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Aquatic Toxicology 176 (2016) 116–127

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

inking the response of endocrine regulated genes to adverse effectsn sex differentiation improves comprehension of aromatasenhibition in a Fish Sexual Development Test

lke Muth-Köhnea,∗, Kathi Westphal-Setteleb, Jasmin Brücknerb, Sabine Konradib,iktoria Schillerc, Christoph Schäfersa, Matthias Teigelera,1, Martina Fensked,1

Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, GermanyGerman Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, GermanyFraunhofer IME, Attract Group UNIFISH, Forckenbeckstraße 6, 52074 Aachen, GermanyFraunhofer IME, Project Group Translational Medicine and Pharmacology TMP, Forckenbeckstraße 6, 52074 Aachen, Germany

r t i c l e i n f o

rticle history:eceived 23 December 2015eceived in revised form 13 April 2016ccepted 19 April 2016vailable online 20 April 2016

eywords:romatase inhibitionebrafishPCRadrozoleish sexual development test (FSDT)dverse outcome pathway (AOP)

a b s t r a c t

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrinedisrupting chemicals. With the present study it was intended to evaluate whether gene expression end-points would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDTaccording to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (testconcentrations: 10 �g/L, 32 �g/L, 100 �g/L) using zebrafish (Danio rerio). Gene expression analyses usingquantitative RT-PCR were included at 48 h, 96 h, 28 days and 63 days post fertilization (hpf, dpf). Theselection of genes aimed at finding molecular endpoints which could be directly linked to the adverseapical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexualdevelopment of zebrafish were a complete sex ratio shift towards males and an acceleration of gonadmaturation already at low fadrozole concentrations (10 �g/L). Due to the specific inhibition of the aro-matase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, thesteroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is theregulation 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 com-pared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls asearly as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlledby endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho)decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), 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 OutcomePathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentia-tion. We were further able to identify MoA-specific marker gene expression which can be instrumentalin 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

. Introduction

Environmental pollutants which interact with the endocrineystem of organisms, and exert a negative impact on devel-pment and reproduction, are commonly known as endocrine

∗ Corresponding author.E-mail address: elke.muth-koehne@ime.fraunhofer.de (E. Muth-Köhne).

1 These authors contributed equally to the project.

ttp://dx.doi.org/10.1016/j.aquatox.2016.04.018166-445X/© 2016 The Author(s). Published by Elsevier B.V. This is an open access articl.0/).

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

disrupting chemicals (EDC). Endocrine disruption manifests inan organism or a population after sub-acute to chronic (long-term) exposure, which makes the experimental identificationof hormone-active substances time- and cost-intensive. Theseelaborative experiments require large numbers of animals, andalternatives 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 weredesigned as screening tools to trigger more complex studies likea fish life cycle test, which are required to assess the endocrine

e under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

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isruption relevant apical endpoints. These shorter and less fishonsuming predictive tests have, in principal, the potential toeduce the number of long-term apical tests. However, apical effectata from screening assays could not be used for regulatory pur-oses.

Most life-stage confined tests like the Fish Early Life Stage Test,FELS; OECD TG 210 (OECD, 2013)) are not designed to provideubstantive information about the chemical mode-of-action (MoA;illeneuve et al., 2014) and are prone to interpretation error, as

hey exclude relevant apical endpoints, like the determination ofhe sex ratio. It seems therefore logical to include quick respondingarameters which may also be indicative of the underlying MoA orinpoint the molecular initiating event (MIE).

Molecular endpoints respond promptly to pollutants andromise to provide information indicative of adverse effects atrganism level (Piersma, 2006). Powerful genomic approaches haveontributed essentially to the determination of molecular mecha-isms resulting in apical adverse endpoints (Ankley et al., 2006;awle et al., 2010). Global transcriptomics allow meaningful anal-ses of adverse effects in cells, organs, and the whole organism atoncentrations potentially below thresholds of the most sensitivendpoints of the “conventional” studies. This higher sensitivity ofolecular analyses is crucial to prevent the overlay of endocrine

ffects by other effects, especially systemic toxicity (Scholz andayer, 2008; Wang et al., 2010). The progress in molecular and

omputer-based (in silico) methods paved the way for the shiftf paradigms in ecotoxicology (NRC, 2007), and for the develop-ent of causal mechanism-based concepts for environmental risk

ssessment, e.g. the Toxicity Pathways (Bradbury et al., 2004) orore recently the Adverse Outcome Pathways (AOPs; Ankley et al.,

010; Villeneuve and Garcia-Reyero, 2011). Specific AOPs aim torovide causal links between the MIE and the adverse outcome (AO)f regulatory concern via well-established, measureable key eventsKEs) and key event relationships (KERs), which are intended toacilitate regulatory decision making (Kramer et al., 2011; Segner,011, 2009). To date several AOPs have already been defined forndocrine disruption (aopkb.org/aopwiki). However, the AOP forromatase inhibition is only applicable to sexually mature femalesh, as the AO at the organism level is reduced fecundity (Ankleyt al., 2010, 2009; aopkb.org/aopwiki/index.php/Aop:25), and noeasureable KE is described at the molecular level.Zebrafish are particularly sensitive to aromatase inhibitors dur-

ng gonadal sex differentiation and maturation, the phase betweenhe early life stage and adulthood (Baumann et al., 2015, 2013;enske and Segner, 2004; Kinnberg et al., 2007). As undifferentiatedonochorists, zebrafish’ gonads develop via a stage described ason-functional protogyne gonad (Maack and Segner, 2003). Testisifferentiation and male maturation are triggered by increasing lev-ls of androgens, while maturation of ovaries requires higher levelsf estrogens. Aromatase inhibitors block the synthesis of estrogensy inhibition of the conversion of C19-androgens to C18-estrogensCallard et al., 1978). The surplus of androgens consequently favors

asculinization in the population (Baumann et al., 2015, 2013;enske and Segner, 2004; Morthorst et al., 2010; Trant et al., 2001).

The Fish Sexual Development Test (FSDT; OECD TG 234, OECD,011) allows the analysis of effects of endocrine disruptors on thearly life stage and sexual development. The FSDT is essentially anxtension of the FELS test. It was originally developed as a “Fish Par-ial Life Cycle Test” and considered an alternative to specifically testndocrine disruptors in fish without the requirement of a wholeife span exposure. It covers the endocrine disruption prone devel-pmental period of sexual differentiation of particularly zebrafish

nd allows the assessment of gonadal differentiation and sex ratios endocrine disruption-associated endpoints (Thorpe et al., 2011).owever, the information on the endocrine system functions which

cology 176 (2016) 116–127 117

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

The conceptual framework of the OECD for Testing and Assess-ment of Endocrine Disruptors (OECD, 2012a) is mainly focused onthe assessment of chemicals disrupting estrogen, androgen, thy-roid signaling processes and steroidogenesis (EATS), but there areseveral other endocrine pathways (e.g. corticosteroid, gestagenic,or retinoid signaling pathways), which additionally may be dis-rupted by chemical exposure. The use of additional new endpointsmay help in better understanding the link between the initiatingendocrine related mechanisms and resulting apical effects. Thiswould strengthen an AOP-driven approach in the context of theweight-of-evidence strategy that is currently pursued in the reg-ulatory context to assess an endocrine disruption hazard (OECD,2012b). The need for more comprehensive methods has beenacknowledged and highlighted in the Review paper of the OECDon Novel In Vitro and In Vivo Screening and Testing Methods andEndpoints for Evaluating Endocrine Disruptors (Kortenkamp et al.,2011; OECD, 2012b).

The rationale of the current study was to demonstrate that geneexpression data could help to increase the evidence on whether andhow a substance may act as an EDC. To this end, an FSDT accord-ing to the OECD guideline 234 was conducted with the aromataseinhibitor fadrozole and refined by mRNA analysis using quantita-tive RT-PCR. The rationale to choose fadrozole as test substance isthat its endocrine disrupting effects are reasonably well defined forfish (Afonso et al., 2000; Ankley et al., 2002; Fenske and Segner,2004; Scholz and Klüver, 2009; Takatsu et al., 2013; Villeneuveet al., 2013; Wang et al., 2010; Zhang et al., 2008). Fadrozole isa potent, selective non-steroid aromatase inhibitor. Although itsinteraction with the enzyme is not yet fully understood, coordina-tion with the iron of the prophyrine core of the CYP19 enzyme islikely. This describes a characteristic general function of type-II-inhibitors, which was first described in mammals (Browne et al.,1991). Fadrozole therefore exhibits a specific aromatase-inhibitingMoA with fewer side effects than e.g. prochloraz, which inhibitssteroidogenesis as well as androgen receptor signaling (Ankleyet al., 2005; Laier et al., 2006).

In this study, a set of endocrine-related genes was measured inearly life stages, and in juvenile and pre-adult fish at the end ofthe 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 previousresults of our own and published studies (Schiller et al., 2013a,2013b; Villeneuve et al., 2009), which indicated their sensitivityto endocrine disruption and especially aromatase inhibition. Thegroup of selected genes covers also several critical steps in steroidbiosynthesis.

Taken together, this study demonstrated that the existingAdverse Outcome Pathway (AOP) for aromatase inhibition in fishcan be translated to the life stage of sexual differentiation. Fur-ther, we were able to identify marker gene transcription involvedin steroid biosynthesis which can be applied as measures of keyevents (KE) at the molecular level.

2. Material and methods

2.1. Test species

Wild-type zebrafish (Danio rerio) originally obtained from WestAquarium GmbH (Bad Lauterberg, Germany) and continuously bred

for several generations in the Fraunhofer IME laboratories, wereused for testing. Zebrafish of this line correspond in their juve-nile/sexual development to the line described by Takahashi (1977)and Maack and Segner (2003).

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Fish of the broodstock were maintained under flow-throughonditions in 150 L tanks at 25 ± 2 ◦C on a 12:12-h photoperiod in aemperature-controlled room. They were fed daily with TetraMin®

ain feed ad libitum and nauplii of Artemia salina.For egg collection, stainless steel spawning trays were inserted

nto the tanks, and spawning was induced at the beginning of theight period (light intensity: 1000 lux). The eggs were microscopi-ally examined for fertilization success. Only healthy eggs in at leastlastula stage were inserted into the test system.

.2. Test substance

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

.3. General exposure conditions

Purified tap water was used according to the OECD-Guideline10 (OECD, 2013). The purification included filtration with acti-ated charcoal. The water was aerated to the level of oxygenaturation. The water quality was monitored in the testing facility.

Water temperature in the test vessels was adjusted to 25 ± 2 ◦C.xygen saturation of the test solutions was between 99.0% and03.4% of air saturation. The pH was between 8.08 and 8.17 in theest tanks.

.4. Assessment of Fish Sexual Development test (OECD TG 234)ndpoints

The Fish Sexual Development Test (FSDT) was performedccording to the OECD Test Guideline 234 (OECD, 2011) in a flow-hrough exposure system. Minor modifications of the protocol wereecessary to include gene expression analysis at different timeoints.

The test was performed with three test concentrations (10 �g/L,2 �g/L, and 100 �g/L.) under flow through conditions, with foureplicate tanks per concentration run in parallel. A number of 85ggs per replicate tank (12 L total volume) were introduced at testtart divided into two stainless steel fry cages. One fry cage of 50ggs provided samples for the gene expression analysis, the otherry cage of 35 eggs remained undisturbed.

The eggs were kept in the fry cages until hatching and theumber of hatched larvae was estimated at 4 and 6 days post fer-ilization (dpf). At 21 dpf, larvae were released from both fry cagesnto the main tank. After the early life stage at 28 dpf, the juvenilesh were photographed for image-based evaluation of survival androwth (ImageTool of the University of Texas Health Science Centert San Antonio, Version 3.0). Fish remained in the test system untilest termination at 63 dpf. At test termination, survival rate, size inerms of length and weight, and the sex of all fish were determined.wenty fish per replicate were analyzed for their vitellogenin (VTG)ontent, and were histopathologically evaluated.

For gene expression analysis, 20 embryos of 48 and 96 h postertilization (hpf) were sampled from each replicate, pooled in a.5 mL Eppendorf tube and snap frozen. At 28 dpf, 4 randomly cho-en larvae per replicate were pooled and snap frozen in a 2.0 mLppendorf tube; at test termination, the trunks of 5 fish per repli-ate (heads removed) were randomly chosen for gene expressionnalysis and snap frozen individually. The heads were removed in

rder to focus on endocrine effects related to gonad development.ll samples were stored at −80 ◦C until further use.

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

cology 176 (2016) 116–127

fish except those dedicated to gene expression analysis. Gonadswere incubated in modified Davidsons’s fixative 20% formalde-hyde (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 orien-tated ventrally to the cutting surface. Sections of 4–5 �m were cutwith a microtome, mounted on glass slides and then stained withhematoxylin-eosin.

Microscopic evaluation of the tissue sections was performedaccording to the OECD Histopathology Guidance Document (OECD2010). 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 pri-mary and secondary diagnostic evaluation criteria.

For determination of the VTG levels, an enzyme-linkedimmunosorbent assay (ELISA) for zebrafish VTG (homologous ELISAkit, Biosense, Bergen, Norway) was used according to the manu-facturers’ instructions. Plasma samples were measured in 96-wellplates 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 ineach assay. The VTG concentrations were normalized to the totalplasma protein content, and data expressed as ng VTG/�g pro-tein (Fenske et al., 2001). Total protein was quantified using a BCAProtein Assay Reagent Kit (Pierce, Rockford, USA).

2.5. Quantitative RT- PCR (QPCR)

Total RNA was isolated from homogenized frozen trunk tis-sue (63 dpf) or pooled embryos/larvae/juveniles using TRIreagent(Sigma-Aldrich, T9424) method followed by a clean-up withRNeasy Mini Spin Columns (QIAGEN, Hilden, Germany) accordingto the manufacturers’ protocols. Prior to cDNA synthesis, RNA wasDNAse (Sigma-Aldrich, AMP-D1) treated and subsequently mea-sured using a NanoDrop 1000 spectrophotometer (Thermo FisherScientific, Schwerte, Germany). Reverse transcription was car-ried out using SuperScriptTM III Reverse Transcriptase (Invitrogen,Carlsbad, USA). Negative controls without reverse transcriptasewere prepared for each sample.

QPCR analysis was performed on three of four replicates ofeach treatment (fadrozole exposures and controls) at each sam-pling time point, using a BioRad iQ5 real-time PCR detectionsystem (BioRadTM, Hercules, USA). The fourth replicate sampleswere excluded from the analysis due to RNA quality issues. For eachreaction, 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 andthe respective reference genes were run for each cDNA sample.Minus template controls were included on each plate; minus RTcontrols were included repeatedly, at least once for each sample,however not in every run. The reference genes 18s and rpl8 wereused to normalize the expression values of the target genes.

The PCR protocol was as follows: After incubating for 2 minat 50 ◦C and for 10 min 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 par-ticular genes) and 60 ◦C for 30 s (for acquisition; 40 cycles); therun was finalized by a melting curve analysis: 95 ◦C for 60 s, then

55 ◦C for 60 s (80 cycles, with +0.5 ◦C increment each cycle and a10 s acquisition). Relative gene expression ratios were calculatedby the comparative delta Ct method (ddCt) (Livak and Schmittgen,2001; Pfaffl, 2001).

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.6. Chemical water analysis

Water samples of 10 mL volume were taken from each tank fromhe mid water body, at test start, and thereafter once a week. Theamples were taken alternating from one of the two test vesselserved by one dosing pump. Water samples were stabilized by addi-ion of acetonitrile (1:1; v + v) containing 0.2% formic acid prior totorage.

Fadrozole analysis was performed by high performance liq-id chromatography tandem mass spectrometry (LC–MS/MS)ith negative ionization. Data were collected on a Waters 2695

eparations module coupled to a Quattro-Micro tandem mass spec-rometer (Waters). Aliquots of 50 �L were injected into a Gemini18 high-performance LC column (150 mm × 3 mm, 5 �m parti-le size; Phenomenex) at a flow rate of 0.5 mL/min and a columnemperature of 30 ◦C. Matrix-free procedural blanks were run eachorking day to exclude possible cross-contaminations during lab-

ratory work. Separation was performed on a binary gradient of0 mmol ammonium acetate solution in (A) methanol and (B) a0:10 water:methanol mixture. The gradient started with 60% A,

ncreasing to 100% A within 3 min, then returning to 60% A and 40% after 6.1 min, and holding initial conditions for 3 min. A Calibra-ion was performed in a concentration range of 0 �g/L to 250 �g/Ladrozole. Coefficient of correlation (r2) of the calibration functionas estimated ≥ 0.99. The substance-specific limit of quantitation

LOQ) was defined as 5 �g/L fadrozole.

.7. Statistical analysis

All physiological endpoints as well as the sex ratio were ana-yzed using ToxRat® Professional 3. Normal distribution of data

as confirmed by the Shapiro-Wilks test, followed by Levene’sest for variance homogeneity. A one-way ANOVA was performed,ith the Williams t-test as post-hoc test, applying a significance

evel of p ≤ 0.05. Data on gonad histopathology were evaluatedith GraphPad Prism (GraphPad Software, La Jolla, USA) including

ne-way ANOVA followed by Dunnett’s multiple t-test procedure, ≤ 0.05. For the biological results, No Observed Effect Concentra-ions (NOEC) and Lowest Observed Effect Concentrations (LOEC)ere determined.

For gene expression, the statistical determination of signif-cant differences of the fadrozole treatments compared to theontrols, one-way ANOVAs followed by Dunnett’s multiple com-arison 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 tran-cript expression values of the three replicates. Control-normalizedata were log2-transformed for presentation.

. Results

.1. Chemical analysis

The mean measured concentrations of the test solutions ofadrozole between day 1 and day 61 of the study ranged between4.4% and 114.8% of nominal concentration per treatment (Table2). Test concentrations during the time course of the study didot vary by more than 20% from nominal concentrations for mostessels with few exceptions (maximum concentration of 147.7% ofominal at a nominal concentration of 32 �g/L). All effect data areepicted according to the nominal fadrozole concentrations.

.2. Effects of fadrozole on the FSDT standard endpoints

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

cology 176 (2016) 116–127 119

a concentration-dependent manner. Post-hatch survival rates at 28dpf 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). The28 dpf post-hatch survival rates of the 32 �g/L and 100 �g/L treat-ment decreased significantly to 70.6% and 67.8% (Fig. 1A; *p < 0.05,Williams t-test, one-sided smaller). The NOEC for post-hatch sur-vival was determined to be at 10 �g/L fadrozole. Survival was notfurther affected by fadrozole at 63 dpf.

Growth in terms of mean total body length at 28 dpf and in termsof mean standard length and wiped wet weight at 63 dpf was foundnot to be affected by fadrozole. At 28 dpf, the average length ofthe control fish was 10.1 mm whereas the treatment lengths variedbetween 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 exposed28 dpf fish was observed. The mean body length of 63 dpf controland exposed fish was 28.0 mm. The mean weight increased slightlywith increasing exposure concentration, from 0.183 g for the con-trols to 0.196 g in the highest treatment condition. However, thedifferences 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 proportionalratio of males to females of 49.9–50.1% across all replicates. Inall fadrozole treatments, the sex ratios were significantly differ-ent from the controls (Fig. 1B; p < 0.05, Williams t-test, two-sided,data arcsine-transformed prior to statistical analysis). At the low-est concentration, all fish were males, except for one female in onereplicate. 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 ovariandevelopment of the control fish was characterized by gonads con-sisting of early stage oocytes. Only one gonad had reached amaturation stage of 2, all other ovaries were at stage 0 or 1. Accord-ing to the maturity index of Baumann et al. (2013), this early ovarymaturation stage corresponded to a maturity index of 1.8. The ovar-ian maturation of the remaining single female at 10 �g/L fadrozolecorresponded to a maturity stage of 0 (maturity index of 1). Thedelay in ovarian maturation was not associated with any patholog-ical alterations of the gonads.

Males of the control groups had an average testis maturity indexof 2.1. The average gonad maturity of males increased with increas-ing fadrozole concentrations (Fig. 1C) and became statisticallysignificant 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 reached2.7.

The gonads of control males were found to be in an undevelopedprotogynous state at 38.2 %. This state marks the transition phasefrom an ovary-like structure to the male testis, which is a typicalphase in zebrafish sexual development (Maack and Segner, 2003).The incidence of the undeveloped gonad stage remained below20% in all fadrozole exposed fish (statistically no difference to thecontrol) (Fig. 1D), consistent with the increasing gonad maturityindices 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 ofthe control females displayed high variation but were on average12.18 ng VTG/�g protein. Females with low amounts in the range≤0.01 ng/�g were also found.

120 E. Muth-Köhne et al. / Aquatic Toxicology 176 (2016) 116–127

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 significantlyreduced at nominal 32 �g/L and 100 �g/L fadrozole compared to control fish (white bar). (B) Sex ratio at 63 dpf (depicted as% males versus females) was fully skewed towardsmales at all fadrozole concentrations. (C) The gonad maturation stage of 63 dpf males, represented by the maturity index (Baumann et al., 2013), increased significantlyf ic stage) gonads after fadrozole treatment. Statistical differences were determined byW with Dunnett’s multiple comparison test, *p < 0.05; **p < 0.01; ***p < 0.001 (C–D); n = 4s

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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-changesversus the control mRNA levels. (A) At 48 hpf, mvd and igf1 were significantly up-regulated and vtg1 was down-regulated. (B) At 96 hpf, lss, vtg1, esr2a, gnrhr2, gnrhr3,were down-regulated and igf1, sox9b, and zgc:64022 up-regulated. Statistical differ-ences of reference-normalized mean transcription values of treatments to controlvalues (Livak and Schmittgen, 2001; Pfaffl, 2001) were determined by one-way

rom 10 �g/L fadrozole. (D) Ratio of males (%) displaying undeveloped (protogynilliams t-test, one-sided smaller, with *p < 0.05 (A–B), and by one-way ANOVA,

amples/treatment.

Male fish displayed VTG concentrations below or close to theOD. A tendency to lower concentrations in treatment conditionshan in the controls was observed but was not quantifiable. Effectsf fadrozole on VTG plasma concentrations of females could note determined with only one single female found in one replicatef the 10 �g fadrozole/L treatment. This female displayed a VTGoncentration of 0.01 ng VTG/�g protein, which was lower by aactor of 1000 than the mean VTG concentration found in controlemales.

.5. Effects of fadrozole on gene expression

Twenty-eight target genes involved in steroid hormone biosyn-hesis and signaling (Table S1) were analyzed by qPCR. MRNA levelsf these genes were assessed for all sampling time points, calcu-ating the log2-fold changes compared to the respective controls.esults are only shown for the most significantly changed genesFig. 2 and Fig. 3).

At the end of embryogenesis (48 hpf), 21 of the 28 selected targetenes were found to be expressed. No expression could be detectedor neuropeptide Y1 receptor (npy1r), gonadotropin-releasing hor-one receptor 1, 2 and 3 (gnrhr1, gnrhr2, gnrhr3), insulin-like growth

actor binding protein 5a (igfbp5a), prospero homeobox 1 (prox1), andromatase a (cyp19a1a) (Fig. 4). Statistically significant differencesetween controls and fadrozole treatments were observed forevalonate (diphospho) decarboxylase (mvd), vitellogenin 1 (vtg1),

nd insulin-like growth factor 1 (igf1). MRNA levels of mvd and

gf1 was found to be up-regulated up to log2-fold changes of 2,

hile vtg1 was found to be down-regulated with log2-fold changesetween 0 and −1 (Fig. 2A). These differences were statisticallyignificant (*p < 0.05) at 10 �g/L fadrozole for mvd and vtg1, and

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 werelog2-transformed prior to presentation.

at 32 �g/L fadrozole for igf1, and highly significant (**p < 0.01) at32 �g/L fadrozole for mvd.

At the larval stage of 96 hpf, 26 genes were found to be expressedand significant differences compared to the control values were

observed for eight genes. No expression was observed for gnrhr1,and follicle stimulating hormone receptor (fshr) (Fig. 4). The genesigf1, SRY (sex determining region Y)-box 9b (sox9b), and zgc:64022were fadrozole concentration-dependently up-regulated, with

E. Muth-Köhne et al. / Aquatic Toxicology 176 (2016) 116–127 121

Fig. 3. MRNA levels in pre-adult zebrafish at 63 dpf. (A) Divergence in the relative vtg1 mRNA amounts in control zebrafish: Five fish out of two control replicates displayedhigh vtg1 mRNA and ten fish out of three control replicates low vtg1 mRNA. Accordingly, the control fish were divided into 5 high and 10 low vtg1 expressers. (B) Significantdifferences in gene expression between low and high vtg1 expressers, depicted as log2-fold change. The gene esr2a, and cyp19a1b showed the same discriminating expressionlevels in control fish than vtg1. (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:d esserso ermint Contro

lahcswTgae

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ark grey bars) compared to (C) low vtg1 expressers (control) and (D) high vtg1 exprf treatments to control values (Livak and Schmittgen, 2001; Pfaffl, 2001) were det-test; *p < 0.05; **p < 0.01; ***p < 0.001; n = 5 fish/replicate; 3 replicates/treatment.

og2-fold changes between 0.5 and up to 2. Differences for sox9bnd zgc:64022 were significant (*p < 0.05) at 32 �g/L and for igf1ighly significant (**p < 0.01) at 10 �g/L and most highly signifi-ant (***p < 0.001) at 32 �g/L and 100 �g/L (Figure 2 B). Lanostyrylynthase (lss), vtg1, estrogen receptor 2a (esr2a), gnrhr2, and gnrhr3ere down-regulated by log2-fold changes in the range of −1.

hese changes were statistically significant (*p < 0.05) for vtg1 andnrhr2 at 10 �g/L of fadrozole, for esr2a at 32 �g/L, and for gnrhr3t 100 �g/L fadrozole. Highly significant (**p < 0.01) was the differ-nce for lss at 32 �g/L (Fig. 2B).

The developmental stage at 28 dpf showed the lowest overallene response. All target transcripts were found but none expressedt significantly different fold-change levels. Particularly high vari-tions were observed between replicates.

The pre-adult 63 dpf fish which were analyzed for gene expres-ion, had not been subjected to gonadal sex determination oristopathology. Thus, the morphological sex of these fish wasnknown. However, these fish showed divergence in the relativeRNA amounts of vtg1 in the control samples (after normaliza-

ion to the reference genes). A group of control fish exhibitingelative vtg1 mRNA amounts of 0.00005–0.0017, separated clearlyby a factor of 500) from a high level vtg1 group with mRNA lev-ls of 0.13–0.46 (Fig. 3A). A sex-dependent expression of the vtg1RNA was assumed and consequently, the control fish divided into

low and a high level vtg1 group for analysis. Accordingly, the

RNA levels of the other target genes were compared between

he “low” and “high” vtg1 expresser control groups. Twenty-five ofhe 28 target genes were expressed in the controls; no transcrip-

(control). Statistical differences of reference-normalized mean transcription valuesed by one-way ANOVA with Dunnett’s multiple comparison test, and by Studentsl-normalized data were log2-transformed prior to presentation.

tion was detected for kiss1 receptor a (kiss1ra), gnrhr1, and gnrhr2.The log2-fold change differences of low vtg1 compared to high vtg1expressers (Fig. 3 B) were highest for vtg1 (−9.0; most highly sig-nificant, ***p < 0.001), at −0.8 for esr2a, and at −3.1 for the brainaromatase (cyp19a1b; both significant, *p < 0.05). Large but non-significant log2-fold change differences were additionally observedfor gnrhr4 (−5.8) and cyp19a1a (approx. 5.0) (Fig. 4).

Gene expression measured in the fadrozole treated fish wascompared to low as well as high vtg1 expressing controls. Accordingto the results of the histopathological examinations, which identi-fied 100% of fadrozole-treated fish at 63 dpf as males (compareskewed sex ratio described in Section 3.3), no statistically diver-gent vtg1 mRNA expression in individual treatments was observed.Compared to the low vtg1 controls, a significant induction wasobserved for lss at 32 �g/L and 100 �g/L of fadrozole, and for starand prox1 at 100 �g/L (all **p < 0.01; Fig. 3C).

Compared to the high vtg1 expressers of the controls, moregenes were found to be differentially expressed in fadrozole treated63 dpf fish. Statistically significant differences were observed forlss, vtg1, star, esr2a, igfbp5a, prox1, cyp19a1b, and zgc:64022. Highlysignificantly (**p < 0.01) up-regulated was lss at fadrozole concen-trations of 32 �g/L and 100 �g/L, and still significant (*p < 0.05) wasstar, igfbp5a, and prox1 at 100 �g/L. Down-regulation was mosthighly significant (***p < 0.001) for vtg1 at all fadrozole concentra-tions, and for esr2a at 32 �g/L. Highly significant down-regulation

was observed for esr2a at 10 �g/L and 100 �g/L; still significantwas cyp19a1b and zgc:64022 at all concentrations (*p < 0.05). Thelog2-fold changes ranged from −9.0 for vtg1 to 2 for lss (Fig. 3D).

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Fig. 4. Heatmap-like illustration of the log2-fold changes of all target genes in relation to control fish at the respective time points. Color and intensity reflect the mean log2 fold-change values of each gene at a given samplingtime 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 strongestregulation 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 thetable. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

E. Muth-Köhne et al. / Aquatic Toxicology 176 (2016) 116–127 123

Fig. 5. MRNA levels of the potential marker genes igf1, star, lss, cyp19a1b, and vtg1 over time. (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, lss, cyp19a1b, and vtg1 compared to thecorresponding control groups at each analysis time point. Statistical differences of reference-normalized mean transcription values of treatments to control values (Livaka nett’sn ent (i to th

2mivpspwcsaa(

nd Schmittgen, 2001; Pfaffl, 2001) were determined by one-way ANOVA with Dun = 3 replicates/treatment (48/96 hpf, 28 dpf); n = 5 fish/replicate; 3 replicates/treatm

nterpretation of the references to colors in this figure legend, the reader is referred

As Fig. 4 illustrates, statistical analysis revealed that 13 out of the8 target genes showed significant regulation for at least one treat-ent level and time point, seven genes were found to be regulated

n more than one exposure at a given time-point and five genes (lss,tg1, esr2a, igf1, zgc:64022) were regulated at more than one timeoint. No gene was significantly regulated at all developmentaltages investigated. Based on the mRNA expression patterns (Fig. 4),otential marker genes for aromatase inhibition were identified,hich responded significantly at least to two fadrozole exposure

oncentrations, or at two developmental stages. Accordingly, igf1,

tar, lss, cyp19a1b, and vtg1 were selected as potential markers andnalyzed in more detail in terms of their transcription over timet control conditions as well as in response to fadrozole treatmentFig. 5). At control conditions, the mRNA of lss, igf1 and cyp19a1b

multiple comparison test, and by Students t-test; *p < 0.05; **p < 0.01; ***p < 0.001;63 dpf). Control-normalized data were log2-transformed prior to presentation. (Fore web version of this article.)

showed a steady increase from 48 hpf to 63 dpf (Fig. 5A) whereasstar 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 vtg1showed a decrease below the 48 hpf level at the juvenile stage(28 dpf) before it increased sharply in the female fish with sexualmaturation at 63 dpf. In male fish, the vtg1 mRNA was on averageslightly above the 48 hpf level. Sex related differences in mRNA lev-els of control fish at 63 dpf were, apart from vtg1, also displayed bycyp19a1b.

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 �g/L fadro-zole) and at 96 hpf (significant at all treatment levels), lss (Fig. 5D)was down-regulated at 96 hpf at a concentration of 32 �g/L, and

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24 E. Muth-Köhne et al. / Aquat

tg1 (Fig. 5F) responded by up-regulation at 48 hpf (200 �g/L) and6 hpf (10 �g/L). On the other hand, a response to fadrozole of starFig. 5C) and cyp19a1b (Fig. 5E) only occurred at 63 dpf, suggest-ng that the reduced levels of estrogen affected the transcriptionf these genes only at a later developmental stage. Most notably,yp19a1b was down-regulated in the 63 dpf exposed fish only inelation to the high vtg1 control group. Most importantly, vtg1as strongly down-regulated in relation to the high vtg1 puta-

ive female controls at 63 d. This result is consistent with results ofistopathology showing that almost all fadrozole treated fish wereorphologically identified as males.

. Discussion

Alternative concepts are currently being pursued in regulatorycotoxicology to discriminate, prioritize, and assess endocrine dis-upting chemicals (EDCs). These concepts integrate in silico andolecular data to better explain adverse outcomes of regulatory

oncern (Ankley et al., 2010; Bradbury et al., 2004; Meek et al., 2014;RC, 2007; Perkins et al., 2015; Villeneuve and Garcia-Reyero,011). The knowledge of the responses at the molecular level andow these responses relate to a particular adverse outcome is

nstrumental in predicting endocrine disruption more reliably andossibly earlier than traditionally, where only apical effect end-oints and few physiological biomarkers are used. The aim of thetudy was thus to investigate the value of additional molecularndpoints, like gene expression, for the identification of EDCs andotentially also the mode of endocrine action, prior to the manifes-ation of adverse effects.

Classified as a level 4 study, the FSDT covers early developmen-al stages as well as the phase of sexual maturation of zebrafishMaack and Segner, 2003). It therefore delivers apical and physio-ogical data, as well as the possibility to obtain early molecular datarom treated embryos or larvae.

During the FSDT, we observed a weak, but significant effect onost-hatch survival at 28 dpf (Fig. 1A). This observation suggested

ow systemic toxicity, which could be explained by an elevatedtress susceptibility known for larval stages of fish, especiallyfter chemical treatment (Belanger et al., 2010; Diekmann andagel, 2004; Woltering, 1984). During the larval stage, the energyetabolism changes from intrinsic nutrient absorption from the

olk to external feeding, which often results in increased mor-ality also under control conditions. Supporting this assumptionf increased sensitivity of larval fish, zebrafish embryos exposedo even higher fadrozole concentrations (0.1–1.0 mg fadrozole/L;npublished results) did not display increased mortality. Generally,actors such as systemic toxicity were proposed to influence end-oints known as endocrine indicators in a non-endocrine mannerWheeler et al., 2013). However, it was deemed very unlikely thathe observed effects of fadrozole on sex ratio and gonad maturationFig. 1B–D) in the present study were a result of systemic toxicity.adrozole has a very specific aromatase inhibition MoA, and theame effects on zebrafish sex ratio have already been describedlsewhere (Fenske and Segner, 2004; Takatsu et al., 2013). Accord-ngly, we concluded that the observed effects on gene expression

ere also caused by aromatase inhibition and not by systemic tox-city.

During development, zebrafish gonads respond particularlyensitive to aromatase modulations during the phase of gonadalex differentiation. They are described as undifferentiated gono-horists, which develop via a stage described as non-functional

rotogyne gonad (Maack and Segner, 2003). This undifferentiatedonadal stage is influenced by the estrogen-to-androgen ratio dur-ng steroid biosynthesis. An imbalanced ratio easily results in akewed sex ratio. Thus, treatment with an aromatase inhibitor like

cology 176 (2016) 116–127

fadrozole decreases the estrogen level and consequently resultsin an increased male-to-female ratio (Fenske and Segner, 2004;Takatsu et al., 2013). Additionally, steroid biosynthesis may bestimulated by positive feedback regulation due to low levels ofestrogens via a loop induced. This would lead to even higher testos-terone levels with aromatase conversion being inhibited (Ankleyand Villeneuve, 2015; Villeneuve et al., 2013, 2009). The under-lying regulatory molecular mechanisms are not straightforwardas gene expression regulation can occur directly (e.g. via genespossessing an estrogen receptor-responsive element (ERE) in theirpromoter region) and indirectly (via steroid biosynthesis-relatedgenes at gonadal level). The histopathological results verified thehighly potent masculinizing effect of fadrozole in this study, with99% 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 fromapproximately 2.0 in the control groups to approximately 2.7 atthe highest fadrozole concentration (Fig. 1C). A promoting effecton the maturity index of zebrafish was described by Baumannet al. (2013) also for other masculinizing substances. This observa-tion underpins the assumption that aromatase inhibition elevateslevels of androgen and in turn, stimulates testis maturation. Fur-ther 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 inzebrafish after exposure to 17ß-trenbolone (a strong androgen).

However, particular attention of the study was directed to thegene expression endpoints. Even though mRNA synthesis doesnot unequivocally result in protein expression (i.e. translation oftranscribed genes, compare Nikinmaa and Rytkönen, 2011), tran-scription level changes can serve as measurable markers in thecascade form the initial event to effect manifestation at organismlevel. Thus, transcription of selected genes involved in regula-tory events of hypothalamus-pituitary-gonadal (HPG) signaling,steroidogenesis, steroid signaling, and signaling responses wasanalyzed at key developmental stages during early life (48 hpf, 96hpf) and early (28 dpf) and late puberty (63 dpf). Five of the 28selected genes showed significant regulation by fadrozole at morethan one concentration and time point and were therefore consid-ered promising candidates for biomarkers of aromatase inhibition.These genes are igf1, star, lss, cyp19a1b and vtg1. The correspond-ing proteins are involved in the regulation of transcription factors,steroid/terpenoid synthesis, cholesterol transport, and direct reg-ulation of expression by estradiol (see Fig. 6).

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

The vtg1 gene is a well-established estrogenic biomarker(Muncke and Eggen, 2006) as it contains an ERE in its promoterregion. In addition, vtg1 is expressed at reliably measurable mRNAand protein levels in adult zebrafish only in females (Wang et al.,2005). The differential transcription of vtg1 in control fish at 63dpf was interpreted as sexually dimorphic analogue to the VTGprotein. Therefore, the division into high and low vtg1 mRNA lev-

els of controls at 63 dpf, allowed us to perform a differentialsex-related analysis of the other genes without the knowledge ofthe histopathological sex of these fish. Thus, the results obtainedfor fadrozole-treated fish discerned genes that were significantly

E. Muth-Köhne et al. / Aquatic Toxi

Fig. 6. Role of the selected biomarker genes within the steroid biosynthesis path-way. Fadrozole exposure (pink star) directly inhibited the activity of the aromataseand subsequently also of cyp19a1b (and the other isoform cyp19a1a; not shown).The conversion of testosterone to estradiol (E2) is inhibited and the decrease in E2directly lowers the transcription of the ERE-responsive genes vtg1, and cyp19a1b.Aromatase inhibition also has a stimulating effect on the steroid biosynthesis affect-ing key steps, e.g. IGF-R signaling, the synthesis of fatty acids to cholesterol or thecholesterol transport to the inner membrane of the mitochondrion. The genes igf1,lss, and star are involved and respond either already early (igf1) or later duringpuberty (lss and star) with up-regulation. In contrast to the ERE-responsive genes,rc

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We postulate that the transcription of these genes is applicable as

egulation of these genes is subject to mostly indirect control mechanisms, whichomplicates the prediction of expression changes.

hanged compared to putative females as differentially expressedue to sex. Genes that were significantly changed compared tooth control groups, suggested their involvement in a fadrozoleelated but sex independent steroid biosynthesis dysregulation.xpression of vtg1 mRNA in the fadrozole exposed fish showedown-regulation already in the early developmental stages (48/96pf, Fig. 2), which was not surprising since the estrogen respon-iveness of vtg1 in zebrafish embryos as early as 48 hpf was knownrom previous estrogenic exposures (Schiller et al., 2014, 2013b).t 63 dpf, vtg1 showed a strong down-regulation versus the hightg1 control group, and no regulation compared to the low vtg1ontrol group, likely due to the difference in estrogen levels andonsequently, in VTG concentrations in females and males. Thisesult is consistent with results of histopathology showing thatlmost all fadrozole-treated fish were morphologically identifieds males. Moreover, these results are in line with the in AOP ofromatase inhibition established in adult females in which vtg1own-regulation at mRNA and protein level was identified as KE.

Similar to vtg1, transcription of cyp19a1b is controlled by anRE in its promoter region. Accordingly, its mRNA level was signifi-antly down-regulated, as it was anticipated for genes requiring theinding of estrogen-bound estrogen receptors for activation. Aro-atase inhibition leads to reduced estrogen levels, thus inhibiting

he expression of ERE-possessing genes. Transcriptional regulationf these genes by aromatase inhibitor treatment or other estro-enic disruptors has been described for fish in several studies (Filby

cology 176 (2016) 116–127 125

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

The proposed marker gene igf1 showed a concentration-dependent increase in mRNA expression at all investigated timepoints (compare Figs. 2–4), although the up-regulation was sig-nificant only at early developmental stages (48/96 hpf). The IGF1protein is related to the growth hormone (GH)/insulin-like growthfactor (IGF) axis, which is essential for normal growth and develop-ment in vertebrates. For fish, the expression of igf genes, includingigf1, and subsequent translation into proteins, plays a crucial role innormal gonadal function, and modulation by estrogenic disruptorshas been postulated (Brown et al., 2011; Reinecke, 2010). Further-more, it was shown that Igf1 protein stimulated aromatase activityand also cyp19a1 gene expression in red sea bream (Kagawa et al.,2003). In turn, the observed up-regulation of igf1 mRNA in thepresent study may be interpreted as a compensatory mechanismto the aromatase inhibition of fadrozole. This assumption wouldbe supported by evidence found for brown trout (Marca Pereiraet al., 2014), demonstrating the up-regulation of igf1 mRNA upontreatment with prochloraz, an imidazole fungicide known to act asaromatase inhibitor.

In contrast to igf1, transcription of star was exposureconcentration-dependently up-regulated only at 63 dpf in bothhigh and low vtg1 expressers. A differential expression of star upontreatment with EDCs was already evidenced for early life stage fat-head minnow and adult goldfish (Johns et al., 2011; Sharpe et al.,2007). Since StAR protein coordinates cholesterol transport to theinner mitochondrial membrane, which is a rate-limiting step dur-ing steroidogenesis (Johns et al., 2011), we assumed a functionalrole of star in maintenance of steroid concentrations in adulthoodrather than during development. The up-regulation of star mRNAwould indicate an increased demand in cholesterol shuttling due tothe stimulation of steroidogenesis caused by the aromatase inhibi-tion. The up-regulation of star mRNA was equally strong in putativegenetic male and female zebrafish (low and high vtg1 expressers)and thus, suggested an upstream regulation of star transcriptionrather than through the downstream imbalance in estrogen syn-thesis.

The functional role of the protein lanostyryl synthase dur-ing steroid biosynthesis is the synthesis of cholesterol from fattyacids. Together with sc4mol (see Fig. 4), which is involved in thesame key process, the lss gene was found down-regulated early indevelopment (significant at 96 hpf), and up-regulated at 63 dpf.Positive regulation of lss was already described for estrogenic andanti-androgenic substances in 48 hpf zebrafish and 7 dpf medaka(Schiller et al., 2014). An anti-estrogenic effect of aromatase inhi-bition in the present study could therefore explain the suppressionof lss transcription at 96 hpf. The up-regulation of lss mRNA at 63dpf rather signified a higher demand for cholesterol due to thestimulation of steroid biosynthesis (in compensation of aromataseinhibition), which would be expected to be higher in the putativefemales.

Four of the five genes which we selected as potential biomark-ers, based on their regulation upon fadrozole treatment at differentdevelopmental stages, were considered potential markers of estro-genic disruption already earlier. Johns et al. (2011) demonstratedregulation of igf1, star, vtg1 in embryo and larval fathead minnowafter exposure to estrogenic and anti-estrogenic EDCs. Further-more, Hao et al. (2013) performed a study with larval zebrafish andE2 and found regulation of vtg1 and cyp19a1b starting from 4 dpf.

additional marker endpoint to facilitate the interpretation of FSDTresults. The verification of this hypothesis, however, will requirefurther investigations involving different EDCs.

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26 E. Muth-Köhne et al. / Aquat

The results of the present study did not only provide valu-ble findings on potential gene biomarkers for the FSDT, they alsollowed us to extend the scope of the current AOP of aromatasenhibition. The existing AOP for aromatase inhibition (Ankley et al.,010, 2009; aopkb.org/aopwiki/index.php/Aop:25) was developedased on data obtained by Fish Short Term Reproduction AssaysFSTRAs) with fathead minnow. It describes the gonadal aromatasenhibition in adult females, which results in reduced female fecun-ity due to reduced VTG synthesis. When transferring this AOP ofromatase inhibition to the sensitive life stage of fish sexual devel-pment and gonad maturation, the adverse outcome (AO) changeso an altered sex ratio. The MIE of fadrozole exposure during fishexual development is the effective inhibition of the P450 aro-atase (CYP19), which regulates the conversion of C19-androgens

o C18-estrogens (Callard et al., 1978). This conversion is a key eventf the steroid biosynthesis pathway and its inhibition leads to aeduction of estrogen synthesis and subsequently lowers E2 plasmaevels. In developing zebrafish, this estradiol imbalance promotesestis differentiation in a concentration dependent manner.

In adult females, the reduction on E2 plasma level impactsegatively on ERE-regulated genes like vtg1 and cyp19a1b and con-equently reduces the synthesis of VTG, which in turn diminisheshe oocyte quality and maturation in female fish as the next keyvent, leading to reproductive dysfunction. Both alternative AOPsf aromatase inhibition end in declining population trajectories,ndependent of the developmental stage affected by aromatasenhibition. The mRNA levels that were identified to be regulatedt the different life stages, in particular of the directly estrogen reg-lated genes vtg1 and cyp19a1b, could represent useful biomarkersupporting a KE at the molecular level (OECD, 2014). In this wayhey can be integrated into the existing AOP of aromatase inhibitionaopkb.org/aopwiki/index.php/Aop:25).

In conclusion, this study was able to provide further valu-ble evidence that the implementation of molecular endpointsn current testing procedures for EDCs can provide a feasiblend informative tool for the prioritization of EDCs. Moreover, thedentification of additional marker genes for supporting the char-cterization of the MoAs of endocrine-disrupting chemicals can benstrumental in defining new KEs of existing or new AOPs relatedo endocrine disruption.

cknowledgements

This project was funded by the German Federal Ministryor the Environment, Nature Conservation, Building and Nuclearafety (FKZ 371263418). Martina Fenske was also co-fundedy the Landes-Offensive zur Entwicklung Wissenschaftlich-konomischer Exzellenz (LOEWE) of the Federal State of Hessen,ermany. We thank Helmut Segner and Lisa Baumann, Centre

or Fish and Wildlife Health, University of Bern, for performancef histopathological examinations. We also thank Walter Böhmeror performing the chemical analysis and Maike Lutter und Tomingner (all Fraunhofer IME) for assisting in the qPCR analysis.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.aquatox.2016.04.18.

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