In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish

Toxicology and Applied Pharmacology 248 (2010) 217–225

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish Lingli Sun a,b, Wei Xu b, Jiangyan He b, Zhan Yin b,⁎ a b

Graduate University of Chinese Academy of Sciences, Beijing, 100049, PR China Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, PR China

a r t i c l e

i n f o

Article history: Received 20 May 2010 Revised 29 July 2010 Accepted 14 August 2010 Available online 21 August 2010 Keywords: Endocrine disruption Zebrafish POMC:EGFP transgenic line Interrenal development

a b s t r a c t Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMCencoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo–pituitary–adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effects on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4 days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3′,4,4′5pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg (POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling. © 2010 Elsevier Inc. All rights reserved.

Introduction The POMC gene is predominantly expressed in the anterior and intermediate lobes of the pituitary as well as in several other tissues including the brain, lymphocytes, skin, testis, thyroid, gut, kidney, and liver. It is generally accepted that the vast majority of POMC-derived hormones found in the circulation are synthesized by the pituitary. The POMC gene encodes a polypeptide precursor that undergoes extensive, “tissue-specific” posttranslational modification to generate a range of smaller and biologically active peptides, including adrenocorticotropin (ACTH) and α-, β-, γ-melanocyte-stimulating hormone. For instance, the processing of POMC in anterior pituitary corticotrophs results in the generation of pro-γ-MSH, ACTH, and β-LPH (Bicknell, 2008). These POMC-derived hormones regulate many physiological processes including steroidogenesis, skin pigmentation, food intake, and energy balance (Coll et al., 2004, 2005; Coll and Tung, 2009). ACTH is the most important pituitary-derived peptide-controlling steroidogenesis

⁎ Corresponding author. Fax: + 86 27 6878 0069. E-mail address: [email protected] (Z. Yin). 0041-008X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2010.08.015

in the adult adrenal gland (Hinson and Raven, 2006; Coll and Tung, 2009). ACTH is released into the blood to stimulate the adrenal cortex to produce and secrete glucocorticoids, whereas glucocorticoids produce negative feedback inhibition at the level of both the pituitary and hypothalamus to reduce ACTH output (Fig. 1; To et al., 2007; Raff and Jacobson, 2007). The expression of POMC is believed to be regulated by hypothalamic corticotrophin-releasing hormone and arginine vasopressin. This cascade is known as the HPA axis and is self-regulated by negative feedback from cortisol in both the hypothalamus and pituitary. The anterior pituitary expression domain of POMC precisely regulated HPA signal cascades, which renders the measurement of POMC (or ACTH) expression in the anterior pituitary a potentially useful marker of adrenocortical steroid production and adrenocortical competency. Numerous environmental contaminants and commercial products have the potential to disrupt the endocrine system in humans and wildlife. These chemicals have the potential to cause an extremely wide range of adverse health effects, including reproductive difficulties, tumor development, cell/tissue differentiation and growth issues, and defects in steroidogenesis (Waring and Harris, 2005). In teleosts, steroidogenic cells and intermingled chromaffin cells are embedded in the head kidney forming the interrenal organ, which is homologous to

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Fig. 1. Primary components in the hypothalamo–pituitary–adrenal axis cascade and their feedback regulation. The structures involved in this system include the hypothalamus, the anterior pituitary and the adrenal gland. Anterior pituitary POMC expression is stimulated by hormones secreted by the hypothalamus. Active ACTH is then secreted from the anterior pituitary corticotrophs to promote and sustain regular adrenal steroidogenesis (shown in green arrows). Inhibition of the anterior pituitary POMC expression by the glucocorticoids (such as exogenous dexamethasone) is accomplished by two mechanisms: intermediate feedback regulation to the anterior pituitary and delayed feedback regulation of the hypothalamus (shown in broken lines). Stimulation of anterior pituitary POMC expression by adrenostatic compounds (aminoglutethimide) is achieved by feedback regulation of the hypothalamus (blue arrow), which is caused by impaired adrenal steroidogenesis resulting from treatment (broken blue line). POMC, propiomelanocortin; ACTH, adrenocorticotropin.

the mammalian adrenal gland (Grassi Milano et al., 1997). Zebrafish, Danio rerio, has been shown as an ideal vertebrate model for studying dynamic gene expression in vivo. Previous evidence has demonstrated that the development of zebrafish interrenal glands resembles adrenal development in higher vertebrates (Chai et al., 2003). The zebrafish POMC has been characterized and is expressed mainly in brain and pituitary tissues (Gonzalez-Nunez et al., 2003). It was proven that the expression of green fluorescent protein (GFP) driven by the zebrafish POMC promoter resembles the pattern of endogenous POMC expression in the transgenic fish (Liu et al, 2003). This transgenic fish also demonstrated that zebrafish corticotroph development shares basic conserved mechanisms with development in higher vertebrates (Liu et al., 2003; Liu, 2007). The aim of the current study was to investigate an in vivo screening assay using Tg (POMC:EGFP) zebrafish to prioritize chemicals that would interfere with anterior pituitary POMC expression and fish interrenal steroidogenesis. Our results showed that Tg (POMC:EGFP) fish can be used to assess the effects of Dex, AG, simazine, and PCB126 on anterior pituitary POMC expression and early interrenal development. Our results also suggested that the Tg (POMC:EGFP) zebrafish line could be used as an in vivo screening method to identify chemicals with potential endocrine-disrupting effects on POMC expression and adrenal steroidogenesis. Materials and methods Fish maintenance and breeding. Zebrafish were kept at 28.5 °C with a light–dark cycle of 14:10 hours. All fish were fed three times per day, twice with freshly hatched brine shrimp, and once with commercial flake (Tetra). About 5% of system water was replaced with filtered tap water daily. Water quality parameters such as pH and salinity were monitored daily. A pair of female and male fish was placed in a 3-L fish tank after the last feed of the day, and eggs were collected and kept in egg water with 60 mg/L of “Instant Ocean” salt in a clean Petri dish at 28.5 °C, according to the protocol described by Westerfield (1995).

Production of transgenic fish. The zebrafish POMC:EGFP transgenic construct was previously described (Liu et al., 2003). A 1006-bp fragment amplified from zebrafish genomic DNA containing the entire exon 1 and the first 22 bp of exon 2 of the zebrafish POMC gene (−451 to 60 bp within the POMC locus) was amplified and cloned into pEGFP-N1 (Clontech) to generate the POMC:EGFP reporter construct. The POMC:EGFP reporter construct was linearized with the restriction enzyme BglII and injected into one- or two-cell stage fish embryos at a concentration of 100 μg/ml, as previously described by Ju et al. (1999). Expression of EGFP was observed and photographed under a Leica fluorescence stereomicroscope. The injected embryos showing EGFP expression were raised to adulthood. Individual founder fish were crossed with each other or with wild-type fish, and their offspring embryos were observed under a fluorescence microscope to examine EGFP expression in the pituitary. The presence of the transgenic construct was verified in the genomic DNA of founder fish by PCR. F1 fish from each transgenic founder fish were generated through crosses with wild-type fish and were maintained in our laboratory (Westerfield, 1995). Whole-mount in situ hybridization. Zebrafish POMC was amplified and cloned into pGEMT vector according to the procedure described previously (Liu et al., 2003). The zebrafish ff1b clone was obtained from Dr. Chan at the National University of Singapore (Chai et al, 2003). These plasmids were linearized with restriction enzyme, and antisense probes were synthesized by in vitro transcription reactions. Whole-mount in situ hybridization using digoxigenin (DIG)-labeled riboprobes was carried out as previously described (Li et al., 2009). Briefly, the embryos/larvae up to 5-days post-fertilization (dpf) were fixed in 4% paraformaldehyde in PBS, hybridized with DIG-labeled riboprobe in hybridization buffer [50% formamide, 5 × SSC (1× = 15 mM NaCl, 15 mM sodium citrate, pH 7.6), 50 mg/ml tRNA and 0.1% Tween] at 70 °C, incubated with anti-DIG antibody conjugated to alkaline phosphatase and stained with nitroblue phosphate (NBT) and 5-bromo, 4-chloro, 3-indolil phosphate (BCIP) to obtain purple and insoluble precipitates. Chemical treatment. The chemicals used in this study are listed as follows: dexamethasone (Sigma, D2915), aminoglutethimide (Sigma, A9657), simazine (Fluka, 32059), bisphenol A diglycidyl ether (BADGE, Sigma, SA-D3415), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126, AccuStandard, B3040278), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, Cambridge Isotope Laboratories, ED-901-B), dichlorvos (Dr. Ehrenstofer Gmbh, 2530000), malathion (Dr. Ehrenstofer Gmbh, 4710000), parathion-methyl (Dr. Ehrenstofer Gmbh, 15890000), demeton (Dr. Ehrenstofer Gmbh, 12140000), trichlorfon (Dr. Ehrenstofer Gmbh, 17680000), and tarathion-ethyl (Dr. Ehrenstofer Gmbh, 15880000). PCB126 solution (100°μg/ml in issoctane), Simazine, and trichlorofon were dissolved in sterile distilled water to make stock solutions. AG was dissolved in 0.2 M HCl to make a 0.2 M stock solution, and when the stock solution was applied to embryos, about 0.4 L of 1 M HEPES solution (pH 8.4) was added to every 1 L of the AG stock solution to adjust the pH back to neutral. The rest of the chemicals were either dissolved or diluted with DMSO to the desired concentration as stock solutions. The final chemical concentrations used in this study are listed in Table 1. Although the vehicle solution represented 0.01–0.5% of the egg water and had no visible effects on embryonic development, all experiments were performed side by side with test and vehicle solutions alone. All treatments began at 24 hours post-fertilization (hpf) and continued for 4 days, 50% of the test solution was renewed daily. The embryos were observed under the microscope to determine if any obvious developmental defects were caused by the toxicity of those chemicals. No observable adverse effect on fish development concentration (NOAEC) was observed for any chemical (Table 1). The average proportion of larvae responding for a given endpoint was

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Table 1 Concentrations determined for each chemical exposure. Chemical

NOAECa

Dexamethasone (Dex) (μM)

100

Aminoglutethimide (AG) (mM) Simazine (mg/L) 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) (nM) Bisphenol A diglycidyl ether (BADGE) (mg/L) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (ng/L) Dichlorvos (mg/L) Malathion (mg/L) Parathion-methyl (mg/L) Parathion-ethyl (mg/L) Demeton (mg/L) Trichlorfon (mg/L) a b

1 10 400 5.0 200 0.2 1.6 4 3 2 10

Concentrations used for POMC:EGFP observation

EC50b

–

Suppression of anterior pituitary POMC expression in zebrafish at 40 μM (To et al., 2007) or 50 μM (Liu et al., 2003) Late zebrafish larval ff1b morphant phenotypes are phenocopied by AG treatment at 1 mM (Chai et al., 2003). 96 h LC50 for silver catfish (Rhamdia quelen) was 8.9–12.4 mg/L (Kreutz et al., 2008) Zebrafish embryos treated with 100 nM PCB126 exhibited multiple development defects (Na et al., 2009) Inhibition activity of vtg induction in carp (Cyprinus carpio) at 5.5 μM (1.87 mg/L) (Letcher et al., 2005) N 10 nM caused pericardial edema in zebrafish (Seok et al., 2008)

– – – – – –

96 h LC50 to zebrafish was 0.47 mg/L (Zhang et al., 2010) N 1.5 mg/L caused heart edema in zebrafish larva (Fraysse et al., 2006) 96 h LC50 to Pimephales promelas was 5.4 mg/L (Roex et al., 2002) 96 h LC50 to zebrafish was 1.9 mg/L (Roex et al., 2002) 28 d EC50 to rainbow trout (Oncorhynchus mykiss) was 20 μg/L (Arnold et al., 1996) 96 h LC50 to medaka (Oryzias latipes) was 17.6 mg/L (Yoshimura and Endoh, 2005)

50

35

0.2 1 100 1.0 200 0.2 1.0 2 2 1 10

Previous toxicology study on its effects and concentration

0.2 0.8 100 –

NOAEC, no observable adverse effect concentration on fish development as determined by our 4-day embryo–larval treatment starting from 24 hpf. EC50 for the effects on POMC expression.

calculated for each dose. Effective concentrations at 50% (EC50) were calculated from a linear regression of log-probit transformations of the dose–response data. The expression patterns presented here were taken as the representative images at the EC50. For those treatments that did not clearly affect POMC expression, the exposure concentrations of each chemical were increased gradually until obvious development effects were observed (Table 1). Results The plasmid constructs were linearized with the restriction enzyme BglII and injected into embryos at the one- or two-cell stage at a concentration of 100 μg/ml. The transgenic founders (F0) were screened based on the pituitary eGFP expressed, as observed with a fluorescence microscope. As a result, we obtained 2 F0 fish that exhibited pituitary EGFP expression and transmitted this gene in the germ line. Insertion of the POMC:EGFP reporter construct has been confirmed by PCR performed with genomic DNA samples. These founders were then used to produce the F1 generation. The rate of transgene transmission to the F1 generation varied between 7–12%. As with previous observations (Liu et al., 2003), the eGFP expressed in germ-line transgenic zebrafish is present in both anterior and posterior pituitary corticotroph groups, but not in melanotrophs (Figs. 2A and B). This suggested that the promoter region chosen for our Tg (POMC:EGFP) reporter zebrafish contains the appropriate putative regulatory elements required for POMC expression in the anterior and posterior lobes of the pituitary. Dex and AG are potent synthetic adrenergic agents. To examine the POMC:EGFP expression response to Dex in our transgenic reporter zebrafish, live Tg (POMC:EGFP) zebrafish were treated continuously with 50 μM Dex starting from 24 hpf. By 5 dpf, there was a significant reduction in EGFP in the anterior group of cells (Fig. 2C, arrow). Wholemount in situ hybridization using zebrafish POMC riboprobe on zebrafish treated with Dex also showed selective suppression of POMC expression in the anterior group of corticotrophs (Fig. 2G, arrow), with no effect on posterior corticotrophs (Figs. 2C and G). Our live Tg (POMC: EGFP) zebrafish were treated with 0.2 mM AG starting from 24 hpf. By 5 dpf, there was a significant induction of EGFP expression in the anterior group of cells (Fig. 2D, arrow). Elevated anterior pituitary POMC expression was also confirmed by whole-mount in situ hybridization using a zebrafish POMC riboprobe (Fig. 2H, arrow). Zebrafish ff1b is a homologue of mammalian steroidogenic factor-1 (SF-1). It is one of the earliest molecular markers and required for the

development of the steroidogenic tissue of teleost interrenal organs (Chai et al., 2003). With the zebrafish ff1b riboprobe, we have demonstrated the down-regulation of ff1b expression in zebrafish treated with either Dex or AG (Figs. 2J and K). This suggested that the impaired interrenal development resulted from the exogenous Dex or AG treatment. Certain chemicals are able to interfere with normal endocrine function by altering the steroidogenic process, as determined previously by adrenal toxicological tests. These include simazine, PCB126, BADGE, and TCDD (Hinson and Raven, 2006; Hecker et al., 2006; Villeneuve et al., 2007; Harvey et al., 2007). To examine the effects of these chemicals on POMC:EGFP expression in our transgenic fish as well as endogenous POMC expression in zebrafish, Tg (POMC: EGFP) reporter fish and wild-type zebrafish were treated with those chemicals for 4 days starting at 1 dpf (Table 1). By 5 dpf, no obvious developmental defects in larvae were seen except the pericardiac edema observed in the 50% of larvae in the group treated with TCDD at 200 nM (Figs. 3A–F). However, suppression of the anterior pituitary POMC:EGFP in transgenic zebrafish was observed with simazine or PCB126 treatment (Figs. 3G and H, arrow). The down-regulation of endogenous POMC expression in the anterior pituitary corticotrophs of wild-type zebrafish was also confirmed with the whole-mount in situ technique (Figs. 4C and D, arrow). Using ff1b in situ hybridization, we also found that zebrafish interrenal development was impaired after simazine and PCB126 treatment (Figs. 5C and D, arrow). No significant change was observed among the reporter fish treated with BADGE and TCDD with regard to the expression of POMC:EGFP (Figs. 3I and J) or endogenous POMC mRNA in the anterior pituitary (Figs. 4E and F). BADGE and TCDD caused no significant effects on interrenal development although obvious developmental retardation had been seen after either exposure treatment (Figs. 5E and F, arrow). Many organophosphorus compounds, such as dichlorvos, malathion, parathion-methy, parathion-ethyl, demeton, and trichlorfon, are commonly used OPs in China. In our present studies, the Tg (POMC:EGFP) transgenic zebrafish were also used to detect the potential adrenergic activities of these organophosphorus compounds. However, based on our fluorescent microscopy and whole-mount in situ hybridization assays on the POMC:EGFP transgenic zebrafish treated with the organophosphorus, no obvious effects on anterior pituitary POMC expression have been observed (Figs. 6C–N). Although a fan-shaped malformation of the POMC mRNA expression pattern was visualized by in situ hybridization in the melanotroph domains during treatment with demeton (Fig. 6J), compared with a double-arch-shaped pattern of the melanotroph domains in control group (Fig. 6B). However, the eGFP

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Fig. 3. EGFP expression in transgenic POMC:EGFP fish in response to chemicals . (A, B, C, D, E, F) Views with light maicroscope on the larvae of the isooctane control (167 ppm), DMSO control (167 ppm), and treatments with 1 mg/L simazine, 100 nM PCB126 in 0.02% isooctane solution, 1.0 mg/L BADGE in 0.02% DMSO, and 200 ng/L TCDD in 0.02% DMSO solution. (G, H, I, J) Anterior pituitary GFP expression (arrow) in POMC:EGFP transgenic fish at 5 dpf after 4-day exposure to the corresponding control solution (right) and treatments (left) with 1 mg/L simazine, 100 nM PCB126, 1.0 mg/L BADGE, and 200 ng/L TCDD (for more details, see Materials and methods). A–F, lateral views; G–J, ventral views. Pericardiac edema (red arrow).

expression in the Tg (POMC:EGFP) line only recapitulated that of endogenous anterior pituitary POMC expression. However, based on the theory of the ‘tissue-specific processing’ of endogenous POMC, these observations have suggested that the effects of OPs are not likely due to the interference of ACTH in HPA cascades. Discussion A great amount of attention has been paid to the estrogenic or antiandrogenic properties of these endocrine-disrupting chemicals (EDCs) and their subsequent effects on gender phenotype and reproductive capability. In contrast, little effort has been made to investigate the underlying mechanisms involved in the adverse influences of environmental EDCs on many other interference effects in endocrine systems other than the reproductive system (Hinson and Raven, 2006; Harvey et al., 2007). The adrenal is a relatively neglected organ in aquatic endocrine toxicology in spite of its importance in the function and overall responses of the adrenal gland to toxic chemicals (Harvey et al., 2007). Over the past decade, numerous studies focused

on the estrogenic toxicity of EDCs and utilized the teleost vitellogenin (VTG) assay. However, our recent work demonstrated the effects of adrenergic agents on the expression of teleost VTG, which indicated the need to reevaluate EDCs with regard to their potential adrenal toxicity (Yin et al., 2009). So far, there are no standard assessment assays for the effects of endocrine-disrupting chemicals on adrenal steroidogenesis and cortex development. Measurements of the relative adrenal organ weights and ACTH levels in plasma are normally utilized in the evaluation of adrenal-associated toxicities (Hinson and Raven, 2006; Harvey et al., 2007). An in vitro steroidogenesis assay using H295R human adenocarcinoma cells has also been suggested as a possible Tier I screening assay. However, the agreement of this in vitro assay with the real effects in animal models has always been a concern for endocrine-disrupting toxicological studies (Hecker et al., 2006; Villeneuve et al., 2007). As with the ‘tissue-specific processing’ of endogenous POMC suggested in rodents (Bicknell, 2008), the anterior domain of the pituitary gland is also the key compartment of POMC-expressing cells involved in the production of ACTH and functional regulation of

Fig. 2. Endogenous POMC expression and EGFP expression in transgenic POMC:EGFP fish in response to adrenergic agents. (A and B) Anterior and posterior pituitary EGFP-positive cells at 48 hpf and 5 dpf. (C) Anterior pituitary POMC-EGFP expression at 5 dpf is downregulated by 50 μM Dex (left, arrow). (D) Anterior pituitary POMC-GFP expression is upregulated by 0.2 mM AG (left, arrow) compared to the control embryo (right). (E, F, G, H) Endogenous POMC expression in the anterior pituitary at 5 dpf as shown by in situ hybridization (red arrow) after a 4-day treatment with medium plus 0.1% DMSO (E), egg water alone (F), egg water containing 0.1% DMSO and 50 μM Dex (G), and medium with 0.2 mM AG (H). (I–K) Endogenous ff1b expression in interrenal tissue at 72 hpf as shown by in situ hybridization after a 2-day treatment with egg water containing 0.1% DMSO (I), egg water containing 0.1% DMSO and 50 μM Dex (J), and egg water containing 0.2 mM AG (K). C–H, 5 dpf; I–K, 72 hpf; A–B and I–K, dorsal view; C–D, E–H, ventral view. Melanotrophs are indicated with blue arrows.

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Fig. 4. Endogenous POMC expression in response to chemical treatment. (A-F), Endogenous anterior pituitary POMC expression at 5 dpf after a 4-day chemical treatment (arrow): A, egg water alone; B, egg water with 0.5% DMSO; C, egg water with 1 mg/L simazine; D, egg water with 100 nM PCB126; E, egg water and 0.1% DMSO with 1.0 mg/L BADGE; F, egg water and 0.1% DMSO with 200 ng/L TCDD. A–F, 5 dpf, ventral view, with anterior to left.

zebrafish interrenal steroidogenesis (To et al., 2007). Our results have confirmed that the promoter selected for the regulation elements in this transgenic line contains the proper region for reporting endogenous anterior pituitary POMC mRNA expression (Fig. 2). This indicated that eGFP observation in transgenic fish could be applied as an ideal model for monitoring the defects in the HPA axis cascade caused by the alteration of adrenal steroidogenesis and ACTH levels in vivo. Notably, Dex is a synthetic member of the glucocorticoid class of steroid hormones. The pituitary adenoma has a feedback mechanism that has been reset to a higher level of cortisol. Exogenous Dex treatment could suppress anterior pituitary POMC expression in zebrafish via negative feedback at the level of the pituitary before the initiation of the control of interrenal steroidogenesis (Fig. 1 presents negative feedback regulation of POMC expression in broken lines; Liu et al., 2003, To et al., 2007). The adrenostatic compound AG is an inhibitor of de novo steroid synthesis. It acts mainly by inhibition of P450 scc, the first step in the steroidogenic pathway. This compound has been used for experimental adrenalectomy in previous studies. Normal corticotroph responses to the removal of glucocorticoid feedback resulted in strong increased basal plasma ACTH and anterior pituitary POMC gene expression (Fig. 1, with feedback regulation of AG shown in blue; Raff and Jacobson, 2007). Our results suggested that suppressed anterior pituitary POMC (ACTH) expression might be responsible for the interrenal defects due to Dex treatment, whereas the direct adrenostatic effect of AG causing impaired interrenal development resulted in enhanced POMC expression in anterior

Fig. 5. Endogenous ff1b expression in interrenal tissue after chemical treatment. (A–F) Endogenous ff1b expression in the interrenal tissue at 3 dpf as shown by in situ hybridization after a 2-day treatment with egg water only, egg water plus 0.1% DMSO, egg water with 1 mg/L simazine, egg water with 100 nM PCB126, egg water containing 0.1% DMSO plus 1.0 mg/L BADGE, egg water with 0.1% DMSO plus 200 ng/L TCDD (more details in Materials and methods). A–F, dorsal view.

pituitary corticotrophs. The levels of anterior pituitary POMC expression in zebrafish have been highly relevant to interrenal development and steroidogenesis, as reflected by interrenal ff1b expression in fish. Taken together, the expression of anterior pituitary eGFP in our transgenic reporter zebrafish exhibited proper responsiveness to these adrenergic agents. Therefore, measurement of the anterior pituitary EGFP expression driven by the POMC promoter reflecting the endogenous POMC mRNA expression domain in anterior pituitary corticotrophs could be a quick and powerful in vivo approach to detect the potential effects on critical signals in the HPA cascade of EDCs. Simazine (2-chloro-4,6-bis[ethylamino]-s-triazine) is a member of the triazine family of broad-leaved herbicides. It has been reported that simazine could cause increased expression of cyp19 in H295R cells, which encode aromatase, raising the possibility of increased adrenal estrogen secretion (Sanderson et al., 2000). Lead has also been reported to increase the expression of cyp11B2 in H295R cells treated with PCB126, which might enhance the final step of adrenal aldosterone biosynthesis (Li et al., 2004). There are some previous controversial observations on the toxic adrenal effects of BADGE and TCDD as well. BADGE is a potent inhibitor of VTG synthesis. It has been reported that the increased weight of the adrenal gland relative to the body with no significant histopathological changes in rats could be caused by BADGE treatment (Hyoung et al., 2007). However, no significant effect of BADGE on cyp19 aromatase activity has been

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observed in H295R cells (Letcher et al., 2005). Minor alterations in adrenal steroidogenesis and significant suppressed testicular steroidogenesis in rats caused by TCDD feeding at a dose of 1 μg/kg were observed (Adamsson et al., 2008). As seen in our studies, significant suppression of the anterior pituitary POMC and adverse effects on early zebrafish interrenal development were observed in simazine and PCB126 treatment groups but not in BADGE and TCDD treatment groups (Figs. 3–5). Both PCB126 and TCDD are well-known aryl hydrocarbon receptor (AhR)-binding toxicants. However, on the basis of the effect on animal hepatic gene expression analysis after PCB126 or TCDD treatment, it is possible that PCB126 could activate a unique group of genes not activated during TCDD exposure (Vezina et al., 2004). Further investigations using a variety of approaches, such as the treatment combined them with AhR antagonists, should be carried out in order to ascertain the interfere on POMC expression of PCB126 in AhR-dependent toxicity in developing zebrafish. OPs are the most widely used pesticides worldwide and their metabolites are widespread. The major adverse effects of exposure have been observed in the nervous system, and even more recently, their genotoxicity has been documented (Hreljac and Filipic, 2009). However, some OPs have been positively associated with certain developmental and reproductive effects. Moreover, some have been suggested as potent androgen receptor antagonists (Tamura et al., 2003). This emphasizes the need for further studies on the endocrine potential of the chemicals. Because OPs comprise a group of different chemical substances, it is not surprising that their potentials as EDCs could involve different modes of endocrine action. However, in our present studies, even when various developmental defects were observed in the zebrafish treated at the OP dosages, there was still no obvious and consistent alteration in the anterior pituitary eGFP and POMC expression (Fig. 6). The results suggested that the OPs tested in this study should not be considered as primary adrenal toxic compounds. In our current studies, a Tg (POMC:EGFP) reporter fish was employed to detect the interference effects on the HPA cascade. We have demonstrated that eGFP expression acts as a reliable and accurate indicator of cell-specific anterior pituitary POMC gene expression. EGFP expression in these fish faithfully mimics that of the endogenous anterior pituitary POMC genes during both normal development and chemical treatment conditions. The stable nature of this transgenic line should allow for rapid and reproducible anterior pituitary POMC expression profiling throughout development. Our results have also demonstrated that the anterior pituitary POMC results obtained in the test could be applied as useful indicators reflecting fish interrenal steroidogenesis activity. This approach was supported by its successful confirmation of known adrenergic agents, such as Dex and AG. The adrenal toxicity of simazine and PCB126 observed in this study might also reflect the effects reported previously (Harvey et al., 2007). The EC50 values of Dex, AG, simazine, and PCB126 for anterior pituitary POMC expression as determined in this study were significantly lower than their NOAECs (Table 1), which might also indicate the fact that the adrenal/interrenal gland is one of their primary targets with regard to endocrine disruption effects. Employed as sentinels for aquatic pollution, transgenic zebrafish can provide sensitive, economical, and practical biological monitors for compounds with specific activity. They should also be able to identify the specific biological effects even within a complex mixture. The approach adapted in our present studies was suitable for an in vivo qualitative Pier 1 screening test of EDCs with certain effects on HPA signaling. However, the POMC codes for multipolypeptide precursors that undergo tissue-specific posttranslational modification to generate smaller biologically active peptides other than ACTH, even in the anterior pituitary corticotroph. Other assessments such as adrenal histological studies and expression pattern analysis of key genes in adrenal development might be needed for further validation of the potential tissue toxicity of chemicals identified with this transgenic reporter. Regardless, the findings of this study indicated that the Tg (POMC:EGFP) zebrafish might at least allow for

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extremely rapid and reproducible toxicological profiling of the potential EDCs and their interference effects in the HPA cascade, especially with regard to anterior pituitary POMC (ACTH) expression in vivo, which might reflect adverse influences on interrenal/adrenal steroidogenesis. Acknowledgments This study was supported by grants from the Hi-Tech Research and Development Program of China (no. 2006AA06Z424) and the National Natural Science Foundation of China (nos. 90608017 and 20897013). References Adamsson, A., Simanalinen, U., Viluksela, M., Paranko, J., Toppari, J., 2008. The effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on foetal male rat steroidogenesis. Int. J. Androl. 32, 575–585. Arnold, H., Pluta, H.J., Braunbeck, T., 1996. Sublethal effects of prolonged exposure to disulfoton in rainbow trout (Oncorhynchus mykiss): cytological alterations in the liver by a potent acetylcholine esterase inhibitor. Ecotoxicol. Environ. Saf. 34, 43–55. Bicknell, A.B., 2008. 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Fig. 6. EGFP and endogenous POMC expression in transgenic zebrafish in response to organophosphorus compound treatments. (A, C, E, G, I, K, M) anterior pituitary GFP expression (arrow) in POMC:EGFP transgenic fish at 5 dpf after 4-day treatment with control solution, dichlorvos (0.2 mg/L), parathion-ethyl (2 mg/L), trichlorfon (10 mg/L), demeton (1 mg/L), parathionmethyl (2 mg/L), malathion (1 mg/L) (for more details, see Materials and methods). (B, D, F, H, J, L, N) Endogenous anterior pituitary POMC expression (arrow) detected by in situ hybridization in zebrafish at 5 dpf after 4-day treatment with control solution, dichlorvos (0.2 mg/L), parathion-ethyl (2 mg/L), trichlorfon (10 mg/L), demeton (1 mg/L), parathionmethy (2 mg/L), malathion (1 mg/L). A–N, ventral views. Melanotrophs are indicated with blue arrows.