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Evaluating estrogenic and anti-estrogenic effect of endocrine disrupting chemicals (EDCs) by zebrafish (Danio rerio) embryo-based vitellogenin 1 (vtg1) mRNA expression
Comparative Biochemistry and Physiology, Part C 204 (2018) 45–50
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Evaluating estrogenic and anti-estrogenic effect of endocrine disrupting chemicals (EDCs) by zebrafish (Danio rerio) embryo-based vitellogenin 1 (vtg1) mRNA expression
T
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Minjie Chena,b, Jie Zhangb, , Shaochen Pangc, Chang Wangc, Ling Wangc, Yonghua Sund, ⁎⁎ Maoyong Songe, Yong Lianga,c, a
School of Medicine, Jianghan University, Wuhan 430056, PR China College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, PR China Institute of Environmental Health, Jianghan University, Wuhan 430056,PR China d State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China e State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Zebrafish embryo Vitellogenin 1 Estrogenic effect Endocrine disrupting chemicals
By measuring the vitellogenin 1 (vtg1) expression through quantitative PCR and in situ hybridization, we used the zebrafish embryo as an in vivo model to access the estrogenic or anti-estrogenic effects of several endocrine disrupting chemicals (EDCs), such as natural estrogen 17β-estradiol (E2), estriol (E3), synthetic hormones including diethylstilbestrol (DES), 4-octyl phenol (OP), bisphenol A (BPA), tamoxifen (TMX) and 3-(2,3-dibromopropyl) isocyanurate (TBC). According to our data, the estrogenic effect of the tested chemicals was ranked as: DES > E2 > E3 > OP > BPA, which is consistent with various in vivo and in vitro models. Therefore, the measurement of vtg1 gene expression in zebrafish embryos would be a valuable method for screening EDCs including both environmental estrogens and anti-estrogens.
1. Introduction Endocrine disrupting chemicals (EDCs) are a class of exogenous substance or mixture that alter the normal function of the endocrine system (Diamantikandarakis et al., 2009). Nowadays EDCs are of great concern to the health issue of human and wildlife. Be natural or manmade, increasing numbers of chemicals with estrogenic or anti-estrogenic effects have been defined as EDCs by using various in vivo or in vitro models. 17β-estradiol (E2) is a natural steroid estrogen with strong estrogenic activity to male zebrafish and rainbow trout (Oncorhynchus mykiss) juveniles (Van den Belt et al., 2003; Pawlowski et al., 2000). It has been widely used as a positive control compound in evaluating the estrogenic effects of some new pollutants. Estriol (E3), metabolite of estradiol and estrone, is also a natural estrogen in human body, which could bind to and acts as a weak agonist of estrogen receptor (ER) (Russo et al., 2001; Head, 1998). As a synthesized non-steroidal estrogenic substance, diethylstilbestrol (DES) has been proved to seriously threat human health through the food chain enrichment (Stefanick, 2005; Yin et al., 2006). With toxicity to development of
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fetus' reproductive organs, DES could increase the risk of reproductive system cancer during adulthood (Li et al., 2003; Hendry et al., 2006). 4octylphenol (OP) as a representative EDCs with estrogenic activity was reported to stimulate the proliferation of MCF-7 cell (White et al., 1994). With both weak estrogenic activity and strong anti-androgenic activity (Sohoni and Sumpter, 1998), bisphenol A (BPA) was reported to cause metabolic disorders in various organisms, by interacting with ER (Richter et al., 2007), thyroid hormone receptor (Moriyama et al., 2002) and peroxisome proliferator-activated receptor (Riu et al., 2011). Additionally, BPA exposure was identified to alter the early dorsoventral patterning, segmentation, and brain development in zebrafish embryos (Tse et al., 2013). Tamoxifen (TMX) as a pharmaceutical antagonist of the estrogen could competitively bind to ER in breast cells, playing an important role in the treatment of certain breast cancer and ovarian cancer (Wallen et al., 2005). Tris-(2, 3-dibromopropyl) isocyanurate (TBC), which is released into the environment during the use of flame retardants, is a potential EDC with anti-estrogen activity (Ruan et al., 2009; Zhang et al., 2011). The toxicity of EDCs and related molecular mechanism have been explored in multiple studies. Cultured
Corresponding author. Correspondence to: Y. Liang, School of Medicine, Jianghan University, Wuhan 430056, PR China. E-mail addresses: [email protected] (J. Zhang), [email protected] (Y. Liang).
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https://doi.org/10.1016/j.cbpc.2017.11.010 Received 20 September 2017; Received in revised form 28 November 2017; Accepted 28 November 2017 Available online 01 December 2017 1532-0456/ © 2017 Published by Elsevier Inc.
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and 10 μM of BPA. Co-exposure of TMX (0.5 and 1 μM) and E2 (0.5 μM) as well as TBC (1 and 5 μM) and E2 (0.5 μM) were also performed at the same time point. Additionally, zebrafish embryos were exposed to 0.5 μM E2 at several exposure window including 4 to 28 hpf (1d), 4 to 52 hpf (2d), 4 to 76 hpf (3d), 4 to 100 hpf (4d), 4 to 124 hpf (5d), 4 to 148 hpf (6d), 4 to 172 hpf (7d). 0.5% DMSO was used as vehicle control in each exposure experiment.
cell lines have been widely used, while in vivo models could better reflect the physiological and functional changes caused by EDCs. However, traditional animal models are costly, time-consuming, may have some ethic problems and individual differences (Kuhn et al., 2003; Legler et al., 2002). Therefore, it is necessary to develop a fast approach which could accurately identify EDCs and elucidate the potential molecular mechanism of these pollutants. There are several advantages by using zebrafish (Danio rerio) embryos in scientific studies. Due to the embryo transparency, zebrafish embryos are easily visualized and operated. It is also convenient to utilize their comprehensive heredity feature and different development stages for biological research (Lam et al., 2005). Vitellogenin (VTG) is the yolk precursor protein of vitellin (vn) in oviparous animals like zebrafish, which is predominantly synthesized in liver of mature female fish. VTG expression is regulated by luteinizing hormone and folliclestimulating hormone, also specifically by estrogens and anti-estrogens (Okuzawa, 2002). Previous studies have reported that VTG protein could be induced in male and larval fish in response to exogenous estrogen (Mitsui et al., 2003). Thus, VTG can be a good biomarker of estrogen exposure (Sumpter and Jobling, 1995). Additionally, vitellogenin 1 (vtg1) gene expression is the highest among the seven forms of vtg genes (vtg1-7) in zebrafish genome and has been proposed to be an indicating molecule for estrogenicity (Muncke and Eggen, 2006; Wang et al., 2005). The relative expression pattern of vtg1 gene can be induced by exogenous estrogens in adult male and larval zebrafish (Henry et al., 2009). For zebrafish embryos, vtg1 transcript usually occurs at 24 h post-fertilization (hpf) and at all later time points. It can be significantly up-regulated by 17α-ethinylestradiol, which also appears early at 24 hpf (Muncke and Eggen, 2006). Additionally, though vtg1 mRNA expression in adult zebrafish and adult zebrafish embryos has been used to test the estrogenic effects of emerging chemicals (Chow et al., 2013; Muncke and Eggen, 2006), assessment of the anti-estrogenic effect of certain compound by this model is so far lacking. This study primarily aimed to assess both the estrogenic or antiestrogenic effect of certain compounds by measuring zebrafish embryobased vtg1 mRNA levels. Multiple EDCs including E2, E3, DES, OP, BPA, TMX and TBC were investigated by single exposure as well as co-exposure experiment using zebrafish embryo. Vtg1 mRNA expression measurement assay clearly indicate an estrogenic response and antiestrogenic response by the testing chemicals. The analysis was furthermore verified through a comparison among different in vitro and in vivo experiment models. Therefore, our approach could be considered as an economic and promising evaluation for rapid testing and screening of multiple EDCs.
2.3. RNA extraction and real-time PCR (RT-PCR) Total RNA was extracted from forty pooled zebrafish embryos in each group by using Trizol reagent (Invitrogen), following the manufacturer's instructions. The yield and purity of extracted total RNA was determined by UV spectrophotometry (A260 and A260/A280 ratio). 2 μg of total RNA was used to synthesize cDNA by M-MLV (Moloney murine leukemia virus) RNase H reverse transcriptase (Promega, Fitchburg, WI, USA). Each PCR reaction mixture contained 1 μL cDNA template, 0.1 μmol/L primer, 7 μL water, and 10 μL 2 × SYBR QPCR Master Mix (Toyobo, Osaka, Japan). Reactions were performed in the Bio-Rad iQ5 thermal cycler equipped with a real-time fluorescence detector. The thermal cycling program consisted of a denaturing step (95 °C, 3 min) followed by 40 cycles of denaturation (95 °C for 10 s), annealing (55 °C for 10 s), and extension (72 °C for 20 s). The PCR primers were designed with Primer Premier 5.0 (Premier, Palo Alto, CA, USA): vtg1 forward 5′-AGCTGCTGAGAGGCTTGTTA-3′; vtg1 reverse 5′-GTCCAGGATTTCCCTC AGT-3′; β-actin forward 5′-GTCACACCATCA CCAGAGTCCATCAC-3′; β-actin reverse 5′-CAACAGAGAGAAGATGAC ACAGATCA-3′. β-actin was used as the internal standard. The target gene (vtg1) expression level was calculated by 2–ΔΔCT method (Livak and Schmittgen, 2001) and normalized by β-actin. 2.4. In situ hybridization Whole-mount in situ hybridization was performed using digoxigenin (DIG)-labeled antisense RNA probes and an anti-DIG alkaline phosphatase-conjugated antibody as described (Thisse et al., 2004). The vtg1 probe was synthesized by T3 polymerase (Promega, Fitchburg, WI, USA) using vtg1 (GenBank Accession Number of NM_001044897) PCR product as the templates, which were amplified from cDNA of 500 nM E2 exposed zebrafish embryo using a set of primers 5′-CAGCAGTCG TAACAGTCGC-3′ and 5′-GATCCATTAACCCTCACTAAAGGGAACTCCG CACC CCAAGAAA-3′, which contained T3 promoter sequence (underlined). 2.5. Statistical analyses
2. Materials and methods Statistical analyses were performed in SPSS 16.0 for Windows (IBM Corp. (2006), Somers, NY, USA). Values are presented as group means ± standard error (SE). One-way analysis of variance (ANOVA) was used to analyze the significant difference between the control and treatment groups, with post hoc analysis by Fisher's least significant difference (LSD) test. P values < 0.05 were considered statistically significant, P values < 0.01 were considered very significant.
2.1. Test compounds E2 (≥98% purity), E3 (≥ 97% purity), DES (≥ 99% purity), OP (99% purity), BPA (≥ 99% purity), TMX and TBC (97% purity) were purchased from Sigma-Aldrich (St Louis, MO, USA). All chemicals were dissolved in dimethyl sulfoxide (DMSO, Amresco, Solon, OH, USA) and diluted into fish water.
3. Results 2.2. Zebrafish embryo and exposure experiment 3.1. Dose-dependent expression of endogenous vtg1 gene after E2 exposure in the zebrafish embryos
Zebrafish (wild-type, AB strain) were obtained from China Zebrafish Resource Center (http://zfish.cn; Wuhan, China). The fish were raised in a closed flow-through system with fresh water at 26 ± 1.5 °C under a 14 h light/10 h dark cycle, maintained according to the Zebrafish Book (Westerfield, 2000). Zebrafish embryos were exposed to E2, E3, DES, OP and BPA since 4 hpf for 7 days at series of concentrations as following: 0.05, 0.1, 0.25, 0.5 and 1 μM of E2; 0.1, 0.25, 0.5, 1 and 2.5 μM of E3; 0.05, 0.1, 0.25, 0.5 and 1 μM of DES, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM of OP; 0.5, 1, 2.5, 5
To determine the response of the vtg1 gene to E2 exposure at the zebrafish embryos levels, we primarily detected the transcriptionally expression levels of vtg1 by RT-PCR analysis. By referring to the data from a previous study (Wang et al., 2011), the zebrafish embryos were exposed to various concentrations of E2 for 7 days. A dose-dependent increase of vtg1 mRNA-expression occurred in zebrafish embryos following E2 exposure (Fig. 1A). The relative mRNA levels of vtg1 in the 46
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Fig. 1. Dose-dependent expression of vtg1 in exposed zebrafish embryos after E2 exposure. (A) Dose-dependent transcriptional expression of vtg1 in E2 exposed zebrafish embryos. (B) Temporal expression of vtg1 in the zebrafish embryos exposed to 0.5 μM E2. (C) Temporal and spatial expression of vtg1 in 0.5 μM E2 exposed zebrafish embryos, analyzed by the wholemount in situ hybridization. Vtg1 was transcriptionally expressed both in liver (arrow) and heart (triangle) tissue of zebrafish embryos until 4 day exposure. The percentages of embryos with positive signal were shown in the top right corner. All the data were normalized to DMSO-treated group and represented as mean value ± SE (n = 3). Significant differences between E2 exposure group and control group was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
0.25, 0.5 and 1 μM E2 treated groups were 34.7, 49 and 61.8 folds higher than that in the control group, respectively (P < 0.01). These results indicated the sensitive response of vtg1 transcription to the E2 exposure, which is consistent with previous study (Wang et al., 2011). Aimed to investigate the temporal expression pattern of vtg1 following E2 stimulation, we then analyzed the vtg1 mRNA levels in zebrafish embryos during a 7-day exposure of 0.5 μM E2. Comparing with the controls, increased vtg1 transcript (> 5 folds) was detected on the 6th day of E2 exposure, and was induced by 20 folds on the 7th day (Fig. 1B). The hybridization data indicated that 12.5% of embryos began to weakly transcript vtg1 in the liver and heart on the 4th day of 0.5 μM E2 exposure. The vtg1 levels then continued to be elevated by E2 with the exposure time, the percentage of vtg1-expressed embryos increased from 39.13% to 89.36% and finally 100% on the 5th, 6th and 7th exposure day respectively (Fig. 1C). Therefore, as only a small amount of embryos expressed little vtg1 only in the liver and heart on the 4th and 5th day of E2 exposure, RT-PCR analysis detected no significant vtg1 transcripts until the 6th exposure day. As the exposure time increased, vtg1 expression was strongly up-regulated both in the liver and heart tissue, indicating the temporal effect of E2 exposure (Fig. 1B and C). These results suggested that the vtg1 transcriptional expression is a sensitive and ideal biomarker for assessing the effect of E2 exposure in the zebrafish embryos.
estrogen E2, we further explored the feasibility of using vtg1 transcription to assess the estrogenic potentiality of other EDCs. Zebrafish embryos were exposed to series doses of several compounds including E3, DES, OP and BPA for 7 days, and the relative mRNA levels of vtg1 were determined by RT-PCR (Fig. 2). Comparing with the controls, exposure group with a P value < 0.05 was considered to have significant estrogenic effect. Therefore, the lowest observable effect concentration (LOEC) of each compound here was defined as their lowest dose to produce significant vtg1 induction. According to this definition, the LOEC of E2 was 0.25 μM, and the estrogenic effect was induced with the increasing dose of E2 (Fig. 2). Low doses of E3 caused no significant increase in vtg1 transcription, while obvious estrogenic effect occurred at the concentration of 2.5 μM (Fig. 2), confirming that E3 is a weak natural estrogen. DES caused significant estrogenic effect at a very low concentration of 0.1 μM, vtg1 expression was induced by > 800-fold in 0.25 μM DES-treated embryos (Fig. 2). Interestingly, the vtg1 induction slightly declined after exposure to high dosed DES (0.5 μM and 1 μM). However, though all the doses of OP showed a mild estrogenic effect to zebrafish embryos, there was no statistical difference between these exposure groups and the controls (Fig. 2). Additionally, no significant estrogenic effect appeared after exposed to the various concentrations of BPA (Fig. 2). Therefore, these results indicated the potency of known EDCs to induce vtg1 expression in zebrafish embryos, which reflected their estrogenic effects.
3.2. Assessing the estrogenic effects of estrogenic agonists by vtg1 transcription in the zebrafish embryos
3.3. Anti-estrogenic effects by TMX and TBC EDCs could either act as agonist or antagonist of ER, which shows
Based on the sensitively response of vtg1 expression to natural 47
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the LOEC of E2. Comparison of RP values between several experimental models including our zebrafish embryo test model, cell lines and in vitro binding assay was performed to evaluate estrogenic effect of the tested compounds (Table 1). Accordingly, the RP values of E2, E3 and DES in zebrafish embryos were 1, 0.1 and 2.5 respectively, which was comparable with other test models. Though no LOEC was obtained from the vtg1 response curve of OP, gentle estrogenic effect could be detected following the OP exposure. Therefore, their estrogenic effects were ranked as: DES > E2 > E3 > OP > BPA, which is consistent with other models listed in Table 1. These data suggested that zebrafish embryo-based vtg1 mRNA expression is an efficient model for evaluating estrogenic effect of certain chemicals. 4. Discussion This study aimed to provide substantiating data for zebrafish embryo-based testing strategy suitable for the assessment of estrogenic effects. We demonstrated the vtg1 gene dose-response relationship after 7-day exposure and co-exposure experiment using E2, E3 and known EDCs, which indicated the sensitivity of zebrafish embryo as an evaluation model of estrogenic effect as well as anti-estrogenic effect. In oviparous vertebrates, VTG protein is synthesized in liver and transported to ovaries via blood circulation. Since the main function of VTG is to provide nutrients for the egg development in mature female, there was very little VTG content in early embryonic development. In current study, the experimental results of qPCR and in situ hybridization show that E2 can induce vtg1 gene expression with a dose-response relationship in zebrafish embryos, which is consistent with known knowledge (Wang et al., 2011). And although the vtg1 mRNA was dominantly expressed in liver, it was detected in embryo heart by in situ hybridization. The vtg1 in heart tissue has already been reported, and its function is considered as to unload surplus intracellular lipids in cardiomyocytes for reverse triglyceride transportation (Yin et al., 2009). Therefore, E2 does induce vtg1 expression via some pathway in zebrafish embryos. In order to discuss whether zebrafish embryo-based vtg1 expression can be used as an indicator to evaluate estrogenic effects of certain compounds, here typical EDCs such as E2, E3, DES, OP, BPA, TMX and TBC were chosen to perform exposure experiment by using zebrafish embryos. Exposure of 0.5 μM and 1 μM E2 for 7 days strongly induces vtg1 expression in zebrafish embryo and the maximal induction is 61.8
Fig. 2. The estrogenic effects of estrogenic agonists were assessed by vtg1 expression in the zebrafish embryos. Zebrafish embryos were exposed by E2, E3, DES, OP and BPA at a serial of indicated concentrations for 7 days. All the data were normalized to DMSOtreated group and represented as mean value ± SE (n = 3). Significant differences between each exposure group and control group was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
estrogenic or anti-estrogenic effect. Therefore, to further evaluate the anti-estrogenic response ability of zebrafish embryo-based vtg1 expression, co-exposure test was employed in the zebrafish embryos for 7 days. TMX as an antagonist of ER, co-exposure (0.5 μM and 1 μM) with 0.5 μM E2 strongly inhibited the vtg1 induction effect caused by single E2 exposure (Fig. 3A). As an emerging EDC, single exposure of TBC induced no vtg1 transcript in the zebrafish embryos. However, the vtg1 mRNA levels were significantly decreased by co-exposure of TBC (1 μM and 5 μM) with 0.5 μM E2, when comparing with 0.5 μM E2 single exposure individuals (Fig. 3B). Thus our model could also be used to access the anti-estrogenic effect of certain compound by using coexposure test with E2. 3.4. Comparison between various models In order to obtain the relative potency (RP) value for the estrogenic effect of the compounds, the LOEC of each compound was divided by
Fig. 3. Anti-estrogenic effect of estrogen antagonist was assessed by vtg1 expression in the zebrafish embryos. (A) TMX inhibited vtg1 transcriptional expression stimulated by E2 treatment. Zebrafish embryos were exposed to E2 (0.5 μM) or TMX (0.5, 1 μM), and co-exposed to E2 (0.5 μM) and TMX (0.5, 1 μM) for 7 days. (B) TBC showed anti-estrogenic effect zebrafish embryos. Zebrafish embryos were exposed to E2 (0.5 μM) or TBC (1, 5 μM), and co-exposed to E2 (0.5 μM) and TBC (1, 5 μM) for 7 days. All the data were normalized to DMSOtreated group and represented as mean value ± SE (n = 3). Significant differences between the indicated groups was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
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Table 1 Comparison between several experimental models to evaluate estrogenic effect of compounds. Compound
E2 E3 DES OP BPA Tamoxifen TBC a b c d
zebrafish embryo assay
LOEC (μM)
Relative potencya
0.25 2.5 0.1 NDb NDb N/A N/A
1 0.1 2.5 NDb NDb N/A N/A
Binding to ERαc
Binding to ERβc
MVLN cellsc
HGELN cellsc
E-Screenc
Yeast assayd
Carp VTG assayd
Trout VTG assayd
1
1
1
4.6 × 10− 4 8.1 × 10− 5
2.4 × 10− 3 3.1 × 10− 3
5.1 × 10− 4 6.5 × 10− 4
Relative potencya
1 0.07 1.75 7.0 × 10− 4 2.3 × 10− 4 0.023
1 0.26 1.3 6.5 × 10− 3 2.6 × 10− 3 0.054
1 0.083 1.25 8.33 × 10− 5 2.5 × 10− 5 8.33 × 10− 6
1 0.4 8 8.0 × 10− 4 1.9 × 10− 4 7.1 × 10− 7
1 0.071 2.5 1.0 × 10− 4 2.5 × 10− 5 4.0 × 10− 5
Ratio of LOEC of E2 to that of test compounds. Not detected. Gutendorf and Westendorf, 2001. Segner et al., 2003.
value of DES is higher than E2, confirming that DES is a compound with strong estrogenic-effect, which has not been identified by using in vitro models. In zebrafish embryo, the RP values of E3 and OP are lower than E2, indicating that their estrogenic effects are weaker than E2. According to the ranking of RP value for the testing compounds, the estrogenic effect of EDCs was accurately accessed by using zebrafish embryo-based vtg1 expression measurement. These results provided sufficient evidence for the feasibility of our model here to evaluate the estrogenic or anti-estrogenic effect of certain chemicals, which could be used to screen the emerged EDCs.
times compared to the control group. While E3 can significantly induce vtg1 transcription when the exposure dose increased to 2.5 μM. This is consistent with previous report that the ER affinity of E3 was weaker than that of E2 (Rodan and Martin, 2000; Melamed et al., 1997). DES is known to have strong estrogenic effects, which can disrupt the normal function of the endocrine system (Li et al., 2003; Hendry et al., 2006). Here at the concentration of 0.25 μM, DES exposure highly induced vtg1 expression, which is 895 times higher than that in the control group. However, vtg1 induction slightly declined when the DES exposure concentration increased to 0.5 μM and 1 μM. This might be due to the toxic effects of high dosed DES on zebrafish embryos. OP has been reported as an estrogenic compound, which is present in the environment and exerts reproductive as well as developmental toxicity to wildlife and humans (White et al., 1994). Our study showed that 7-day exposure of OP upon 0.25 μM could induce vtg1 expression in zebrafish embryos, but no statistical significance occurred. Therefore, zebrafish embryo may be less sensitive to OP than other species. Though BPA has a similar chemical structure as estrogen and DES, it binds to ER with much lower affinity (Gaido et al., 1997), this might be the reason for the low estrogenic effect of BPA. Therefore, BPA exposure within the dose range here could not cause significant increase of vtg1 mRNA expression in zebrafish embryos. This is consistent with known findings that BPA's estrogenic activity is 1000 times less than estradiol (Iso et al., 2006). Thus, the vtg1 gene expression in zebrafish embryo is a susceptive indicator for evaluating estrogenic effect of various compounds. TMX is a well-known competitive antagonist of estrogen (Coezy et al., 1982). Though TMX single exposure caused no significant increase of vtg1 level, adding of 0.5 μM and 1 μM TMX can strongly reverse the vtg1 induction caused by E2 exposure. Thus our results suggested that TMX could interfere with the E2 pathway in zebrafish embryo with the anti-estrogenic effect, which is consistent with the known working mechanism of TMX (Jobling and Sumpter, 1993; Liu et al., 2007). TBC is a persistent environmental pollutant with great concern due to its potential anti-estrogenic effect. In current study, 1 μM and 5 μM TBC completely suppressed the E2-induced vtg1 transcription in zebrafish embryos, while single TBC exposure showed no anti-estrogenic effect. Similar result was obtained when using male adult zebrafish, where TBC significantly slowed down the increase of relative fatness, sperm development retardation and vtg1 expression in liver which caused by E2 (Zhang et al., 2011). Therefore, the vtg1 level in zebrafish embryos could also reflect the anti-estrogenic effect caused by estrogen antagonists. Using E2 as a reference, we further performed a parallel comparison between our results and other studies using different models (Gutendorf and Westendorf, 2001; Segner et al., 2003). The RP value for the estrogenic effect of tested chemicals indicated that zebrafish embryo as an in vivo model is superior to in vitro models. In current study, the RP
5. Conclusions In summary, this study suggests an approach using zebrafish embryo and affordable quantitative PCR to assess estrogenic or the antiestrogenic effect of certain compounds. As a complex multi-cellular system, zebrafish embryo is facile to manipulate and relatively inexpensive. Therefore, the vtg1 gene measurement assay in zebrafish embryos could be a valuable alternative method for of the screening EDCs including both environmental estrogens and anti-estrogens. Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21477049), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030501) and the State Key Laboratory of Freshwater Ecology and Biotechnology (Grant No. 2014FB07). References Chow, W.S., Chan, W.K., Chan, K.M., 2013. Toxicity assessment and vitellogenin expression in zebrafish (Danio rerio) embryos and larvae acutely exposed to bisphenol A, endosulfan, heptachlor, methoxychlor and tetrabromobisphenol A. J. Appl. Toxicol. 33 (7), 670–678. Coezy, E., Borgna, J.L., Rochefort, H., 1982. Tamoxifen and metabolites in MCF7 cells: correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res. 42 (1), 317. Diamantikandarakis, E., Bourguignon, J., Giudice, L.C., Hauser, R., Prins, G.S., Soto, A.M., Zoeller, R.T., Gore, A.C., 2009. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr. Rev. 30 (4), 293–342. Gaido, K.W., Leonard, L.S., Lovell, S., Gould, J.C., Babai, D., Portier, C.J., Mcdonnell, D.P., 1997. Evaluation of chemicals with endocrine modulating activity in a yeastbased steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 143 (1), 205–212. Gutendorf, B., Westendorf, J., 2001. Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology 166 (1), 79–89. Head, K.A., 1998. Estriol: safety and efficacy. Altern. Med. Rev. 3 (2), 101. Hendry, W.J., Weaver, B.P., Naccarato, T.R., Khan, S.A., 2006. Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle. Reprod. Toxicol. 21 (3), 225–240.
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Henry, T.B., Mcpherson, J.T., Rogers, E.D., Heah, T.P., Hawkins, S.A., Layton, A.C., Sayler, G.S., 2009. Changes in the relative expression pattern of multiple vitellogenin genes in adult male and larval zebrafish exposed to exogenous estrogens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 154 (1), 119–126. Iso, T., Watanabe, T., Iwamoto, T., Shimamoto, A., Furuichi, Y., 2006. DNA damage caused by bisphenol A and estradiol through estrogenic activity. Biol. Pharm. Bull. 29 (2), 206–210. Jobling, S., Sumpter, J.P., 1993. Detergent components in sewage effluent are weakly oestrogenic to fish: an in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 361–372. Kuhn, D.M., Balkis, M., Chandra, J., Mukherjee, P.K., Ghannoum, M.A., 2003. Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J. Clin. Microbiol. 41 (1), 506–508. Lam, C.S., Korzh, V., Strahle, U., 2005. Zebrafish embryos are susceptible to the dopaminergic neurotoxin MPTP. Eur. J. Neurosci. 21 (6), 1758–1762. Legler, J., Dennekamp, M., Vethaak, A.D., Brouwer, A., Koeman, J.H., Van Der Burg, B., Murk, A.J., 2002. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Sci. Total Environ. 293 (1–3), 69–83. Li, S., Hursting, S.D., Davis, B.J., Mclachlan, J.A., Barrett, J.C., 2003. Environmental exposure, DNA methylation, and gene regulation. Ann. N. Y. Acad. Sci. 983 (1), 161–169. Liu, C., Du, Y., Zhou, B., 2007. Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes. Aquat. Toxicol. 85 (4), 267–277. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2–ΔΔCT method. Methods 25 (4), 402–408. Melamed, M., Castaño, E., Notides, A.C., Sasson, S., 1997. Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol. Mol. Endocrinol. 11 (12), 1868–1878. Mitsui, N., Tooi, O., Kawahara, A., 2003. Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 [beta] effects on whole animals and primary-cultured hepatocytes. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 135 (3), 305–313. Moriyama, K., Tagami, T., Akamizu, T., Usui, T., Saijo, M., Kanamoto, N., Hataya, Y., Shimatsu, A., Kuzuya, H., Nakao, K., 2002. Thyroid hormone action is disrupted by bisphenol A as an antagonist. J. Clin. Endocrinol. Metab. 87, 5185–5190. Muncke, J., Eggen, R.I., 2006. Vitellogenin 1 mRNA as an early molecular biomarker for endocrine disruption in developing zebrafish (Danio rerio). Environ. Toxicol. Chem. 25 (10), 2734–2741. Okuzawa, K., 2002. Puberty in teleosts. Fish Physiol. Biochem. 26 (1), 31–41. Pawlowski, S., Islinger, M., Volkl, A., Braunbeck, T., 2000. Temperature-dependent vitellogenin-mRNA expression in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes at 14 and 18 °C. Toxicol. in Vitro 14 (6), 531–540. Richter, C.A., Taylor, J.A., Ruhlen, R.L., Welshons, W.V., Saal, F.S., 2007. Estradiol and bisphenole A stimulate androgen receptor and estrogen receptor gene expression in fetal mouse prostate mesenchyme cells. Environ. Health Perspect. 115, 902–908. Riu, A., Grimaldi, M., Maire, A.L., Bey, G., Phillips, K., Boulahtouf, A., Perdu, E., Zalko, D., Bourguet, W., Balaguer, P., 2011. Peroxisome proliferator-activated receptor γ is a target for halogenated analogs of bisphenol A. Environ. Health Perspect. 119, 1227–1232.
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Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Evaluating estrogenic and anti-estrogenic effect of endocrine disrupting chemicals (EDCs) by zebrafish (Danio rerio) embryo-based vitellogenin 1 (vtg1) mRNA expression
T
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Minjie Chena,b, Jie Zhangb, , Shaochen Pangc, Chang Wangc, Ling Wangc, Yonghua Sund, ⁎⁎ Maoyong Songe, Yong Lianga,c, a
School of Medicine, Jianghan University, Wuhan 430056, PR China College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, PR China Institute of Environmental Health, Jianghan University, Wuhan 430056,PR China d State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China e State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Zebrafish embryo Vitellogenin 1 Estrogenic effect Endocrine disrupting chemicals
By measuring the vitellogenin 1 (vtg1) expression through quantitative PCR and in situ hybridization, we used the zebrafish embryo as an in vivo model to access the estrogenic or anti-estrogenic effects of several endocrine disrupting chemicals (EDCs), such as natural estrogen 17β-estradiol (E2), estriol (E3), synthetic hormones including diethylstilbestrol (DES), 4-octyl phenol (OP), bisphenol A (BPA), tamoxifen (TMX) and 3-(2,3-dibromopropyl) isocyanurate (TBC). According to our data, the estrogenic effect of the tested chemicals was ranked as: DES > E2 > E3 > OP > BPA, which is consistent with various in vivo and in vitro models. Therefore, the measurement of vtg1 gene expression in zebrafish embryos would be a valuable method for screening EDCs including both environmental estrogens and anti-estrogens.
1. Introduction Endocrine disrupting chemicals (EDCs) are a class of exogenous substance or mixture that alter the normal function of the endocrine system (Diamantikandarakis et al., 2009). Nowadays EDCs are of great concern to the health issue of human and wildlife. Be natural or manmade, increasing numbers of chemicals with estrogenic or anti-estrogenic effects have been defined as EDCs by using various in vivo or in vitro models. 17β-estradiol (E2) is a natural steroid estrogen with strong estrogenic activity to male zebrafish and rainbow trout (Oncorhynchus mykiss) juveniles (Van den Belt et al., 2003; Pawlowski et al., 2000). It has been widely used as a positive control compound in evaluating the estrogenic effects of some new pollutants. Estriol (E3), metabolite of estradiol and estrone, is also a natural estrogen in human body, which could bind to and acts as a weak agonist of estrogen receptor (ER) (Russo et al., 2001; Head, 1998). As a synthesized non-steroidal estrogenic substance, diethylstilbestrol (DES) has been proved to seriously threat human health through the food chain enrichment (Stefanick, 2005; Yin et al., 2006). With toxicity to development of
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fetus' reproductive organs, DES could increase the risk of reproductive system cancer during adulthood (Li et al., 2003; Hendry et al., 2006). 4octylphenol (OP) as a representative EDCs with estrogenic activity was reported to stimulate the proliferation of MCF-7 cell (White et al., 1994). With both weak estrogenic activity and strong anti-androgenic activity (Sohoni and Sumpter, 1998), bisphenol A (BPA) was reported to cause metabolic disorders in various organisms, by interacting with ER (Richter et al., 2007), thyroid hormone receptor (Moriyama et al., 2002) and peroxisome proliferator-activated receptor (Riu et al., 2011). Additionally, BPA exposure was identified to alter the early dorsoventral patterning, segmentation, and brain development in zebrafish embryos (Tse et al., 2013). Tamoxifen (TMX) as a pharmaceutical antagonist of the estrogen could competitively bind to ER in breast cells, playing an important role in the treatment of certain breast cancer and ovarian cancer (Wallen et al., 2005). Tris-(2, 3-dibromopropyl) isocyanurate (TBC), which is released into the environment during the use of flame retardants, is a potential EDC with anti-estrogen activity (Ruan et al., 2009; Zhang et al., 2011). The toxicity of EDCs and related molecular mechanism have been explored in multiple studies. Cultured
Corresponding author. Correspondence to: Y. Liang, School of Medicine, Jianghan University, Wuhan 430056, PR China. E-mail addresses: [email protected] (J. Zhang), [email protected] (Y. Liang).
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https://doi.org/10.1016/j.cbpc.2017.11.010 Received 20 September 2017; Received in revised form 28 November 2017; Accepted 28 November 2017 Available online 01 December 2017 1532-0456/ © 2017 Published by Elsevier Inc.
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and 10 μM of BPA. Co-exposure of TMX (0.5 and 1 μM) and E2 (0.5 μM) as well as TBC (1 and 5 μM) and E2 (0.5 μM) were also performed at the same time point. Additionally, zebrafish embryos were exposed to 0.5 μM E2 at several exposure window including 4 to 28 hpf (1d), 4 to 52 hpf (2d), 4 to 76 hpf (3d), 4 to 100 hpf (4d), 4 to 124 hpf (5d), 4 to 148 hpf (6d), 4 to 172 hpf (7d). 0.5% DMSO was used as vehicle control in each exposure experiment.
cell lines have been widely used, while in vivo models could better reflect the physiological and functional changes caused by EDCs. However, traditional animal models are costly, time-consuming, may have some ethic problems and individual differences (Kuhn et al., 2003; Legler et al., 2002). Therefore, it is necessary to develop a fast approach which could accurately identify EDCs and elucidate the potential molecular mechanism of these pollutants. There are several advantages by using zebrafish (Danio rerio) embryos in scientific studies. Due to the embryo transparency, zebrafish embryos are easily visualized and operated. It is also convenient to utilize their comprehensive heredity feature and different development stages for biological research (Lam et al., 2005). Vitellogenin (VTG) is the yolk precursor protein of vitellin (vn) in oviparous animals like zebrafish, which is predominantly synthesized in liver of mature female fish. VTG expression is regulated by luteinizing hormone and folliclestimulating hormone, also specifically by estrogens and anti-estrogens (Okuzawa, 2002). Previous studies have reported that VTG protein could be induced in male and larval fish in response to exogenous estrogen (Mitsui et al., 2003). Thus, VTG can be a good biomarker of estrogen exposure (Sumpter and Jobling, 1995). Additionally, vitellogenin 1 (vtg1) gene expression is the highest among the seven forms of vtg genes (vtg1-7) in zebrafish genome and has been proposed to be an indicating molecule for estrogenicity (Muncke and Eggen, 2006; Wang et al., 2005). The relative expression pattern of vtg1 gene can be induced by exogenous estrogens in adult male and larval zebrafish (Henry et al., 2009). For zebrafish embryos, vtg1 transcript usually occurs at 24 h post-fertilization (hpf) and at all later time points. It can be significantly up-regulated by 17α-ethinylestradiol, which also appears early at 24 hpf (Muncke and Eggen, 2006). Additionally, though vtg1 mRNA expression in adult zebrafish and adult zebrafish embryos has been used to test the estrogenic effects of emerging chemicals (Chow et al., 2013; Muncke and Eggen, 2006), assessment of the anti-estrogenic effect of certain compound by this model is so far lacking. This study primarily aimed to assess both the estrogenic or antiestrogenic effect of certain compounds by measuring zebrafish embryobased vtg1 mRNA levels. Multiple EDCs including E2, E3, DES, OP, BPA, TMX and TBC were investigated by single exposure as well as co-exposure experiment using zebrafish embryo. Vtg1 mRNA expression measurement assay clearly indicate an estrogenic response and antiestrogenic response by the testing chemicals. The analysis was furthermore verified through a comparison among different in vitro and in vivo experiment models. Therefore, our approach could be considered as an economic and promising evaluation for rapid testing and screening of multiple EDCs.
2.3. RNA extraction and real-time PCR (RT-PCR) Total RNA was extracted from forty pooled zebrafish embryos in each group by using Trizol reagent (Invitrogen), following the manufacturer's instructions. The yield and purity of extracted total RNA was determined by UV spectrophotometry (A260 and A260/A280 ratio). 2 μg of total RNA was used to synthesize cDNA by M-MLV (Moloney murine leukemia virus) RNase H reverse transcriptase (Promega, Fitchburg, WI, USA). Each PCR reaction mixture contained 1 μL cDNA template, 0.1 μmol/L primer, 7 μL water, and 10 μL 2 × SYBR QPCR Master Mix (Toyobo, Osaka, Japan). Reactions were performed in the Bio-Rad iQ5 thermal cycler equipped with a real-time fluorescence detector. The thermal cycling program consisted of a denaturing step (95 °C, 3 min) followed by 40 cycles of denaturation (95 °C for 10 s), annealing (55 °C for 10 s), and extension (72 °C for 20 s). The PCR primers were designed with Primer Premier 5.0 (Premier, Palo Alto, CA, USA): vtg1 forward 5′-AGCTGCTGAGAGGCTTGTTA-3′; vtg1 reverse 5′-GTCCAGGATTTCCCTC AGT-3′; β-actin forward 5′-GTCACACCATCA CCAGAGTCCATCAC-3′; β-actin reverse 5′-CAACAGAGAGAAGATGAC ACAGATCA-3′. β-actin was used as the internal standard. The target gene (vtg1) expression level was calculated by 2–ΔΔCT method (Livak and Schmittgen, 2001) and normalized by β-actin. 2.4. In situ hybridization Whole-mount in situ hybridization was performed using digoxigenin (DIG)-labeled antisense RNA probes and an anti-DIG alkaline phosphatase-conjugated antibody as described (Thisse et al., 2004). The vtg1 probe was synthesized by T3 polymerase (Promega, Fitchburg, WI, USA) using vtg1 (GenBank Accession Number of NM_001044897) PCR product as the templates, which were amplified from cDNA of 500 nM E2 exposed zebrafish embryo using a set of primers 5′-CAGCAGTCG TAACAGTCGC-3′ and 5′-GATCCATTAACCCTCACTAAAGGGAACTCCG CACC CCAAGAAA-3′, which contained T3 promoter sequence (underlined). 2.5. Statistical analyses
2. Materials and methods Statistical analyses were performed in SPSS 16.0 for Windows (IBM Corp. (2006), Somers, NY, USA). Values are presented as group means ± standard error (SE). One-way analysis of variance (ANOVA) was used to analyze the significant difference between the control and treatment groups, with post hoc analysis by Fisher's least significant difference (LSD) test. P values < 0.05 were considered statistically significant, P values < 0.01 were considered very significant.
2.1. Test compounds E2 (≥98% purity), E3 (≥ 97% purity), DES (≥ 99% purity), OP (99% purity), BPA (≥ 99% purity), TMX and TBC (97% purity) were purchased from Sigma-Aldrich (St Louis, MO, USA). All chemicals were dissolved in dimethyl sulfoxide (DMSO, Amresco, Solon, OH, USA) and diluted into fish water.
3. Results 2.2. Zebrafish embryo and exposure experiment 3.1. Dose-dependent expression of endogenous vtg1 gene after E2 exposure in the zebrafish embryos
Zebrafish (wild-type, AB strain) were obtained from China Zebrafish Resource Center (http://zfish.cn; Wuhan, China). The fish were raised in a closed flow-through system with fresh water at 26 ± 1.5 °C under a 14 h light/10 h dark cycle, maintained according to the Zebrafish Book (Westerfield, 2000). Zebrafish embryos were exposed to E2, E3, DES, OP and BPA since 4 hpf for 7 days at series of concentrations as following: 0.05, 0.1, 0.25, 0.5 and 1 μM of E2; 0.1, 0.25, 0.5, 1 and 2.5 μM of E3; 0.05, 0.1, 0.25, 0.5 and 1 μM of DES, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM of OP; 0.5, 1, 2.5, 5
To determine the response of the vtg1 gene to E2 exposure at the zebrafish embryos levels, we primarily detected the transcriptionally expression levels of vtg1 by RT-PCR analysis. By referring to the data from a previous study (Wang et al., 2011), the zebrafish embryos were exposed to various concentrations of E2 for 7 days. A dose-dependent increase of vtg1 mRNA-expression occurred in zebrafish embryos following E2 exposure (Fig. 1A). The relative mRNA levels of vtg1 in the 46
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Fig. 1. Dose-dependent expression of vtg1 in exposed zebrafish embryos after E2 exposure. (A) Dose-dependent transcriptional expression of vtg1 in E2 exposed zebrafish embryos. (B) Temporal expression of vtg1 in the zebrafish embryos exposed to 0.5 μM E2. (C) Temporal and spatial expression of vtg1 in 0.5 μM E2 exposed zebrafish embryos, analyzed by the wholemount in situ hybridization. Vtg1 was transcriptionally expressed both in liver (arrow) and heart (triangle) tissue of zebrafish embryos until 4 day exposure. The percentages of embryos with positive signal were shown in the top right corner. All the data were normalized to DMSO-treated group and represented as mean value ± SE (n = 3). Significant differences between E2 exposure group and control group was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
0.25, 0.5 and 1 μM E2 treated groups were 34.7, 49 and 61.8 folds higher than that in the control group, respectively (P < 0.01). These results indicated the sensitive response of vtg1 transcription to the E2 exposure, which is consistent with previous study (Wang et al., 2011). Aimed to investigate the temporal expression pattern of vtg1 following E2 stimulation, we then analyzed the vtg1 mRNA levels in zebrafish embryos during a 7-day exposure of 0.5 μM E2. Comparing with the controls, increased vtg1 transcript (> 5 folds) was detected on the 6th day of E2 exposure, and was induced by 20 folds on the 7th day (Fig. 1B). The hybridization data indicated that 12.5% of embryos began to weakly transcript vtg1 in the liver and heart on the 4th day of 0.5 μM E2 exposure. The vtg1 levels then continued to be elevated by E2 with the exposure time, the percentage of vtg1-expressed embryos increased from 39.13% to 89.36% and finally 100% on the 5th, 6th and 7th exposure day respectively (Fig. 1C). Therefore, as only a small amount of embryos expressed little vtg1 only in the liver and heart on the 4th and 5th day of E2 exposure, RT-PCR analysis detected no significant vtg1 transcripts until the 6th exposure day. As the exposure time increased, vtg1 expression was strongly up-regulated both in the liver and heart tissue, indicating the temporal effect of E2 exposure (Fig. 1B and C). These results suggested that the vtg1 transcriptional expression is a sensitive and ideal biomarker for assessing the effect of E2 exposure in the zebrafish embryos.
estrogen E2, we further explored the feasibility of using vtg1 transcription to assess the estrogenic potentiality of other EDCs. Zebrafish embryos were exposed to series doses of several compounds including E3, DES, OP and BPA for 7 days, and the relative mRNA levels of vtg1 were determined by RT-PCR (Fig. 2). Comparing with the controls, exposure group with a P value < 0.05 was considered to have significant estrogenic effect. Therefore, the lowest observable effect concentration (LOEC) of each compound here was defined as their lowest dose to produce significant vtg1 induction. According to this definition, the LOEC of E2 was 0.25 μM, and the estrogenic effect was induced with the increasing dose of E2 (Fig. 2). Low doses of E3 caused no significant increase in vtg1 transcription, while obvious estrogenic effect occurred at the concentration of 2.5 μM (Fig. 2), confirming that E3 is a weak natural estrogen. DES caused significant estrogenic effect at a very low concentration of 0.1 μM, vtg1 expression was induced by > 800-fold in 0.25 μM DES-treated embryos (Fig. 2). Interestingly, the vtg1 induction slightly declined after exposure to high dosed DES (0.5 μM and 1 μM). However, though all the doses of OP showed a mild estrogenic effect to zebrafish embryos, there was no statistical difference between these exposure groups and the controls (Fig. 2). Additionally, no significant estrogenic effect appeared after exposed to the various concentrations of BPA (Fig. 2). Therefore, these results indicated the potency of known EDCs to induce vtg1 expression in zebrafish embryos, which reflected their estrogenic effects.
3.2. Assessing the estrogenic effects of estrogenic agonists by vtg1 transcription in the zebrafish embryos
3.3. Anti-estrogenic effects by TMX and TBC EDCs could either act as agonist or antagonist of ER, which shows
Based on the sensitively response of vtg1 expression to natural 47
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the LOEC of E2. Comparison of RP values between several experimental models including our zebrafish embryo test model, cell lines and in vitro binding assay was performed to evaluate estrogenic effect of the tested compounds (Table 1). Accordingly, the RP values of E2, E3 and DES in zebrafish embryos were 1, 0.1 and 2.5 respectively, which was comparable with other test models. Though no LOEC was obtained from the vtg1 response curve of OP, gentle estrogenic effect could be detected following the OP exposure. Therefore, their estrogenic effects were ranked as: DES > E2 > E3 > OP > BPA, which is consistent with other models listed in Table 1. These data suggested that zebrafish embryo-based vtg1 mRNA expression is an efficient model for evaluating estrogenic effect of certain chemicals. 4. Discussion This study aimed to provide substantiating data for zebrafish embryo-based testing strategy suitable for the assessment of estrogenic effects. We demonstrated the vtg1 gene dose-response relationship after 7-day exposure and co-exposure experiment using E2, E3 and known EDCs, which indicated the sensitivity of zebrafish embryo as an evaluation model of estrogenic effect as well as anti-estrogenic effect. In oviparous vertebrates, VTG protein is synthesized in liver and transported to ovaries via blood circulation. Since the main function of VTG is to provide nutrients for the egg development in mature female, there was very little VTG content in early embryonic development. In current study, the experimental results of qPCR and in situ hybridization show that E2 can induce vtg1 gene expression with a dose-response relationship in zebrafish embryos, which is consistent with known knowledge (Wang et al., 2011). And although the vtg1 mRNA was dominantly expressed in liver, it was detected in embryo heart by in situ hybridization. The vtg1 in heart tissue has already been reported, and its function is considered as to unload surplus intracellular lipids in cardiomyocytes for reverse triglyceride transportation (Yin et al., 2009). Therefore, E2 does induce vtg1 expression via some pathway in zebrafish embryos. In order to discuss whether zebrafish embryo-based vtg1 expression can be used as an indicator to evaluate estrogenic effects of certain compounds, here typical EDCs such as E2, E3, DES, OP, BPA, TMX and TBC were chosen to perform exposure experiment by using zebrafish embryos. Exposure of 0.5 μM and 1 μM E2 for 7 days strongly induces vtg1 expression in zebrafish embryo and the maximal induction is 61.8
Fig. 2. The estrogenic effects of estrogenic agonists were assessed by vtg1 expression in the zebrafish embryos. Zebrafish embryos were exposed by E2, E3, DES, OP and BPA at a serial of indicated concentrations for 7 days. All the data were normalized to DMSOtreated group and represented as mean value ± SE (n = 3). Significant differences between each exposure group and control group was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
estrogenic or anti-estrogenic effect. Therefore, to further evaluate the anti-estrogenic response ability of zebrafish embryo-based vtg1 expression, co-exposure test was employed in the zebrafish embryos for 7 days. TMX as an antagonist of ER, co-exposure (0.5 μM and 1 μM) with 0.5 μM E2 strongly inhibited the vtg1 induction effect caused by single E2 exposure (Fig. 3A). As an emerging EDC, single exposure of TBC induced no vtg1 transcript in the zebrafish embryos. However, the vtg1 mRNA levels were significantly decreased by co-exposure of TBC (1 μM and 5 μM) with 0.5 μM E2, when comparing with 0.5 μM E2 single exposure individuals (Fig. 3B). Thus our model could also be used to access the anti-estrogenic effect of certain compound by using coexposure test with E2. 3.4. Comparison between various models In order to obtain the relative potency (RP) value for the estrogenic effect of the compounds, the LOEC of each compound was divided by
Fig. 3. Anti-estrogenic effect of estrogen antagonist was assessed by vtg1 expression in the zebrafish embryos. (A) TMX inhibited vtg1 transcriptional expression stimulated by E2 treatment. Zebrafish embryos were exposed to E2 (0.5 μM) or TMX (0.5, 1 μM), and co-exposed to E2 (0.5 μM) and TMX (0.5, 1 μM) for 7 days. (B) TBC showed anti-estrogenic effect zebrafish embryos. Zebrafish embryos were exposed to E2 (0.5 μM) or TBC (1, 5 μM), and co-exposed to E2 (0.5 μM) and TBC (1, 5 μM) for 7 days. All the data were normalized to DMSOtreated group and represented as mean value ± SE (n = 3). Significant differences between the indicated groups was analyzed with one-way ANOVA, Fisher's LSD test (*P < 0.05, **P < 0.01).
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Table 1 Comparison between several experimental models to evaluate estrogenic effect of compounds. Compound
E2 E3 DES OP BPA Tamoxifen TBC a b c d
zebrafish embryo assay
LOEC (μM)
Relative potencya
0.25 2.5 0.1 NDb NDb N/A N/A
1 0.1 2.5 NDb NDb N/A N/A
Binding to ERαc
Binding to ERβc
MVLN cellsc
HGELN cellsc
E-Screenc
Yeast assayd
Carp VTG assayd
Trout VTG assayd
1
1
1
4.6 × 10− 4 8.1 × 10− 5
2.4 × 10− 3 3.1 × 10− 3
5.1 × 10− 4 6.5 × 10− 4
Relative potencya
1 0.07 1.75 7.0 × 10− 4 2.3 × 10− 4 0.023
1 0.26 1.3 6.5 × 10− 3 2.6 × 10− 3 0.054
1 0.083 1.25 8.33 × 10− 5 2.5 × 10− 5 8.33 × 10− 6
1 0.4 8 8.0 × 10− 4 1.9 × 10− 4 7.1 × 10− 7
1 0.071 2.5 1.0 × 10− 4 2.5 × 10− 5 4.0 × 10− 5
Ratio of LOEC of E2 to that of test compounds. Not detected. Gutendorf and Westendorf, 2001. Segner et al., 2003.
value of DES is higher than E2, confirming that DES is a compound with strong estrogenic-effect, which has not been identified by using in vitro models. In zebrafish embryo, the RP values of E3 and OP are lower than E2, indicating that their estrogenic effects are weaker than E2. According to the ranking of RP value for the testing compounds, the estrogenic effect of EDCs was accurately accessed by using zebrafish embryo-based vtg1 expression measurement. These results provided sufficient evidence for the feasibility of our model here to evaluate the estrogenic or anti-estrogenic effect of certain chemicals, which could be used to screen the emerged EDCs.
times compared to the control group. While E3 can significantly induce vtg1 transcription when the exposure dose increased to 2.5 μM. This is consistent with previous report that the ER affinity of E3 was weaker than that of E2 (Rodan and Martin, 2000; Melamed et al., 1997). DES is known to have strong estrogenic effects, which can disrupt the normal function of the endocrine system (Li et al., 2003; Hendry et al., 2006). Here at the concentration of 0.25 μM, DES exposure highly induced vtg1 expression, which is 895 times higher than that in the control group. However, vtg1 induction slightly declined when the DES exposure concentration increased to 0.5 μM and 1 μM. This might be due to the toxic effects of high dosed DES on zebrafish embryos. OP has been reported as an estrogenic compound, which is present in the environment and exerts reproductive as well as developmental toxicity to wildlife and humans (White et al., 1994). Our study showed that 7-day exposure of OP upon 0.25 μM could induce vtg1 expression in zebrafish embryos, but no statistical significance occurred. Therefore, zebrafish embryo may be less sensitive to OP than other species. Though BPA has a similar chemical structure as estrogen and DES, it binds to ER with much lower affinity (Gaido et al., 1997), this might be the reason for the low estrogenic effect of BPA. Therefore, BPA exposure within the dose range here could not cause significant increase of vtg1 mRNA expression in zebrafish embryos. This is consistent with known findings that BPA's estrogenic activity is 1000 times less than estradiol (Iso et al., 2006). Thus, the vtg1 gene expression in zebrafish embryo is a susceptive indicator for evaluating estrogenic effect of various compounds. TMX is a well-known competitive antagonist of estrogen (Coezy et al., 1982). Though TMX single exposure caused no significant increase of vtg1 level, adding of 0.5 μM and 1 μM TMX can strongly reverse the vtg1 induction caused by E2 exposure. Thus our results suggested that TMX could interfere with the E2 pathway in zebrafish embryo with the anti-estrogenic effect, which is consistent with the known working mechanism of TMX (Jobling and Sumpter, 1993; Liu et al., 2007). TBC is a persistent environmental pollutant with great concern due to its potential anti-estrogenic effect. In current study, 1 μM and 5 μM TBC completely suppressed the E2-induced vtg1 transcription in zebrafish embryos, while single TBC exposure showed no anti-estrogenic effect. Similar result was obtained when using male adult zebrafish, where TBC significantly slowed down the increase of relative fatness, sperm development retardation and vtg1 expression in liver which caused by E2 (Zhang et al., 2011). Therefore, the vtg1 level in zebrafish embryos could also reflect the anti-estrogenic effect caused by estrogen antagonists. Using E2 as a reference, we further performed a parallel comparison between our results and other studies using different models (Gutendorf and Westendorf, 2001; Segner et al., 2003). The RP value for the estrogenic effect of tested chemicals indicated that zebrafish embryo as an in vivo model is superior to in vitro models. In current study, the RP
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