Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations

Chemosphere 144 (2016) 1162–1169

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Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations Qiyu Chen a, Ai Jia b, Shane A. Snyder b, Zhiyuan Gong c, Siew Hong Lam a,c,∗ a b c

NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1, 117411, Singapore University of Arizona, 1133 E. James E. Rogers Way, Harshbarger 108, Tucson, AZ 85721-0011, USA Department of Biological Science, National University of Singapore, 14 Science Drive 4, 117543, Singapore

h i g h l i g h t s • • • • •

Zebrafish genes are responsive to glucocorticoids at environmental concentrations. Identified robust glucocorticoid-responsive genes for in vivo zebrafish assay. In vivo and in vitro detection of glucocorticoid activity in environmental sample. Analytical chemistry confirmed glucocorticoids in environmental sample. Combined approaches useful for environmental glucocorticoid monitoring.

a r t i c l e

i n f o

Article history: Received 13 July 2015 Received in revised form 21 September 2015 Accepted 23 September 2015 Available online 23 October 2015 Handling editor: David C. Volz Keywords: Environmental glucocorticoid contaminants In vivo zebrafish assay Gene expression In vitro glucocorticoid receptor bioassay

a b s t r a c t Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction

∗ Corresponding author. Department of Biological Sciences, S3-Level 5, 14 Science Drive 4, National University of Singapore, 117543, Singapore. E-mail address: [email protected] (S.H. Lam).

http://dx.doi.org/10.1016/j.chemosphere.2015.09.089 0045-6535/© 2015 Elsevier Ltd. All rights reserved.

Glucocorticoids are a group of steroidal compounds commonly prescribed for treatment of inflammation, allergies and autoimmune disorders. Depending on the therapy, type and preparation of the glucocorticoid drug, the daily average dose can vary from

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100 μg to 500 mg (Kugathas et al., 2012). Due to the large amount of glucocorticoid drugs being prescribed, they have found their ways into environmental waters through treated and untreated wastewater discharges (Fan et al., 2011; Brausch et al., 2012; Kugathas et al., 2012). In Beijing, China, total glucocorticoid levels of 0.9–3.4 ng/L have been detected in effluents of seven WWTPs, however, the total glucocorticoids of up to 4.8–9.0 ng/L detected in two of the receiving rivers suggested that glucocorticoids were also entering the rivers via untreated wastewater discharge (Chang et al., 2007). A follow up study of 45 river samples in Beijing region, detected total glucocorticoids up to 52 ng/L (Chang et al., 2009). Incomplete removal of glucocorticoids have been reported in Guangdong, China where glucocorticoids were detected at levels of 2.2–6.3 ng/L in the effluent of two WWTPs (Liu et al., 2012). Besides China, in vitro glucocorticoid activity of Dex-EQ at several ng/L range was detected in surface waters used for water production in the Netherland (Schriks et al., 2013). High levels of synthetic glucocorticoids were detected at 23000 ng/L (dexamethasone) and 300 ng/L (prednisone) in the effluent of an industrial WWTP in France and water samples from the receiving river were extrapolated to contain 1−2900 ng/L (dexamethasone) and 50– 1260 ng/L (6 α -methylprednisolone) (Creusot et al., 2014). Total glucocorticoid levels between 30 and 850 ng/L have been predicted in the best and worst case scenario, respectively, in River Thames, UK, based on a modelling approach (Kugathas et al., 2012). Prednisone at 20 ng/L was detected in the effluent of one WWTP and 7 ng/L of hydrocortisone was detected in one of the receiving surface water in Arizona, USA (Anumol et al., 2013). Recently, glucocorticoid activities of DEX-EQ to 16–24 ng/L were also detected in the effluents from two WWTPs and two processed water samples out of the nine water samples tested in Arizona, USA (Jia et al., 2015). The incomplete removal of glucocorticoids by wastewater treatments and their presence in environmental waters will inevitably expose aquatic organisms to glucocorticoids. This has led to the rising concerns on the biological effects of glucocorticoids at environmental concentrations. To detect glucocorticoid activity and provide biological perspective, cell-based in vitro bioassays with specific reporter genes fused to a promoter with glucocorticoid responsive elements have been used to detect glucocorticoid transcriptional activity in environmental samples (Van der Linden et al., 2008; Schriks et al., 2013; Creusot et al., 2014; Jia et al., 2015). Therefore, in vitro glucocorticoid receptor bioassays are able to provide specific biological activity information without a priori knowledge of glucocorticoids present. However, biological activity detected in vitro by cell-based assays does not necessary mean that similar in vivo biological activity will be detected in a whole organism. In other words, glucocorticoid transcriptional activity detected in vitro may not be recapitulated in vivo, and thus may lack biological relevance in wholeorganism. One of the major concern is that in vitro cell-based assays lack whole-organism organ-systems and physiological relevance, and therefore in vitro activity may not translate to in vivo effects (Bale et al., 2014). The zebrafish is a popular model for in vivo aquatic toxicology (Sipes et al., 2011) and environmental risk assessment (Scholz et al., 2008). Earlier studies have been done to characterize the expression of glucocorticoid receptors (Schaaf et al., 2008, 2009; Chatzopoulou et al., 2015) and glucocorticoid responsiveness similarity to mammalian including induction of gene expression by exogenous glucocorticoids at μg/L to mg/L (high nanomolar to micromolar) range (Tseng et al., 2005; Elo et al., 2007; Mathew et al., 2007; Hillegass et al., 2008). Due to its small size, availability, ease of maintenance and amenability to molecular techniques, the zebrafish is in a strategic position to bridge the in vitro and in vivo platforms with medium- to high-throughput capacity for large scale chemical screening (Sukardi et al., 2011). Transgenic ze-

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brafish lines with a reporter gene fused to a promoter with multiple glucocorticoid responsive elements were developed for monitoring in vivo glucocorticoid activity or screening of glucocorticoid compounds (Weger et al., 2012; Benato et al., 2014; Krug et al., 2014). However, due to the strong background signal induced by endogenous glucocorticoids, they lack sensitivity in detecting low glucocorticoid activity in environmental samples. This study aimed to determine if glucocorticoids at environmental relevant concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and investigate their potentials as in vivo zebrafish assays in complementing in vitro glucocorticoid bioassay for environmental monitoring of glucocorticoid contaminants. In this study, we employed realtime PCR to determine the relative changes in expression level of eleven selected glucocorticoid-responsive genes in larval zebrafish and the liver of adult male zebrafish exposed to three representative synthetic glucocorticoids (dexamethasone, prednisolone, and triamcinolone) at 50 pM, 500 pM, 5 nM or 50 nM (from 18– 19.7 ng/L to 18,000–19,700 ng/L) comparable with the total glucocorticoid concentrations estimated in environmental waters from several and tens of ng/L range (Chang et al., 2007, 2009; Schriks et al., 2013; Anumol et al., 2013; Jia et al., 2015) to more than hundreds and thousands of ng/L range (Kugathas et al., 2012; Creusot et al., 2014). The selected genes that were identified to be responsive to glucocorticoids at environmental relevant concentrations were further assayed for altered expressions in both adult and larval zebrafish that were exposed to a reconstituted extract of a secondary wastewater effluent that was detected having in vitro glucocorticoid activity and confirmed to contain low concentration of the targeted glucocorticoids by analytical chemistry. The study demonstrated the different but complementary roles of in vivo zebrafish assay and in vitro bioassay coupled with analytical chemistry approach in environmental monitoring of glucocorticoid contaminants. 2. Material and methods 2.1. Glucocorticoids and secondary wastewater effluent extract preparation The glucocorticoids used for the exposure experiments, dexamethasone (DEX), prednisolone (PRE), triamcinolone (TRI) and the vehicle dimethyl sulfoxide (DMSO) were purchased from Sigma– Aldrich (MO, USA). The glucocorticoids were prepared as 10 mM stock in DMSO and stored at −20 °C. Fresh working solutions diluted from the stock solution were prepared before each exposure. An environmental extract was prepared from secondary effluent of a wastewater plant in Tucson, Arizona, USA, following the procedure described by Anumol et al. (2013) (Supplementary materials). 2.2. Glucocorticoid and secondary wastewater effluent exposure experiments The animal experiments were conducted under the Institutional Animal Care and Use Committee of the National University of Singapore (IACUC protocols 079/07 and 027/11). For glucocorticoid exposure, 40 zebrafish embryos of wild type AB strain aged 3 h postfertilization (hpf) were placed into each well of a 6-well plate containing 50 pM, 500 pM, 5 nM or 50 nM of glucocorticoid (DEX, PRE or TRI) in 0.05% (v/v) DMSO at final concentration in E3 medium [5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 ·2H2 O, 0.33 mM MgSO4 ·7H2 O, 0.0001% Methylene blue in Milli-Q water, pH 7.1–7.2]. For secondary wastewater effluent exposure, 40 zebrafish embryos per well were exposed to sample extract reconstituted to 1-fold and 0.5-fold of the original concentration in E3 medium with final concentration of 0.05% (v/v) DMSO. The respective control groups

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were exposed to E3 medium with final concentration of 0.05% (v/v) DMSO. The duration of the exposure experiment was 120 h and the developing fish were maintained at 28 ± 0.5 °C throughout the experiment. All working solutions and media were freshly prepared and renewed daily. At the end of the exposure experiment, larvae pooled from each well were considered as one sample replicate and were snap-frozen in liquid nitrogen before storing at −80 °C until sample processing. Wild-type adult male zebrafish were acclimatized for one week in dechlorinated freshwater at 29.0 ± 1.0 °C and 12 h illumination time and fed twice a day with artemia and tropical fish food flakes (TetraMin® , Germany). The water was dechlorinated by activatedcarbon column and thereafter allowing it to age under aeration for more than 24 h in the open before used. For glucocorticoid exposure, adult zebrafish were maintained for four days in medium consisted of 50 nM, 5 nM, 500 pM or 50 pM of glucocorticoid (DEX, PRE or TRI) in 0.05% (v/v) DMSO at final concentration in dechlorinated fresh water. For secondary wastewater effluent exposure, acclimatized fish were exposed for 4 days to sample extract prepared from a secondary wastewater effluent reconstituted to the original concentration (1-fold) in dechlorinated fresh water with final concentration of 0.05% (v/v) DMSO. The control group were maintained in dechlorinated freshwater with final concentration of 0.05% (v/v) DMSO. All working solution and media were freshly prepared and renewed daily. Fish were not fed during the experiment and were maintained at density of 1 fish/200 ml. By the end of the 4 day exposure experiment, liver tissues were sampled from individual fish and snap-frozen in liquid nitrogen before storing at −80 °C until sample processing. 2.3. Total RNA extraction and cDNA synthesis Total RNAs from zebrafish larvae and adult male livers of each group were extracted according to the protocol of TRIzol® Reagent (Ambion® , USA) and was followed by DNase I (InvitrogenTM , CA, USA) treatment to remove DNA contamination. The quality and quantity of RNA samples were determined by a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) followed by RNA gel electrophoresis. Only RNA samples with purity of 1.8–2.0 (260 nm/280 nm ratio) and clear bands showing 2:1 (28S:18S) ratio on the gel were selected for cDNA synthesis using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, USA). All the RNA and cDNA stock samples were stored at −80 °C until used. 2.4. Quantitative real-time PCR and analysis The cDNA of zebrafish larvae and adult male liver samples were subjected to real-time PCR analysis using EXPRESS SYBR® GreenERTM qPCR SuperMix Kit (InvitrogenTM , USA). Primer information for target genes: brain-specific angiogenesis inhibitor 1associated protein 2 (baiap2), cyclin-dependent kinase inhibitor 1C (cdkn1c), cytochrome P450, family 3, subfamily A, polypeptide 65 (cyp3a65), FK506 binding protein 5 (fkbp5), glucocorticoid-induced leucine zipper, TSC22D3 (gilz), matrix metalloproteinase-2 (mmp2), matrix metalloproteinase-9 (mmp-9), matrix metalloproteinase13 (mmp-13), phosphoenolpyruvate carboxykinase (pepck), pregnane X receptor (pxr), SRY-box containing gene 9b (sox9b), and housekeeping gene beta-actin are listed in Supplementary materials. The genes were selected based on their reported glucocorticoid-responsiveness and/or presence of glucocorticoidresponsive elements (Supplementary materials). Real-time PCR reactions were performed on a StepOnePlusTM Real-Time PCR system (Applied Biosystems® , USA) under the program of 20 s at 95 °C, 40 cycles 95 °C (3 s) and 60 °C (30 s), followed by melting curve post run at 95 °C (15 s), 60 °C (1 min) and 95 °C (15 s).

The threshold cycle (Ct) values of target genes were obtained from real-time PCR analysis, and were then normalized by subtracting Ct values of respective housekeeping gene of each sample to obtain Ct values. The beta-actin (actb) was used as housekeeping gene for zebrafish larvae and aminolevulinate synthase 2 (alas2) was used as housekeeping gene for liver of adult male as both genes were relatively more stable and did not change significantly (P > 0.05) between glucocorticoid treatment and control groups. The primer sequences for actb and alas2 are provided in the Supplementary materials. Relative fold-change in expression level of treated samples against DMSO controls were calculated based on method described by Livak and Schmittgen (2001). The data were analysed using non-parametric Kruskal–Wallis test followed by post-hoc Mann–Whitney U test for paired comparison between treatment and control groups. For all the samples, the Ct values from each sample were adopted and analysed using IBM SPSS statistics (IBM Corporation, USA). 2.5. In vitro glucocorticoid receptor bioassay The GeneBLAzer® glucocorticoid receptor assay kit (Life Technologies, USA) was used as described in Jia et al. (2015) (Supplementary materials). 2.6. Detection of three targeted glucocorticoids in secondary wastewater effluent extract The three targeted glucocorticoids (dexamethasone, prednisolone and triamcinolone) were analysed in sample extract of secondary wastewater effluent using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLCMS/MS) as described in Anumol et al. (2013) (Supplementary materials). 3. Results 3.1. Expression of selected glucocorticoid-responsive genes in zebrafish larvae exposed to glucocorticoids Less than 10% cumulative mortality with no apparent differences in mortality were observed in all groups and no apparent morphological abnormality was observed in the developing fish suggesting that the concentrations used did not induce acute toxicity and were not teratogenic. The expression of the eleven selected glucocorticoid-responsive genes in developing zebrafish embryos exposed to 50 pM–50 nM of representative glucocorticoids (DEX, PRE and TRI), was determined using real-time PCR and summarized in Fig. 1. Among the eleven genes, pepck, baiap2 and pxr were the top three genes being significantly up-regulated in the most exposure groups. The expression of pepck was significantly up-regulated in all the 12 exposure groups (100%) while baiap2 was significantly up-regulated in 10 of the 12 exposure groups (83.3%) and pxr was significantly up-regulated in 8 of the 12 exposure groups (66.63%). It is worth highlighting that the expression of pepck, baiap2 and pxr were significantly perturbed by the glucocorticoids at concentrations (50 pM–5 nM) equivalent to total glucocorticoids or in vitro DEX-EQ glucocorticoid activity that have been detected in the environment. The expression of mmp-13 was significantly up-regulated in 7 of the 12 exposure groups (58.3%) and appeared to be more responsive to PRE and TRI exposures and less to DEX, suggesting selective response to different glucocorticoids. The remaining genes were not consistently responsive to glucocorticoid perturbation in zebrafish larvae. Although less consistent, down-regulation occurred more frequent for sox9b, gilz and mmp-9. Moreover, there appeared to be differences in the overall transcriptional responsiveness to the three glucocorticoids where

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Fig. 1. Heat-map representing expression profiles of eleven selected glucocorticoid-responsive genes in zebrafish larvae exposed to dexamethasone, prednisolone and triamcinolone at 50 pM, 500 pM, 5 nM and 50 nM relative to control group. The data were analysed using non-parametric Kruskal–Wallis test followed by Mann–Whitney U test (n = 5 to 7 replicates). Asterisks indicated significant deregulation compared to DMSO control group.

TRI and PRE exposures significantly deregulated more treatment groups, a total of 27 (61.4%) and 20 (45.5%), respectively, compared to 15 (34%) in DEX exposure groups, possibly due to different glucocorticoid receptor binding activity, different pharmacokinetics and/or pharmacodynamics of these drugs in the zebrafish larvae.

of adult male fish than in the larvae where both TRI and PRE exposures significantly deregulated 15 (34.1%) treatment groups compared to 8 (18.2%) in DEX exposure groups in adult male liver.

3.2. Expression of selected glucocorticoid-responsive genes in the liver of adult male zebrafish exposed to glucocorticoids

3.3. Expression of selected glucocorticoid-responsive genes in larvae and liver of adult male zebrafish exposed to secondary wastewater effluent extract

Mortality was minimal where four treatment groups having one fish dead while no mortality was observed in the remaining nine groups, suggesting that the concentrations used did not induce acute toxicity. The expression of the eleven selected glucocorticoid-responsive genes in liver of adult male zebrafish exposed to 50 pM–50 nM of representative glucocorticoids (DEX, PRE and TRI) was determined using real-time PCR and summarized in Fig. 2. Among the eleven genes, baiap2, mmp-2 and pxr were the top three genes being significantly up-regulated in most of the exposure groups. The expression of baiap2 was significantly up-regulated in 11 of the 12 exposure groups (91.7%) followed by mmp-2 and pxr which were significantly up-regulated in 9 of the 12 exposure groups (75%) and 8 of the 12 exposure groups (66.6%), respectively. The expression of the three genes was significantly perturbed at concentrations equivalent to total glucocorticoids or total in vitro DEX-EQ activity found in the environment. The remaining genes were either not consistently responsive or unresponsive to glucocorticoid perturbation in the liver of adult male zebrafish. The overall transcriptional responsiveness to the three glucocorticoids were lower in the liver

The average cumulative mortality was about 11% and 13% in larvae exposed to media reconstituted to 0.5-fold and 1-fold of the original concentration of the secondary wastewater effluent, respectively, compared to the control (DMSO) group (4%) suggesting slight increase in acute toxicity exerted by the reconstituted secondary wastewater effluent. However, no adult fish died during the experiments suggesting that the adult fish may be more tolerant to the reconstituted secondary effluent extract when compared to early developing fish. The expression of pepck, baiap2 and pxr in zebrafish larvae and baiap2, pxr and mmp-2 in liver of adult male zebrafish was determined using real-time PCR and summarized in Fig. 3. In zebrafish larvae, the expression of pepck, baiap2 and pxr were significantly up-regulated in both of the 1-fold and 0.5-fold exposure groups, which were consistent with the three targeted glucocorticoid exposure. However, baiap2, pxr and mmp2 were not significantly deregulated in the liver of adult male zebrafish (Fig. 3). The findings suggest that the original, or half the diluted, concentration of the secondary wastewater effluent released to the environment, can exert acute toxicity and up-regulate specific glucocorticoid-responsive genes in zebrafish larvae.

Fig. 2. Heat-map representing expression profiles of eleven selected glucocorticoid-responsive genes in the liver of adult male zebrafish larvae exposed to dexamethasone, prednisolone and triamcinolone at 50 pM, 500 pM, 5 nM and 50 nM relative to control group. The data were analysed using non-parametric Kruskal–Wallis test followed by Mann–Whitney U test (n = 4 to 6 replicates). Asterisks indicated significant deregulation compared to DMSO control group.

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Fig. 3. Relative expression of (A) baiap2, pxr and pepck in zebrafish larvae (n = 14–18 replicates) exposed to 0.5-fold and 1-fold reconstituted secondary wastewater effluent and (B) baiap2, pxr and mmp-2 in liver of adult male zebrafish (n = 8 replicates) exposed to 1-fold reconstituted secondary wastewater effluent. Results are shown as mean fold-change ± standard error (SE) relative to control group. The data were analysed using non-parametric Kruskal–Wallis test followed by Mann–Whitney U test. Asterisks indicated significant up-regulation compared to DMSO control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 for adult male zebrafish.

same level as the EC10 of direct dexamethasone exposure (Table 1). However, further UHPLC-MS/MS analysis of the secondary wastewater effluent extract detected all the three target glucocorticoids to be 0.2 ng/L or less (total calculated DEX equivalent concentration = ∼0.6 pM) which could only account for 0.1% of the in vitro glucocorticoid activity (DEX-EQ = 478 pM) (Table 1). Taken together, the findings suggested that there were other non-detected or unknown glucocorticoid active compounds present in the effluent. 4. Discussion

Fig. 4. Dose–response curve of dexamethasone (DEX), prednisolone (PRE) and triamcinolone (TRI) detected by in vitro glucocorticoid receptor GR GeneBlazer® bioassay. The percentage (%) effect (mean ± standard deviation; n = 3 replicates) at different concentrations (76 pM–500 nM with a 1:3 serial dilutions for 9 dose points) of the three glucocorticoids are shown in the line graph. Detail description of the method and calculations are provided in the Supplementary Materials.

3.4. In vitro glucocorticoid activity and targeted glucocorticoids detected in secondary wastewater effluent extract The dose–response curve of the three glucocorticoids is shown in Fig. 4. The EC50 of DEX, PRE and TRI were 1.8, 17.7, and 11.8 nM, respectively (Table 1). The calculated relative equivalent potency to DEX (REPDEX ) using the ratio of EC50 indicated that PRE and TRI were about 10-fold and 6.6-fold, respectively, less potent than DEX as detected by the in vitro glucocorticoid bioassay. The EC10 value can be considered as the lowest glucocorticoid concentration that induce detectable in vitro glucocorticoid receptor activity and the EC10 for DEX, PRE and TRI were 0.5, 6.7 and 4.2 nM, respectively. The EC10 values DEX, PRE and TRI suggested that the in vitro glucocorticoid receptor bioassay was less sensitive than the in vivo zebrafish assays which were responsive to 50–500 pM of glucocorticoids. For the secondary wastewater effluent, the sample response reached EC10 effect only at 1.09-fold of the original concentration of the effluent, suggesting that it is possible for the effluent to induce the in vitro glucocorticoid activity in its original form when released to the environment. The in vitro glucocorticoid activity was detected at DEX-EQ of 478 pM which was almost the

This study demonstrated that exposure of glucocorticoids equivalent to total glucocorticoids at environmental relevant concentrations from several to thousands ng/L (pM to several nM range) can perturbed the expression of selected glucocorticoid genes in zebrafish larvae and adult male liver. Although the eleven genes were selected based on previous reports of their glucocorticoidresponsiveness and/or presence of glucocorticoid-responsive elements, not all of them have similar responsiveness in the zebrafish. The differences in their glucocorticoid-responsiveness may be due to developmental and tissue-organs specificity as well as species differences with respect to the expression of these genes. Nevertheless, the study identified the expression of pepck, baiap2 and pxr to be perturbed in zebrafish larvae and the expression of baiap2, pxr and mmp-2 to be perturbed in liver of male adult zebrafish when exposed to environmental-relevant concentration of 50 and/or 500 pM (from 18–19.7 to 180–197 ng/L) of glucocorticoids. The genes, however, showed little to no dose–response or nonmonotonic response relationships. Low dose effects and the occurrence of nonmonotonic responses where relationship between dose and effect is nonlinear have been reviewed to be common in studies involving natural hormones and endocrine disruptions (Vandenberg et al., 2012). Nonmonotonic responses could be due to toxicity, differences in tissue-specific receptors and cofactors, receptor down-regulation and desensitization, receptor selectivity, competition and cross-talk, endocrine negative feedback loops and other downstream mechanisms (Vandenberg et al., 2012). It is well known that binding of glucocorticoid mediates the degradation and down-regulation of its receptor and mRNA transcript, respectively, which in turn acts as a homeostatic adaptive mechanism to restrict and regulate transcription signalling by glucocorticoids (Dong et al., 1988; Wallace and Cidlowski, 2001). Moreover, differences in gene expression profiles affected by low doses of hormone compared with higher doses have been reported (Coser et al., 2003; Vandenberg et al., 2012). Therefore, depending on how

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Table 1 Glucocorticoid activity of targeted glucocorticoids and secondary wastewater effluent detected by in vitro GR GeneBlazer bioassay and their concentrations detected by UHPLC-MS/MS analysis. Target glucocorticoids

Secondary wastewater effluent

EC10 (nM)

Dexamethasone (DEX) Prednisolone (PRE) Triamcinolone (TRI)

0.5

1.8

1.0

0.2

6.7

17.7

0.10

<0.04

<0.01

4.2

11.8

0.15

<0.2

<0.08

a b c d

EC50 (nM)

REPDEX a

Name

UHPLC-MS/MS concentration (ng/L)b

DEX equivalent concentration (pM)c

0.51

EC10

In vitro glucocorticoid activity equivalent to DEX concentration DEX-EQ (pM)d

1.09X

478

Relative equivalent potency to DEX (REPDEX ) = EC50 (DEX)/EC50 (sample). Concentration determined by UHPLC-MS/MS. DEX equivalent concentration (pM) = UHPLC-MS/MS Concentration (in pM) x REPDEX . Inferred from the dose–response curve of dexamethasone at EC10 .

these genes are regulated by glucocorticoid receptor and also by other factors, they may contribute to the nonmonotonic or little to absence of dose–response relationships observed in this study. The baiap2 is an insulin receptor substrate that plays a role in insulin signalling and cytoskeletal reorganization involved in neuronal growth (Oda et al., 1999). Insulin signalling is modulated by glucocorticoids and baiap2 was among genes identified to be induced by glucocorticoids and suppressed by glucocorticoid receptor antagonist (Muzikar et al., 2009). The pxr encoded nuclear receptor has multiple roles in xenobiotic metabolism, as well as steroidal and bile homeostasis (Ma et al., 2008). The upregulation of pxr at the mRNA and protein levels by glucocorticoids that synergistically induces other drug metabolic enzymes has been reported (Shi et al., 2010). The pepck encoded enzyme plays a key step in gluconeogenesis (Hanson and Reshef, 1997). Glucocorticoids are known to regulate pepck (Cassuto et al., 2005) in promoting gluconeogenesis and inducing hyperglycemia (Kwon and Hermayer, 2013). The mmp-2 encoded enzyme is involved in breaking down extracellular matrix during remodelling of tissue including disease process such as liver injury (Han, 2006) and its up-regulation in the liver as detected in this study may be associated with glucocorticoid-induced liver anomaly (Kuhlenschmidt et al., 1991; Marinó et al., 2004). The deregulation of these selected glucocorticoid-responsive genes in the larval and adult zebrafish, however, does not necessary mean that these fish experienced or will experience altered physiology or pathology. Nevertheless, significant up-regulation of pepck followed by corresponding increase of plasma glucose level have been reported in adult fathead minnow exposed to 21 days of the glucocorticoid beclomethasone dipropionate at 100 ng/L (∼192 pM) while decreased in leucocyte counts and reduced secondary sexual characteristics in fish were detected at 1 and 10 μg/L (∼1.9–19 nM range) (Kugathas and Sumpter, 2011; Kugathas et al., 2013). Our findings taken together with Kugathas et al. (2013) suggested that glucocorticoid exposure at high ng/L concentrations which are relevant for total glucocorticoids reported in the environment could perturb expression of specific genes, blood parameters, carbohydrate metabolism and exert reproductive effects in fish. The present study showed that the in vivo responsiveness of the selected zebrafish genes to glucocorticoid at 50–500 pM range was more sensitive than the in vitro glucocorticoid receptor GRGeneBLAzer bioassay where the EC50 of DEX, PRE and TRI were detected at 1.8, 17.7, and 11.8 nM, respectively. Likewise, this was also the case when compared to other in vitro glucocorticoid receptor assays such as GR-CALUX bioassay (Schriks et al., 2013), GR-Switchgear bioassay (Jia et al., 2015) and transfected zebrafish cell line AB.9 GRE:Luc bioassay (Weger et al., 2012) which had re-

ported EC50 of DEX at 1.6, 2.6 and 13 nM, respectively. This further demonstrated that the glucocorticoid transcriptional activity detected by in vitro glucocorticoid receptor bioassays at low nM range has in vivo relevance as it could also up-regulate the expression of specific glucocorticoid-responsive genes in zebrafish larvae and adult male liver. This is important as there are valid concerns that biological activities and effects detected in vitro are not necessary similar to, hence may not be recapitulated, in vivo because in vitro bioassays lacks whole-organism physiology and differs in glucocorticoid absorption, distribution, metabolism, excretion and toxicity (ADMET) (Sukardi et al., 2011; Bale et al., 2014). Difference in the overall transcriptional responsiveness was observed where TRI and PRE exposures significantly deregulated more treatment groups than DEX although DEX is known to be more potent in mammals/humans. This could be due to the differences in GR binding activities, pharmacodynamics and/or pharmacokinetics of the different synthetic glucocorticoids in zebrafish compared to mammals/humans. Species differences in pharmacokinetics and pharmacodynamics are common (Toutain et al., 2010) and differences in potency and response to drugs between zebrafish and human have been reported (Redfern et al., 2008; Sukardi et al., 2011). This may suggest that chemically monitoring environmental samples for glucocorticoids that are more potent in fish than in human would have more biological or even ecological relevance. Moreover, commercially available in vitro glucocorticoid receptor bioassays (such as GR-GeneBLAzer, GR-CALUX, GRSwitchgear) reported for environmental glucocorticoid monitoring used human transformed cell lines bearing human glucocorticoid response elements and therefore have little aquatic organismal and eco-environmental relevance as compared to fish. Since fish is positioned in the extreme opposite end of the vertebrate taxon when compared to humans or mammals based on their evolutionary distance and phylogenetic relationships in the animal kingdom, glucocorticoid responsiveness observed in fish and mammals would imply conservation across vertebrates (Schaaf et al., 2009), thus perturbation of in vivo glucocorticoid activity at environmental concentration in fish would further raise concerns of potential perturbation in other aquatic vertebrates. Longer term study on other aquatic organisms would be necessary if wider ecological implication were to be drawn. The glucocorticoid transcriptional activity detected in the extract of secondary wastewater effluent by in vitro glucocorticoid receptor GR-GeneBLAzer bioassay was also detected in vivo by the selected genes in zebrafish larvae although not in adult male liver. The expression of pepck, baiap2 and pxr were significantly up-regulated in zebrafish larvae exposed to media reconstituted to 0.5-fold and 1-fold of the original concentration of the sec-

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ondary wastewater effluent while the in vitro glucocorticoid activity was detected at DEX-EQ of 478 pM. The findings further reiterated that the glucocorticoid transcriptional activity detected by in vitro glucocorticoid receptor bioassay in the environmental sample had in vivo relevance in up-regulating specific glucocorticoidresponsive genes in zebrafish larvae. However, the low levels of the three targeted glucocorticoids detected by analytical chemistry suggested that there were other glucocorticoid-active compounds in the complex wastewater effluent. In addition to glucocorticoid activity, wastewater effluents usually consist of complex mixtures that can exert similar effects such as retinoic acid receptor and aryl hydrocarbon receptor activities (Allinson et al., 2011), multiple hormonal activities (Bain et al., 2014), and as detected from the same WWTP of the present study are aryl hydrocarbon receptor, estrogenic, genotoxic, mutagenic, oxidative stress, cytotoxic and phytotoxic activities (Jia et al., 2015). The mixture of bio-active compounds may have exerted toxicity as observed in the increase mortality of early developing zebrafish exposed to the reconstituted secondary wastewater effluent when compared to the control group. This may further suggest greater vulnerability of early developing fish to the effluent when released to the environment. While the perturbed gene expressions were detected significantly in the larvae despite the presence of a mixture of bioactive compounds, albeit at weaker intensity likely due to the toxicity or mixture effects, they were not detected to be significant in the liver of adult male fish. The liver is a major organ for xenobiotic metabolism hence the homeostatic responses to cope with the toxicity and mixture effects exerted by the complex environmental sample may have affected the transcription of the selected genes in the liver. This suggested that the selected glucocorticoid-responsive genes were more robust in zebrafish larvae than the adult male liver in responding to glucocorticoid transcriptional activity in complex environmental samples. In comparison, in vitro bioassays may be even more robust in detecting glucocorticoid transcriptional activity in complex environmental samples since cell lines, while still susceptible to cytotoxicity, are not subjected to the in vivo complex regulation of multiple biological signals in whole-organism. In conclusion, this study has demonstrated that the expression of specific glucocorticoid-responsive genes can be perturbed by glucocorticoids at concentrations equivalent to total glucocorticoids or in vitro DEX-EQ detected in environmental samples. The study has successfully identified glucocorticoid-responsive genes in zebrafish larvae that was shown to be useful for environmental glucocorticoid monitoring by providing in vivo relevance for the glucocorticoid transcriptional activity detected by in vitro bioassay. The study also demonstrated the different but important complementary roles of in vitro and in vivo bioassays as well as analytical chemistry approach in environmental monitoring of glucocorticoid contaminants. Acknowledgement This study is supported by a grant from the Singapore National Research Foundation PUB 2P 21100/36/4 under its Environmental & Water Technologies Strategic Research Programme and administered by the Environment & Water Industry Programme Office (EWI) of the PUB. We thank the NUS Environmental Research Institute (NERI) for their laboratory and administrative support. Appendix A. Supplementary data Supplementary data related to this article can be found at 10. 1016/j.chemosphere.2015.09.089.

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