Scholarly article on topic 'Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test'

Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test Academic research paper on "Biological sciences"

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{"Aromatase inhibition" / Zebrafish / qPCR / Fadrozole / "Fish sexual development test (FSDT)" / "Adverse outcome pathway (AOP)"}

Abstract of research paper on Biological sciences, author of scientific article — Elke Muth-Köhne, Kathi Westphal-Settele, Jasmin Brückner, Sabine Konradi, Viktoria Schiller, et al.

Abstract The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.

Academic research paper on topic "Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test"

AQUATIC

TOXICOLOGY

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Aquatic Toxicology

journal homepage www.elsevier.com/locate/aquatox

Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test

Elke Muth-Köhnea*, Kathi Westphal-Setteleb, Jasmin Brücknerb, Sabine Konradib, Viktoria Schillerc, Christoph Schäfersa, Matthias Teigelera1, Martina Fensked1

a Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1,57392 Schmallenberg, Germany b German Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, Germany c Fraunhofer IME, Attract Group UNIFISH, Forckenbeckstraße 6,52074 Aachen, Germany

d Fraunhofer IME, Project Group Translational Medicine and Pharmacology TMP, Forckenbeckstraße 6,52074 Aachen, Germany

ARTICLE INFO

Article history:

Received 23 December 2015

Received in revised form 13 April 2016

Accepted 19 April 2016

Available online 20 April 2016

Keywords:

Aromatase inhibition

Zebrafish

Fadrozole

Fish sexual development test (FSDT) Adverse outcome pathway (AOP)

ABSTRACT

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10 ^g/L, 32 ^g/L, 100 ^g/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63 days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10 ^g/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtgl were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; Iss), methylsterol monooxy-genase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.

© 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Environmental pollutants which interact with the endocrine system of organisms, and exert a negative impact on development and reproduction, are commonly known as endocrine

* Corresponding author.

E-mail address: elke.muth-koehne@ime.fraunhofer.de (E. Muth-Kohne). 1 These authors contributed equally to the project.

disrupting chemicals (EDC). Endocrine disruption manifests in an organism or a population after sub-acute to chronic (long-term) exposure, which makes the experimental identification of hormone-active substances time- and cost-intensive. These elaborative experiments require large numbers of animals, and alternatives approaches according to the principles of the 3R (Replacement, Reduction, and Refinement) after Russell and Burch (1959), are highly desired. Shorter, life-stage confined tests were designed as screening tools to trigger more complex studies like a fish life cycle test, which are required to assess the endocrine

http://dx.doi.org/10.1016/j.aquatox.2016.04.018

0166-445X/© 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

disruption relevant apical endpoints. These shorter and less fish consuming predictive tests have, in principal, the potential to reduce the number of long-term apical tests. However, apical effect data from screening assays could not be used for regulatory purposes.

Most life-stage confined tests like the Fish Early Life Stage Test, (FELS; OECD TG 210 (OECD, 2013)) are not designed to provide substantive information about the chemical mode-of-action (MoA; Villeneuve et al., 2014) and are prone to interpretation error, as they exclude relevant apical endpoints, like the determination of the sex ratio. It seems therefore logical to include quick responding parameters which may also be indicative of the underlying MoA or pinpoint the molecular initiating event (MIE).

Molecular endpoints respond promptly to pollutants and promise to provide information indicative of adverse effects at organism level (Piersma, 2006). Powerful genomic approaches have contributed essentially to the determination of molecular mechanisms resulting in apical adverse endpoints (Ankley et al., 2006; Sawle et al., 2010). Global transcriptomics allow meaningful analyses of adverse effects in cells, organs, and the whole organism at concentrations potentially below thresholds of the most sensitive endpoints of the "conventional" studies. This higher sensitivity of molecular analyses is crucial to prevent the overlay of endocrine effects by other effects, especially systemic toxicity (Scholz and Mayer, 2008; Wang et al., 2010). The progress in molecular and computer-based (in silico) methods paved the way for the shift of paradigms in ecotoxicology (NRC, 2007), and for the development of causal mechanism-based concepts for environmental risk assessment, e.g. the Toxicity Pathways (Bradbury et al., 2004) or more recently the Adverse Outcome Pathways (AOPs; Ankley et al., 2010; Villeneuve and Garcia-Reyero, 2011). Specific AOPs aim to provide causal links between the MIE and the adverse outcome (AO) of regulatory concern via well-established, measureable key events (KEs) and key event relationships (KERs), which are intended to facilitate regulatory decision making (Kramer et al., 2011; Segner, 2011, 2009). To date several AOPs have already been defined for endocrine disruption (aopkb.org/aopwiki). However, the AOP for aromatase inhibition is only applicable to sexually mature female fish, as the AO at the organism level is reduced fecundity (Ankley et al., 2010, 2009; aopkb.org/aopwiki/index.php/Aop:25), and no measureable KE is described at the molecular level.

Zebrafish are particularly sensitive to aromatase inhibitors during gonadal sex differentiation and maturation, the phase between the early life stage and adulthood (Baumann et al., 2015, 2013; Fenske and Segner, 2004; Kinnberg et al., 2007). As undifferentiated gonochorists, zebrafish' gonads develop via a stage described as non-functional protogyne gonad (Maack and Segner, 2003). Testis differentiation and male maturation are triggered by increasing levels of androgens, while maturation of ovaries requires higher levels of estrogens. Aromatase inhibitors block the synthesis of estrogens by inhibition of the conversion of C19-androgens to C18-estrogens (Callard et al., 1978). The surplus of androgens consequently favors masculinization in the population (Baumann et al., 2015, 2013; Fenske and Segner, 2004; Morthorst et al., 2010; Trant et al., 2001).

The Fish Sexual Development Test (FSDT; OECD TG 234, OECD, 2011) allows the analysis of effects of endocrine disruptors on the early life stage and sexual development. The FSDT is essentially an extension of the FELS test. It was originally developed as a "Fish Partial Life Cycle Test" and considered an alternative to specifically test endocrine disruptors in fish without the requirement of a whole life span exposure. It covers the endocrine disruption prone developmental period of sexual differentiation of particularly zebrafish and allows the assessment of gonadal differentiation and sex ratio as endocrine disruption-associated endpoints (Thorpe et al., 2011). However, the information on the endocrine system functions which

is being disrupted is limited to EDCs affecting vitellogenin and the phenotypic gonadal sex ratio.

The conceptual framework of the OECD for Testing and Assessment of Endocrine Disruptors (OECD, 2012a) is mainly focused on the assessment of chemicals disrupting estrogen, androgen, thyroid signaling processes and steroidogenesis (EATS), but there are several other endocrine pathways (e.g. corticosteroid, gestagenic, or retinoid signaling pathways), which additionally may be disrupted by chemical exposure. The use of additional new endpoints may help in better understanding the link between the initiating endocrine related mechanisms and resulting apical effects. This would strengthen an AOP-driven approach in the context of the weight-of-evidence strategy that is currently pursued in the regulatory context to assess an endocrine disruption hazard (OECD, 2012b). The need for more comprehensive methods has been acknowledged and highlighted in the Review paper of the OECD on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors (Kortenkamp et al., 2011; OECD, 2012b).

The rationale of the current study was to demonstrate that gene expression data could help to increase the evidence on whether and how a substance may act as an EDC. To this end, an FSDT according to the OECD guideline 234 was conducted with the aromatase inhibitor fadrozole and refined by mRNA analysis using quantitative RT-PCR. The rationale to choose fadrozole as test substance is that its endocrine disrupting effects are reasonably well defined for fish (Afonso et al., 2000; Ankley et al., 2002; Fenske and Segner, 2004; Scholz and Klüver, 2009; Takatsu et al., 2013; Villeneuve et al., 2013; Wang et al., 2010; Zhang et al., 2008). Fadrozole is a potent, selective non-steroid aromatase inhibitor. Although its interaction with the enzyme is not yet fully understood, coordination with the iron of the prophyrine core of the CYP19 enzyme is likely. This describes a characteristic general function of type-11-inhibitors, which was first described in mammals (Browne et al., 1991). Fadrozole therefore exhibits a specific aromatase-inhibiting MoA with fewer side effects than e.g. prochloraz, which inhibits steroidogenesis as well as androgen receptor signaling (Ankley et al., 2005; Laier et al., 2006).

In this study, a set of endocrine-related genes was measured in early life stages, and in juvenile and pre-adult fish at the end of the test, in addition to the default endpoints of the FSDT (hatch, juvenile growth, sex ratio, plasma vitellogenin, and histopathol-ogy).The selection of potential marker genes was based on previous results of our own and published studies (Schiller et al., 2013a, 2013b; Villeneuve et al., 2009), which indicated their sensitivity to endocrine disruption and especially aromatase inhibition. The group of selected genes covers also several critical steps in steroid biosynthesis.

Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life stage of sexual differentiation. Further, we were able to identify marker gene transcription involved in steroid biosynthesis which can be applied as measures of key events (KE) at the molecular level.

2. Material and methods

2.1. Test species

Wild-type zebrafish (Danio rerio) originally obtained from West Aquarium GmbH (Bad Lauterberg, Germany) and continuously bred for several generations in the Fraunhofer IME laboratories, were used for testing. Zebrafish of this line correspond in their juvenile/sexual development to the line described by Takahashi (1977) and Maack and Segner (2003).

Fish of the broodstock were maintained under flow-through conditions in 150 L tanks at 25 ±2 °C on a 12:12-h photoperiod in a temperature-controlled room. They were fed daily with TetraMin® main feed ad libitum and nauplii of Artemia salina.

For egg collection, stainless steel spawning trays were inserted into the tanks, and spawning was induced at the beginning of the light period (light intensity: 1000 lux). The eggs were microscopically examined for fertilization success. Only healthy eggs in at least blastula stage were inserted into the test system.

2.2. Test substance

Fadrozole (IUPAC-Name 4-(5,6,7,8-Tetrahydroimidazo[1,5-a]pyridine-5-yl)-benzonitrile), an aromatase inhibitor, was purchased from Adooq Bioscience LLC. (Irvine, Canada). The substance was directly dissolved in water for the preparation of the dosing solutions.

2.3. General exposure conditions

Purified tap water was used according to the OECD-Guideline 210 (OECD, 2013). The purification included filtration with activated charcoal. The water was aerated to the level of oxygen saturation. The water quality was monitored in the testing facility.

Water temperature in the test vessels was adjusted to 25 ± 2 °C. Oxygen saturation of the test solutions was between 99.0% and 103.4% of air saturation. The pH was between 8.08 and 8.17 in the test tanks.

2.4. Assessment of Fish Sexual Development test (OECD TG 234) endpoints

The Fish Sexual Development Test (FSDT) was performed according to the OECD Test Guideline 234 (OECD, 2011) in a flow-through exposure system. Minor modifications of the protocol were necessary to include gene expression analysis at different time points.

The test was performed with three test concentrations (10 |ig/L, 32 |g/L, and 100 |g/L.) under flow through conditions, with four replicate tanks per concentration run in parallel. A number of 85 eggs per replicate tank (12 L total volume) were introduced at test start divided into two stainless steel fry cages. One fry cage of 50 eggs provided samples for the gene expression analysis, the other fry cage of 35 eggs remained undisturbed.

The eggs were kept in the fry cages until hatching and the number of hatched larvae was estimated at 4 and 6 days post fertilization (dpf). At 21 dpf, larvae were released from both fry cages into the main tank. After the early life stage at 28 dpf, the juvenile fish were photographed for image-based evaluation of survival and growth (ImageTool of the University ofTexas Health Science Center at San Antonio, Version 3.0). Fish remained in the test system until test termination at 63 dpf. At test termination, survival rate, size in terms of length and weight, and the sex of all fish were determined. Twenty fish per replicate were analyzed for their vitellogenin (VTG) content, and were histopathologically evaluated.

For gene expression analysis, 20 embryos of 48 and 96 h post fertilization (hpf) were sampled from each replicate, pooled in a 1.5 mL Eppendorf tube and snap frozen. At 28 dpf, 4 randomly chosen larvae per replicate were pooled and snap frozen in a 2.0 mL Eppendorf tube; at test termination, the trunks of 5 fish per replicate (heads removed) were randomly chosen for gene expression analysis and snap frozen individually. The heads were removed in order to focus on endocrine effects related to gonad development. All samples were stored at -80 °C until further use.

For histopathology and VTG measurements, blood samples were collected (by cardiac puncture) and gonads dissected from all

fish except those dedicated to gene expression analysis. Gonads were incubated in modified Davidsons's fixative 20% formaldehyde (37%), 10% glycerol, 30% ethanol, and 10% acetic acid (100%) overnight, then transferred to 10% neutral buffered formalin (according to the OECD Histopathology guidance document (OECD, 2010)). Embedding was performed with paraffin and trunks orientated ventrally to the cutting surface. Sections of 4-5 |im were cut with a microtome, mounted on glass slides and then stained with hematoxylin-eosin.

Microscopic evaluation of the tissue sections was performed according to the OECD Histopathology Guidance Document (OECD 2010). Each fish was either sexed or recorded as undifferenti-ated and the maturation stage of the gonads were categorized. Endocrine-related effects were determined according to the primary and secondary diagnostic evaluation criteria.

For determination of the VTG levels, an enzyme-linked immunosorbent assay (ELISA) for zebrafish VTG (homologous ELISA kit, Biosense, Bergen, Norway) was used according to the manufacturers' instructions. Plasma samples were measured in 96-well plates by a microplate reader (iEMS Reader, Labsystems, Helsinki, Finland). The assay was calibrated against purified zebrafish VTG (provided with the kit) as a standard. A blank control was run in each assay. The VTG concentrations were normalized to the total plasma protein content, and data expressed as ng VTG/|g protein (Fenske et al., 2001). Total protein was quantified using a BCA Protein Assay Reagent Kit (Pierce, Rockford, USA).

2.5. Quantitative RT-PCR(QPCR)

Total RNA was isolated from homogenized frozen trunk tissue (63 dpf) or pooled embryos/larvae/juveniles using TRIreagent (Sigma-Aldrich, T9424) method followed by a clean-up with RNeasy Mini Spin Columns (QIAGEN, Hilden, Germany) according to the manufacturers' protocols. Prior to cDNA synthesis, RNA was DNAse (Sigma-Aldrich, AMP-D1) treated and subsequently measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany). Reverse transcription was carried out using SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, USA). Negative controls without reverse transcriptase were prepared for each sample.

QPCR analysis was performed on three of four replicates of each treatment (fadrozole exposures and controls) at each sampling time point, using a BioRad iQ5 real-time PCR detection system (BioRadTM, Hercules, USA). The fourth replicate samples were excluded from the analysis due to RNA quality issues. For each reaction, 40 ng of cDNA were added to Sybr®Green qPCR Super Mix (ThermoFisher Scientific/Invitrogen, Waltham, USA) in white 96-well PCR plates. Two technical replicates of each target gene and the respective reference genes were run for each cDNA sample. Minus template controls were included on each plate; minus RT controls were included repeatedly, at least once for each sample, however not in every run. The reference genes 18s and rpl8 were used to normalize the expression values of the target genes.

The PCR protocol was as follows: After incubating for 2 min at 50 °C and for 10min at 95 °C (for DNA polymerase activation), targets were amplified using the following cycle: 95 °C for 15 s, then 54, 54.5 or 60 °C for 30 s (annealing temperatures for particular genes) and 60 °C for 30 s (for acquisition; 40 cycles); the run was finalized by a melting curve analysis: 95 °C for 60 s, then 55°C for 60s (80 cycles, with +0.5°C increment each cycle and a 10 s acquisition). Relative gene expression ratios were calculated by the comparative delta Ct method (ddCt) (Livak and Schmittgen, 2001; Pfaffl, 2001).

2.6. Chemical water analysis

Water samples of 10 mL volume were taken from each tank from the mid water body, at test start, and thereafter once a week. The samples were taken alternating from one of the two test vessels served by one dosing pump. Water samples were stabilized by addition of acetonitrile (1:1; v + v) containing 0.2% formic acid prior to storage.

Fadrozole analysis was performed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) with negative ionization. Data were collected on a Waters 2695 separations module coupled to a Quattro-Micro tandem mass spectrometer (Waters). Aliquots of 50 |L were injected into a Gemini C18 high-performance LC column (150mm x 3mm, 5 |im particle size; Phenomenex) at a flow rate of 0.5mL/min and a column temperature of 30 °C. Matrix-free procedural blanks were run each working day to exclude possible cross-contaminations during laboratory work. Separation was performed on a binary gradient of 20 mmol ammonium acetate solution in (A) methanol and (B) a 90:10 water:methanol mixture. The gradient started with 60% A, increasing to 100% A within 3 min, then returning to 60% A and 40% B after 6.1 min, and holding initial conditions for 3 min. A Calibration was performed in a concentration range of 0 | g/L to 250 | g/L fadrozole. Coefficient of correlation (r2) of the calibration function was estimated > 0.99. The substance-specific limit of quantitation (LOQ) was defined as 5 |g/L fadrozole.

2.7. Statistical analysis

All physiological endpoints as well as the sex ratio were analyzed using ToxRat® Professional 3. Normal distribution of data was confirmed by the Shapiro-Wilks test, followed by Levene's test for variance homogeneity. A one-way ANOVA was performed, with the Williams t-test as post-hoc test, applying a significance level of p < 0.05. Data on gonad histopathology were evaluated with GraphPad Prism (GraphPad Software, LaJolla, USA) including one-way ANOVA followed by Dunnett's multiple t-test procedure, p < 0.05. For the biological results, No Observed Effect Concentrations (NOEC) and Lowest Observed Effect Concentrations (LOEC) were determined.

For gene expression, the statistical determination of significant differences of the fadrozole treatments compared to the controls, one-way ANOVAs followed by Dunnett's multiple comparison test (*p<0.05; **p<0.01; ***p<0.01), or Students t-test (*p < 0.05; **p <0.01; ***p < 0.01), were performed on relative transcript expression values of the three replicates. Control-normalized data were log2-transformed for presentation.

3. Results

3.1. Chemical analysis

The mean measured concentrations of the test solutions of fadrozole between day 1 and day 61 of the study ranged between 94.4% and 114.8% of nominal concentration per treatment (Table S2). Test concentrations during the time course of the study did not vary by more than 20% from nominal concentrations for most vessels with few exceptions (maximum concentration of 147.7% of nominal at a nominal concentration of 32 |g/L). All effect data are depicted according to the nominal fadrozole concentrations.

3.2. Effects of fadrozole on the FSDT standard endpoints

The hatching success of larvae was not influenced by fadrozole. Complete hatch was observed at 6 dpf in controls as well as in treatments. Survival during early life, however, was diminished in

a concentration-dependent manner. Post-hatch survival rates at 28 dpf were 80.6% and 77.8% for control respectively the 10 |g/L fadro-zole treatment, with controls meeting the validity criterion of 75% post-hatch survival recommended by the TG 234 (OECD, 2011). The 28 dpf post-hatch survival rates of the 32 |g/L and 100 |g/L treatment decreased significantly to 70.6% and 67.8% (Fig. 1A; *p < 0.05, Williams t-test, one-sided smaller). The NOEC for post-hatch survival was determined to be at 10 |g/L fadrozole. Survival was not further affected by fadrozole at 63 dpf.

Growth in terms of mean total body length at 28 dpf and in terms of mean standard length and wiped wet weight at 63 dpf was found not to be affected by fadrozole. At 28 dpf, the average length of the control fish was 10.1 mm whereas the treatment lengths varied between 10.6 mm (100 |g/L) and 10.7 mm (at 10 |g/L and 32 |g/L). Thus, no significant difference between the control and exposed 28 dpf fish was observed. The mean body length of 63 dpf control and exposed fish was 28.0 mm. The mean weight increased slightly with increasing exposure concentration, from 0.183 g for the controls to 0.196 g in the highest treatment condition. However, the differences in the mean values were not significant.

3.3. Histopathology and sex ratio

Gonadal sex ratios were determined during the histopatho-logical evaluation. The controls displayed a mean proportional ratio of males to females of 49.9-50.1% across all replicates. In all fadrozole treatments, the sex ratios were significantly different from the controls (Fig. 1B; p< 0.05, Williams t-test, two-sided, data arcsine-transformed prior to statistical analysis). At the lowest concentration, all fish were males, except for one female in one replicate. At the higher concentrations, only males were identified.

As no females were present in the treatment groups, histopathology of ovaries was limited to controls. The ovarian development of the control fish was characterized by gonads consisting of early stage oocytes. Only one gonad had reached a maturation stage of 2, all other ovaries were at stage 0 or 1. According to the maturity index of Baumann et al. (2013), this early ovary maturation stage corresponded to a maturity index of 1.8. The ovarian maturation of the remaining single female at 10 |g/L fadrozole corresponded to a maturity stage of 0 (maturity index of 1). The delay in ovarian maturation was not associated with any pathological alterations of the gonads.

Males of the control groups had an average testis maturity index of 2.1. The average gonad maturity of males increased with increasing fadrozole concentrations (Fig. 1C) and became statistically significant already from 10 |g fadrozole/L (**p<0.01, ***p<0.01; one-way ANOVA followed by Dunnett's multiple comparison test). At 100 |g fadrozole/L the highest average maturity index reached 2.7.

The gonads of control males were found to be in an undeveloped protogynous state at 38.2 %. This state marks the transition phase from an ovary-like structure to the male testis, which is a typical phase in zebrafish sexual development (Maack and Segner, 2003). The incidence of the undeveloped gonad stage remained below 20% in all fadrozole exposed fish (statistically no difference to the control) (Fig. 1D), consistent with the increasing gonad maturity indices of the treatment groups.

3.4. Vitellogenin measurement

VTG plasma concentration analysis was performed in pre-adult development (i.e., 63 dpf). The plasma concentrations of the control females displayed high variation but were on average 12.18 ng VTG/| g protein. Females with low amounts in the range <0.01 ng/|g were also found.

100 80

1 40 «

Post-hatch survival 28 dpf

Sex ratio

males □ females

з га E

0 10 32 100

Fadrozole concentration [pg/L]

Male gonad maturity index

10 32 100

Fadrozole concentration [|jg/L]

0 10 32 100

Fadrozole concentration [|jg/L]

D Males with undeveloped gonads

10 32 100

Fadrozole concentration [|jg/L]

Fig. 1. Physiological and histopathological effects of fadrozole exposure. (A) Post-hatch survival (depicted as% of hatched larvae at 6 dpf) of 28 dpf zebrafish was significantly reduced at nominal 32 |g/Land 100 |g/L fadrozole compared to control fish (white bar). (B) Sex ratio at 63 dpf (depicted as% males versus females) was fully skewed towards males at all fadrozole concentrations. (C) The gonad maturation stage of 63 dpf males, represented by the maturity index (Baumann et al., 2013), increased significantly from 10 |g/L fadrozole. (D) Ratio of males (%) displaying undeveloped (protogynic stage) gonads after fadrozole treatment. Statistical differences were determined by Williams t-test, one-sided smaller, with *p<0.05 (A-B), and by one-way ANOVA, with Dunnett's multiple comparison test, *p<0.05; **p<0.01; ***p<0.001 (C-D); n = 4 samples/treatment.

Male fish displayed VTG concentrations below or close to the LOD. A tendency to lower concentrations in treatment conditions than in the controls was observed but was not quantifiable. Effects of fadrozole on VTG plasma concentrations of females could not be determined with only one single female found in one replicate of the 10 |ig fadrozole/L treatment. This female displayed a VTG concentration of 0.01 ng VTG/|g protein, which was lower by a factor of 1000 than the mean VTG concentration found in control females.

3.5. Effects of fadrozole on gene expression

Twenty-eight target genes involved in steroid hormone biosynthesis and signaling (Table S1) were analyzed by qPCR. MRNA levels of these genes were assessed for all sampling time points, calculating the log2-fold changes compared to the respective controls. Results are only shown for the most significantly changed genes (Fig. 2 and Fig. 3).

At the end of embryogenesis (48 hpf), 21 of the 28 selected target genes were found to be expressed. No expression could be detected for neuropeptide Y1 receptor (npy1r), gonadotropin-releasing hormone receptor 1, 2 and 3 (gnrhr1, gnrhr2, gnrhr3), insulin-like growth factor binding protein 5a (igfbp5a), prospero homeobox 1 (prox1), and aromatase a (cyp19a1a) (Fig. 4). Statistically significant differences between controls and fadrozole treatments were observed for mevalonate (diphospho) decarboxylase (mvd), vitellogenin 1 (vtg1), and insulin-like growth factor 1 (igf1). MRNA levels of mvd and igf1 was found to be up-regulated up to log2-fold changes of 2, while vtg1 was found to be down-regulated with log2-fold changes between 0 and -1 (Fig. 2A). These differences were statistically significant (*p<0.05) at 10 |g/L fadrozole for mvd and vtg1, and

A 48 hpf В 96 hpf

Fig. 2. Significantly changed genes in embryo (48 hpf) and larval stage (96 hpf) zebrafish, after exposure to fadrozole at nominal 10 |g/L (light grey bars) 32 |g/L (medium grey bars) and 100 |g/L (dark grey bars). Bars depict log2-fold-changes versus the control mRNA levels. (A) At 48 hpf, mvd and igfl were significantly up-regulated and vtgl was down-regulated. (B) At 96 hpf, Iss, vtgl, esr2a, gnrhr2, gnrhr3, were down-regulated and igfl, sox9b, and zgc:64022 up-regulated. Statistical differences of reference-normalized mean transcription values of treatments to control values (Livak and Schmittgen, 2001; Pfaffl, 2001) were determined by one-way ANOVA with Dunnett's multiple comparison test, and by Students t-test; *p < 0.05; **p<0.01; ***p< 0.001; n=3 replicates/treatment. Control-normalized data were log2-transformed prior to presentation.

at 32 |ig/L fadrozole for igfl, and highly significant (**p<0.01) at 32 |g/L fadrozole for mvd.

At the larval stage of 96 hpf, 26 genes were found to be expressed and significant differences compared to the control values were observed for eight genes. No expression was observed for gnrhrl, and follicle stimulating hormone receptor (fshr) (Fig. 4). The genes igfl, SRY (sex determining region Y)-box 9b (sox9b), and zgc:64022 were fadrozole concentration-dependently up-regulated, with

vtgl expression in controls

B low vtgl vs. high vtgl

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10"5 J

factor of 500

C low vtgl

D high vtgl

T3 % -2

o _4. -5 -10 -15

c „ ra 0

-5 t -10 -15

Fig. 3. MRNA levels in pre-adult zebrafish at 63 dpf. (A) Divergence in the relative vtgl mRNA amounts in control zebrafish: Five fish out of two control replicates displayed high vtgl mRNA and ten fish out of three control replicates low vtgl mRNA. Accordingly, the control fish were divided into 5 high and 10 low vtgl expressers. (B) Significant differences in gene expression between low and high vtgl expressers, depicted as log2-fold change. The gene esr2a, and cyp19a1b showed the same discriminating expression levels in control fish than vtgl. (C + D) Significantly changed gene transcripts in fadrozole exposed zebrafish (10 |g/L: light grey bars; 32 |g/L: medium grey bars; 100 |g/L: dark grey bars) compared to (C) low vtgl expressers (control) and (D) high vtgl expressers (control). Statistical differences of reference-normalized mean transcription values of treatments to control values (Livak and Schmittgen, 2001; Pfaffl, 2001) were determined by one-way ANOVA with Dunnett's multiple comparison test, and by Students t-test; *p<0.05; **p<0.01; ***p<0.001; n = 5 fish/replicate; 3 replicates/treatment. Control-normalized data were log2-transformed priorto presentation.

log2-fold changes between 0.5 and up to 2. Differences for sox9b and zgc:64022 were significant (*p<0.05) at 32 |ig/L and for igfl highly significant (**p<0.01) at 10 |ig/L and most highly significant (***p < 0.001) at 32 |g/L and 100 |g/L (Figure 2 B). Lanostyryl synthase (Iss), vtgl, estrogen receptor2a (esr2a), gnrhr2, and gnrhr3 were down-regulated by log2-fold changes in the range of -1. These changes were statistically significant (*p< 0.05) for vtgl and gnrhr2 at 10 |g/L of fadrozole, for esr2a at 32 |g/L, and for gnrhr3 at 100 |g/L fadrozole. Highly significant (**p <0.01) was the difference for lss at 32 | g/L (Fig. 2B).

The developmental stage at 28 dpf showed the lowest overall gene response. All target transcripts were found but none expressed at significantly different fold-change levels. Particularly high variations were observed between replicates.

The pre-adult 63 dpf fish which were analyzed for gene expression, had not been subjected to gonadal sex determination or histopathology. Thus, the morphological sex of these fish was unknown. However, these fish showed divergence in the relative mRNA amounts of vtgl in the control samples (after normalization to the reference genes). A group of control fish exhibiting relative vtgl mRNA amounts of 0.00005-0.0017, separated clearly (by a factor of 500) from a high level vtgl group with mRNA levels of 0.13-0.46 (Fig. 3A). A sex-dependent expression of the vtgl mRNA was assumed and consequently, the control fish divided into a low and a high level vtgl group for analysis. Accordingly, the mRNA levels of the other target genes were compared between the "low" and "high" vtgl expresser control groups. Twenty-five of the 28 target genes were expressed in the controls; no transcrip-

tion was detected for kiss1 receptora (kiss1ra), gnrhr1, and gnrhr2. The log2-fold change differences of low vtg1 compared to high vtg1 expressers (Fig. 3 B) were highest for vtg1 (-9.0; most highly significant, ***p< 0.001), at -0.8 for esr2a, and at -3.1 for the brain aromatase (cyp19a1b; both significant, *p<0.05). Large but nonsignificant log2-fold change differences were additionally observed for gnrhr4 (-5.8) and cyp19a1a (approx. 5.0) (Fig. 4).

Gene expression measured in the fadrozole treated fish was compared to low as well as high vtg1 expressing controls. According to the results of the histopathological examinations, which identified 100% of fadrozole-treated fish at 63 dpf as males (compare skewed sex ratio described in Section 3.3), no statistically divergent vtg1 mRNA expression in individual treatments was observed. Compared to the low vtg1 controls, a significant induction was observed for Iss at 32 |ig/L and 100 |ig/L of fadrozole, and for star and prox1 at 100 |g/L(all **p<0.01; Fig. 3C).

Compared to the high vtg1 expressers of the controls, more genes were found to be differentially expressed in fadrozole treated 63 dpf fish. Statistically significant differences were observed for Iss, vtg1, star, esr2a, igfbp5a, prox1, cyp19a1b, and zgc:64022. Highly significantly (**p <0.01) up-regulated was Iss at fadrozole concentrations of 32 |g/L and 100 |g/L, and still significant (*p < 0.05) was star, igfbp5a, and prox1 at 100 |g/L. Down-regulation was most highly significant (***p < 0.001) for vtg1 at all fadrozole concentrations, and for esr2a at 32 | g/L. Highly significant down-regulation was observed for esr2a at 10 |g/L and 100 |g/L; still significant was cyp19a1b and zgc:64022 at all concentrations (*p<0.05). The log2-fold changes ranged from -9.0 for vtg1 to 2 for Iss (Fig. 3D).

48 hpf 96 hpf 28 dpf 63 dpf low VTG 63 dpf high VTG

10ug/L 32 pg/L 100uq/L 10 ug/L 32 Mg/L ioo ug/L 10 ug/L 32 Mg/L 100 pg/L 10(ig/L 32 pg/L 100 ug/L 10 pg/L 32 ug/L ioo ug/L

Steroid and terpenoid synthesis mvd * **

Iss ** ** ** ** **

sc4mol

Lipid transport/ lipid metabolic process vtgl * * * *** *** ***

star ** *

apoAla

Steroid receptors esr1

esr2a * *** **

Neuro-peptide receptor activity kisslrb

kisslra

npySbr

gnrhrl

gnrhr2 *

gnrhr3 *

gnrhrf

Cell growth igfbp5a *

igf 1 * ** *** ***

Transcription sox9b *

Hematopolesis proxl ** *

Aromatases cyp19a1a

cyp19a1b * * *

Others fshr

zgc:64022 * * * *

* statistically significantly different from control; significance levels: *p<0.05;**p<0.01; "*p<0.001

Colours based on log2-fold expression compared to the control at the respective time point.

Log2 fold change

upregulation

downregulation

no expression

Fig. 4. Heatmap-like illustration of the Iog2-fold changes of all target genes in relation to control fish at the respective time points. Color and intensity reflect the mean Iog2 fold-change values of each gene at a given sampling time point and treatment group, with red shades representing down-regulation (negative log2-fold values) and green shades representing up-regulation (positive log2-fold values); the highest color intensity reflects the strongest regulation at the given sampling time point. Light yellow denotes no regulation. White cells indicate that no transcription was detected. Asterisks (*) in the cells denote levels of significance according to the footnote below the table. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A control expression

CM (1. O) u

igfl star Iss

cyp19a1b

48 hpf 96 hpf 28 dpf 63 dpf 63 dpf low VTG high VTG

48 hpf 96 hpf 28 dpf 63 dpf

E 1 cyp19a1b

a> a> c

I— 1

48 hpf 96 hpf 28 dpf

63 dpf

ft It *f

48 hpf 96 hpf 28 dpf 63 dpf

n vtg1

48 hpf 96 hpf 28 dpf

63 dpf

48 hpf 96 hpf 28 dpf 63 dpf

□ 10 pg/L

□ 32 pg/L H 100 pg/L

□ □ □

compared to control low vtgl expression compared to control high vtgl expression compared to control low vtgl expression compared to control high vtgl expression

10 pg/L 32 pg/L

100 ua/L comParecl ,0 control low vtgl expression pg'L compared to control high vtgl expression

Fig. 5. MRNA levels of the potential marker genes igf1, star, Iss, cyp19a1b, and vtg1 overtime. (A) Average expression levels in control fish at the different analysis time points. Data depict relative fold-changes normalized to the mRNA level at 48 hpf. (B-F) Fadrozole-induced log2 fold- changes of igf1, star, Iss, cyp19a1b, and vtg1 compared to the corresponding control groups at each analysis time point. Statistical differences of reference-normalized mean transcription values of treatments to control values (Livak and Schmittgen, 2001; Pfaffl, 2001) were determined by one-way ANOVA with Dunnett's multiple comparison test, and by Students t-test; *p<0.05; **p<0.01; ***p< 0.001; n = 3 replicates/treatment (48/96 hpf, 28 dpf); n = 5 fish/replicate; 3 replicates/treatment (63 dpf). Control-normalized data were log2-transformed prior to presentation. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

As Fig. 4 illustrates, statistical analysis revealed that 13 out of the 28 target genes showed significant regulation for at least one treatment level and time point, seven genes were found to be regulated in more than one exposure at a given time-point and five genes (Iss, vtg1, esr2a, igf1, zgc:64022) were regulated at more than one time point. No gene was significantly regulated at all developmental stages investigated. Based on the mRNA expression patterns (Fig. 4), potential marker genes for aromatase inhibition were identified, which responded significantly at least to two fadrozole exposure concentrations, or at two developmental stages. Accordingly, igf1, star, Iss, cyp19a1b, and vtg1 were selected as potential markers and analyzed in more detail in terms of their transcription over time at control conditions as well as in response to fadrozole treatment (Fig. 5). At control conditions, the mRNA of lss, igf1 and cyp19a1b

showed a steady increase from 48 hpf to 63 dpf (Fig. 5A) whereas star was down-regulated at 96 hpf and 28 dpf compared to 48 hpf, before returning to the 48 hpf level at 63 dpf. Expression of vtg1 showed a decrease below the 48 hpf level at the juvenile stage (28 dpf) before it increased sharply in the female fish with sexual maturation at 63 dpf. In male fish, the vtg1 mRNA was on average slightly above the 48 hpf level. Sex related differences in mRNA levels of control fish at 63 dpf were, apart from vtg1, also displayed by cyp19a1b.

Fadrozole exposure affected the five marker genes differently in the course of development (Fig. 5B-F). During early life, igf1 (Fig. 5B) was up-regulated at 48 hpf (significantly at 32 |ig/L fadrozole) and at 96 hpf (significant at all treatment levels), Iss (Fig. 5D) was down-regulated at 96 hpf at a concentration of 32 |ig/L, and

vtg1 (Fig. 5F) responded by up-regulation at 48 hpf (200 |ig/L) and 96 hpf (10 |g/L). On the other hand, a response to fadrozole of star (Fig. 5C) and cyp19a1b (Fig. 5E) only occurred at 63 dpf, suggesting that the reduced levels of estrogen affected the transcription of these genes only at a later developmental stage. Most notably, cyp19a1b was down-regulated in the 63 dpf exposed fish only in relation to the high vtg1 control group. Most importantly, vtg1 was strongly down-regulated in relation to the high vtg1 putative female controls at 63 d. This result is consistent with results of histopathology showing that almost all fadrozole treated fish were morphologically identified as males.

4. Discussion

Alternative concepts are currently being pursued in regulatory ecotoxicology to discriminate, prioritize, and assess endocrine disrupting chemicals (EDCs). These concepts integrate in silico and molecular data to better explain adverse outcomes of regulatory concern (Ankley et al., 2010; Bradbury et al., 2004; Meeket al., 2014; NRC, 2007; Perkins et al., 2015; Villeneuve and Garcia-Reyero, 2011). The knowledge of the responses at the molecular level and how these responses relate to a particular adverse outcome is instrumental in predicting endocrine disruption more reliably and possibly earlier than traditionally, where only apical effect endpoints and few physiological biomarkers are used. The aim of the study was thus to investigate the value of additional molecular endpoints, like gene expression, for the identification of EDCs and potentially also the mode of endocrine action, prior to the manifestation of adverse effects.

Classified as a level 4 study, the FSDT covers early developmental stages as well as the phase of sexual maturation of zebrafish (Maack and Segner, 2003). It therefore delivers apical and physiological data, as well as the possibility to obtain early molecular data from treated embryos or larvae.

During the FSDT, we observed a weak, but significant effect on post-hatch survival at 28 dpf (Fig. 1A). This observation suggested low systemic toxicity, which could be explained by an elevated stress susceptibility known for larval stages of fish, especially after chemical treatment (Belanger et al., 2010; Diekmann and Nagel, 2004; Woltering, 1984). During the larval stage, the energy metabolism changes from intrinsic nutrient absorption from the yolk to external feeding, which often results in increased mortality also under control conditions. Supporting this assumption of increased sensitivity of larval fish, zebrafish embryos exposed to even higher fadrozole concentrations (0.1-1.0 mg fadrozole/L; unpublished results) did not display increased mortality. Generally, factors such as systemic toxicity were proposed to influence endpoints known as endocrine indicators in a non-endocrine manner (Wheeler et al., 2013). However, it was deemed very unlikely that the observed effects of fadrozole on sex ratio and gonad maturation (Fig. 1B-D) in the present study were a result of systemic toxicity. Fadrozole has a very specific aromatase inhibition MoA, and the same effects on zebrafish sex ratio have already been described elsewhere (Fenske and Segner, 2004; Takatsu et al., 2013). Accordingly, we concluded that the observed effects on gene expression were also caused by aromatase inhibition and not by systemic tox-icity.

During development, zebrafish gonads respond particularly sensitive to aromatase modulations during the phase of gonadal sex differentiation. They are described as undifferentiated gono-chorists, which develop via a stage described as non-functional protogyne gonad (Maack and Segner, 2003). This undifferentiated gonadal stage is influenced by the estrogen-to-androgen ratio during steroid biosynthesis. An imbalanced ratio easily results in a skewed sex ratio. Thus, treatment with an aromatase inhibitor like

fadrozole decreases the estrogen level and consequently results in an increased male-to-female ratio (Fenske and Segner, 2004; Takatsu et al., 2013). Additionally, steroid biosynthesis may be stimulated by positive feedback regulation due to low levels of estrogens via a loop induced. This would lead to even higher testosterone levels with aromatase conversion being inhibited (Ankley and Villeneuve, 2015; Villeneuve et al., 2013, 2009). The underlying regulatory molecular mechanisms are not straightforward as gene expression regulation can occur directly (e.g. via genes possessing an estrogen receptor-responsive element (ERE) in their promoter region) and indirectly (via steroid biosynthesis-related genes at gonadal level). The histopathological results verified the highly potent masculinizing effect of fadrozole in this study, with 99% males already at the lowest fadrozole concentration of 10 |g/L (Fig. 1B-D). It also indicated a more rapid maturation of the gonads. The average maturity index of the male gonads increased from approximately 2.0 in the control groups to approximately 2.7 at the highest fadrozole concentration (Fig. 1C). A promoting effect on the maturity index of zebrafish was described by Baumann et al. (2013) also for other masculinizing substances. This observation underpins the assumption that aromatase inhibition elevates levels of androgen and in turn, stimulates testis maturation. Further strengthening evidence is provided e.g., by Seki et al. (2006), who reported elevated gonadosomatic indices, and Morthorst et al. (2010), who reported a stimulating effect on testis maturation in zebrafish after exposure to 17£-trenbolone (a strong androgen).

However, particular attention of the study was directed to the gene expression endpoints. Even though mRNA synthesis does not unequivocally result in protein expression (i.e. translation of transcribed genes, compare Nikinmaa and Rytkonen, 2011), transcription level changes can serve as measurable markers in the cascade form the initial event to effect manifestation at organism level. Thus, transcription of selected genes involved in regulatory events of hypothalamus-pituitary-gonadal (HPG) signaling, steroidogenesis, steroid signaling, and signaling responses was analyzed at key developmental stages during early life (48 hpf, 96 hpf) and early (28 dpf) and late puberty (63 dpf). Five of the 28 selected genes showed significant regulation by fadrozole at more than one concentration and time point and were therefore considered promising candidates for biomarkers of aromatase inhibition. These genes are igfl, star, Iss, cypl9alb and vtgl. The corresponding proteins are involved in the regulation of transcription factors, steroid/terpenoid synthesis, cholesterol transport, and direct regulation of expression by estradiol (see Fig. 6).

Significant responses to fadrozole were not restricted to these genes but the discussion primarily focuses on these genes in terms of their function as biomarkers for the identification of endocrine disruptive properties of tested substances. In order to put the tran-scriptional changes caused by aromatase inhibition into a broader perspective, also the role of the corresponding proteins in steroido-genesis was contemplated. Alterations at the transcription level often results in changes at the translational level, even though post-transcriptional processing and regulation disallow a one-to-one transfer from transcription to translation (Nikinmaa and Rytkonen, 2011).

The vtgl gene is a well-established estrogenic biomarker (Muncke and Eggen, 2006) as it contains an ERE in its promoter region. In addition, vtgl is expressed at reliably measurable mRNA and protein levels in adult zebrafish only in females (Wang et al., 2005). The differential transcription of vtgl in control fish at 63 dpf was interpreted as sexually dimorphic analogue to the VTG protein. Therefore, the division into high and low vtgl mRNA levels of controls at 63 dpf, allowed us to perform a differential sex-related analysis of the other genes without the knowledge of the histopathological sex of these fish. Thus, the results obtained for fadrozole-treated fish discerned genes that were significantly

Testosterone *-► 17p-estradiol (E2)

Fig. 6. Role of the selected biomarker genes within the steroid biosynthesis pathway. Fadrozole exposure (pink star) directly inhibited the activity of the aromatase and subsequently also of cypl9alb (and the other isoform cypl9ala; not shown). The conversion of testosterone to estradiol (E2) is inhibited and the decrease in E2 directly lowers the transcription of the ERE-responsive genes vtgl, and cypl9alb. Aromatase inhibition also has a stimulating effect on the steroid biosynthesis affecting key steps, e.g. IGF-R signaling, the synthesis of fatty acids to cholesterol or the cholesterol transport to the inner membrane of the mitochondrion. The genes igfl, lss, and star are involved and respond either already early (igfl) or later during puberty (lss and star) with up-regulation. In contrast to the ERE-responsive genes, regulation of these genes is subject to mostly indirect control mechanisms, which complicates the prediction of expression changes.

changed compared to putative females as differentially expressed due to sex. Genes that were significantly changed compared to both control groups, suggested their involvement in a fadrozole related but sex independent steroid biosynthesis dysregulation. Expression of vtgl mRNA in the fadrozole exposed fish showed down-regulation already in the early developmental stages (48/96 hpf, Fig. 2), which was not surprising since the estrogen responsiveness of vtgl in zebrafish embryos as early as 48 hpf was known from previous estrogenic exposures (Schiller et al., 2014, 2013b). At 63 dpf, vtgl showed a strong down-regulation versus the high vtgl control group, and no regulation compared to the low vtgl control group, likely due to the difference in estrogen levels and consequently, in VTG concentrations in females and males. This result is consistent with results of histopathology showing that almost all fadrozole-treated fish were morphologically identified as males. Moreover, these results are in line with the in AOP of aromatase inhibition established in adult females in which vtgl down-regulation at mRNA and protein level was identified as KE.

Similar to vtgl, transcription of cypl9alb is controlled by an ERE in its promoter region. Accordingly, its mRNA level was significantly down-regulated, as it was anticipated for genes requiring the binding of estrogen-bound estrogen receptors for activation. Aro-matase inhibition leads to reduced estrogen levels, thus inhibiting the expression of ERE-possessing genes. Transcriptional regulation of these genes by aromatase inhibitor treatment or other estro-genic disruptors has been described for fish in several studies (Filby

et al., 2007; Heppell et al., 1995; Schiller et al., 2014; Wang et al., 2010). Quantification of mRNA of ERE-controlled genes can thus be considered a direct marker of aromatase inhibition in pubescent zebrafish.

The proposed marker gene igfl showed a concentration-dependent increase in mRNA expression at all investigated time points (compare Figs. 2-4), although the up-regulation was significant only at early developmental stages (48/96 hpf). The IGF1 protein is related to the growth hormone (GH)/insulin-like growth factor (IGF) axis, which is essential for normal growth and development in vertebrates. For fish, the expression of igf genes, including igfl , and subsequent translation into proteins, plays a crucial role in normal gonadal function, and modulation by estrogenic disruptors has been postulated (Brown et al., 2011; Reinecke, 2010). Furthermore, it was shown that Igf1 protein stimulated aromatase activity and also cypl9al gene expression in red sea bream (Kagawa et al., 2003). In turn, the observed up-regulation of igfl mRNA in the present study may be interpreted as a compensatory mechanism to the aromatase inhibition of fadrozole. This assumption would be supported by evidence found for brown trout (Marca Pereira et al., 2014), demonstrating the up-regulation of igfl mRNA upon treatment with prochloraz, an imidazole fungicide known to act as aromatase inhibitor.

In contrast to igfl , transcription of star was exposure concentration-dependently up-regulated only at 63 dpf in both high and low vtgl expressers. A differential expression of star upon treatment with EDCs was already evidenced for early life stage fathead minnow and adult goldfish (Johns et al., 2011; Sharpe et al., 2007). Since StAR protein coordinates cholesterol transport to the inner mitochondrial membrane, which is a rate-limiting step during steroidogenesis (Johns et al., 2011), we assumed a functional role of star in maintenance of steroid concentrations in adulthood rather than during development. The up-regulation of star mRNA would indicate an increased demand in cholesterol shuttling due to the stimulation of steroidogenesis caused by the aromatase inhibition. The up-regulation of star mRNA was equally strong in putative genetic male and female zebrafish (low and high vtgl expressers) and thus, suggested an upstream regulation of star transcription rather than through the downstream imbalance in estrogen synthesis.

The functional role of the protein lanostyryl synthase during steroid biosynthesis is the synthesis of cholesterol from fatty acids. Together with sc4mol (see Fig. 4), which is involved in the same key process, the lss gene was found down-regulated early in development (significant at 96 hpf), and up-regulated at 63 dpf. Positive regulation of lss was already described for estrogenic and anti-androgenic substances in 48 hpf zebrafish and 7 dpf medaka (Schiller et al., 2014). An anti-estrogenic effect of aromatase inhibition in the present study could therefore explain the suppression of lss transcription at 96 hpf. The up-regulation of lss mRNA at 63 dpf rather signified a higher demand for cholesterol due to the stimulation of steroid biosynthesis (in compensation of aromatase inhibition), which would be expected to be higher in the putative females.

Four of the five genes which we selected as potential biomark-ers, based on their regulation upon fadrozole treatment at different developmental stages, were considered potential markers of estrogenic disruption already earlier. Johns et al. (2011) demonstrated regulation of igfl, star, vtgl in embryo and larval fathead minnow after exposure to estrogenic and anti-estrogenic EDCs. Furthermore, Hao et al. (2013) performed a study with larval zebrafish and E2 and found regulation of vtgl and cypl9alb starting from 4 dpf. We postulate that the transcription of these genes is applicable as additional marker endpoint to facilitate the interpretation of FSDT results. The verification of this hypothesis, however, will require further investigations involving different EDCs.

The results of the present study did not only provide valuable findings on potential gene biomarkers for the FSDT, they also allowed us to extend the scope of the current AOP of aromatase inhibition. The existing AOP for aromatase inhibition (Ankley et al., 2010, 2009; aopkb.org/aopwiki/index.php/Aop:25) was developed based on data obtained by Fish Short Term Reproduction Assays (FSTRAs) with fathead minnow. It describes the gonadal aromatase inhibition in adult females, which results in reduced female fecundity due to reduced VTG synthesis. When transferring this AOP of aromatase inhibition to the sensitive life stage of fish sexual development and gonad maturation, the adverse outcome (AO) changes to an altered sex ratio. The MIE of fadrozole exposure during fish sexual development is the effective inhibition of the P450 aromatase (CYP19), which regulates the conversion of C19-androgens to C18-estrogens (Callard et al., 1978). This conversion is a key event of the steroid biosynthesis pathway and its inhibition leads to a reduction of estrogen synthesis and subsequently lowers E2 plasma levels. In developing zebrafish, this estradiol imbalance promotes testis differentiation in a concentration dependent manner.

In adult females, the reduction on E2 plasma level impacts negatively on ERE-regulated genes like vtgl and cypl9alb and consequently reduces the synthesis of VTG, which in turn diminishes the oocyte quality and maturation in female fish as the next key event, leading to reproductive dysfunction. Both alternative AOPs of aromatase inhibition end in declining population trajectories, independent of the developmental stage affected by aromatase inhibition. The mRNA levels that were identified to be regulated at the different life stages, in particular of the directly estrogen regulated genes vtgl and cyp19a1b, could represent useful biomarkers supporting a KE at the molecular level (OECD, 2014). In this way they can be integrated into the existing AOP of aromatase inhibition (aopkb.org/aopwiki/index.php/Aop:25).

In conclusion, this study was able to provide further valuable evidence that the implementation of molecular endpoints in current testing procedures for EDCs can provide a feasible and informative tool for the prioritization of EDCs. Moreover, the identification of additional marker genes for supporting the characterization of the MoAs of endocrine-disrupting chemicals can be instrumental in defining new KEs of existing or new AOPs related to endocrine disruption.

Acknowledgements

This project was funded by the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (FKZ 371263418). Martina Fenske was also co-funded by the Landes-Offensive zur Entwicklung Wissenschaftlichökonomischer Exzellenz (LOEWE) of the Federal State of Hessen, Germany. We thank Helmut Segner and Lisa Baumann, Centre for Fish and Wildlife Health, University of Bern, for performance of histopathological examinations. We also thank Walter Böhmer for performing the chemical analysis and Maike Lutter und Tom Lingner (all Fraunhofer IME) for assisting in the qPCR analysis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016Zj.aquatox.2016.04. 018.

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