, G.; Klaren, P. H. M. Central and peripheral integration of interrenal and thyroid axes signals in common carp (Cyprinuscarpio L.). J. Endocrinol. 2009, 200, 117–123. (28) Birnbaum, L. S.; Staskal, D. F. Brominated flame retardants: Cause for concern? Environ. Health Perspect. 2004, 112, 9–17. (29) Hallgren, S.; Darnerud, P. O. Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and chlorinated paraffins (CPs) in rats-testing interactions and mechanisms for thyroid hormone effects. Toxicology 2002, 177, 227–243. (30) Orozco, A.; Valverde, R. C. Thyroid hormone deiodination in fish. Thyroid 2005, 15, 799–813. (31) Van der Geyten, S.; Byamungu, N.; Reyns, G. E.; Kuhn, E. R.; Darras, V. M. Iodothyronine deiodinases and the control of plasma and tissue thyroid hormone levels in hyperthyroid tilapia (Oreochromis niloticus). J. Endocrinol. 2005, 184, 467–479. (32) Walpita, C. N.; Crawford, A. D.; Janssens, E. D.; Van der Geyten, S.; Darras, V. M. Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish. Endocrinology 2009, 150, 530–539. (33) Kuriyama, S. N.; Wanner, A.; Fidalgo-Neto, A. A.; Talsness, C. E.; Koerner, W.; Chahoud, I. Developmental exposure to low-dose PBDE-99: Tissue distribution and thyroid hormone levels. Toxicology 2007, 242, 80–90. (34) Ayson, F. G.; Lam, T. J. Thyroxine injection of female rabbitfish (Siganus guttatus) broodstock: Changes in thyroid hormone levels in plasma, eggs, and yolk-sac larvae, and its effect on larval growth and survival. Aquaculture 1993, 109, 83–93. (35) Mylonas, C. C.; Sullivan, C. V.; Hinshaw, J. M. Thyroid hormones in brown trout (Salmo trutta) reproduction and early development. Fish Physiol. Biochem. 1994, 13, 485–493. (36) Brown, D. D. The role of thyroid hormone in zebrafish and axolotl development. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 13011–13016. (37) Wendl, T.; Lun, K.; Mione, M.; Favor, J.; Brand, M.; Wilson, S. W.; Rohr, K. B. Pax2.1 is required for the development of thyroid follicles in zebrafish. Development 2002, 129, 3751–3760. (38) Liu, Y. W.; Chan, W. K. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 2002, 70, 36–45. (39) Blake, C. A.; McCoy, G. L.; Hui, Y. Y.; LaVoie, H. A. Perinatal exposure to low-dose DE-71 increases serum thyroid hormones and gonadal osteopontin gene expression. Exp. Biol. Med. 2011, 236, 445–455.
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