Aquatic Toxicology 176 (2016) 116–127

Contents lists available at ScienceDirect

Aquatic Toxicology

j ournal 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

a,∗ b b b

Elke Muth-Köhne , Kathi Westphal-Settele , Jasmin Brückner , Sabine Konradi ,

c a a,1 d,1

Viktoria Schiller , Christoph Schäfers , Matthias Teigeler , Martina Fenske

Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany

German Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, Germany

Fraunhofer IME, Attract Group UNIFISH, Forckenbeckstraße 6, 52074 Aachen, Germany

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

a r a

t i c l e i n f o b s t r a c t

Article history: The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine

Received 23 December 2015

disrupting chemicals. With the present study it was intended to evaluate whether gene expression end-

Received in revised form 13 April 2016

points would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT

Accepted 19 April 2016

according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test

Available online 20 April 2016

concentrations: 10 ␮g/L, 32 ␮g/L, 100 ␮g/L) using zebraﬁsh (Danio rerio). Gene expression analyses using

quantitative RT-PCR were included at 48 h, 96 h, 28 days and 63 days post fertilization (hpf, dpf). The

Keywords:

selection of genes aimed at ﬁnding molecular endpoints which could be directly linked to the adverse

Aromatase inhibition

Zebraﬁsh apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual

qPCR development of zebraﬁsh were a complete sex ratio shift towards males and an acceleration of gonad

Fadrozole maturation already at low fadrozole concentrations (10 ␮g/L). Due to the speciﬁc inhibition of the aro-

Fish sexual development test (FSDT) matase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the

Adverse outcome pathway (AOP) 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 com-

pared 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 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 differentia-

tion. 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.

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

1. Introduction disrupting chemicals (EDC). Endocrine disruption manifests in

an organism or a population after sub-acute to chronic (long-

Environmental pollutants which interact with the endocrine term) exposure, which makes the experimental identiﬁcation

system of organisms, and exert a negative impact on devel- of hormone-active substances time- and cost-intensive. These

opment and reproduction, are commonly known as endocrine 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 ∗

Corresponding author.

designed as screening tools to trigger more complex studies like

E-mail address: [email protected] (E. Muth-Köhne).

1 a ﬁsh life cycle test, which are required to assess the endocrine

These authors contributed equally to the project.

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

E. Muth-Köhne et al. / Aquatic Toxicology 176 (2016) 116–127 117

disruption relevant apical endpoints. These shorter and less ﬁsh is being disrupted is limited to EDCs affecting vitellogenin and the

consuming predictive tests have, in principal, the potential to phenotypic gonadal sex ratio.

reduce the number of long-term apical tests. However, apical effect The conceptual framework of the OECD for Testing and Assess-

data from screening assays could not be used for regulatory pur- ment of Endocrine Disruptors (OECD, 2012a) is mainly focused on

poses. the assessment of chemicals disrupting estrogen, androgen, thy-

Most life-stage conﬁned tests like the Fish Early Life Stage Test, roid signaling processes and steroidogenesis (EATS), but there are

(FELS; OECD TG 210 (OECD, 2013)) are not designed to provide several other endocrine pathways (e.g. corticosteroid, gestagenic,

substantive information about the chemical mode-of-action (MoA; or retinoid signaling pathways), which additionally may be dis-

Villeneuve et al., 2014) and are prone to interpretation error, as rupted by chemical exposure. The use of additional new endpoints

they exclude relevant apical endpoints, like the determination of may help in better understanding the link between the initiating

the sex ratio. It seems therefore logical to include quick responding endocrine related mechanisms and resulting apical effects. This

parameters which may also be indicative of the underlying MoA or would strengthen an AOP-driven approach in the context of the

pinpoint the molecular initiating event (MIE). weight-of-evidence strategy that is currently pursued in the reg-

Molecular endpoints respond promptly to pollutants and ulatory context to assess an endocrine disruption hazard (OECD,

promise to provide information indicative of adverse effects at 2012b). The need for more comprehensive methods has been

organism level (Piersma, 2006). Powerful genomic approaches have acknowledged and highlighted in the Review paper of the OECD

contributed essentially to the determination of molecular mecha- on Novel In Vitro and In Vivo Screening and Testing Methods and

nisms resulting in apical adverse endpoints (Ankley et al., 2006; Endpoints for Evaluating Endocrine Disruptors (Kortenkamp et al.,

Sawle et al., 2010). Global transcriptomics allow meaningful anal- 2011; OECD, 2012b).

yses of adverse effects in cells, organs, and the whole organism at The rationale of the current study was to demonstrate that gene

concentrations potentially below thresholds of the most sensitive expression data could help to increase the evidence on whether and

endpoints of the “conventional” studies. This higher sensitivity of how a substance may act as an EDC. To this end, an FSDT accord-

molecular analyses is crucial to prevent the overlay of endocrine ing to the OECD guideline 234 was conducted with the aromatase

effects by other effects, especially systemic toxicity (Scholz and inhibitor fadrozole and reﬁned by mRNA analysis using quantita-

Mayer, 2008; Wang et al., 2010). The progress in molecular and tive RT-PCR. The rationale to choose fadrozole as test substance is

computer-based (in silico) methods paved the way for the shift that its endocrine disrupting effects are reasonably well deﬁned for

of paradigms in ecotoxicology (NRC, 2007), and for the develop- ﬁsh (Afonso et al., 2000; Ankley et al., 2002; Fenske and Segner,

ment of causal mechanism-based concepts for environmental risk 2004; Scholz and Klüver, 2009; Takatsu et al., 2013; Villeneuve

assessment, e.g. the Toxicity Pathways (Bradbury et al., 2004) or et al., 2013; Wang et al., 2010; Zhang et al., 2008). Fadrozole is

more recently the Adverse Outcome Pathways (AOPs; Ankley et al., a potent, selective non-steroid aromatase inhibitor. Although its

2010; Villeneuve and Garcia-Reyero, 2011). Speciﬁc AOPs aim to interaction with the enzyme is not yet fully understood, coordina-

provide causal links between the MIE and the adverse outcome (AO) tion with the iron of the prophyrine core of the CYP19 enzyme is

of regulatory concern via well-established, measureable key events likely. This describes a characteristic general function of type-II-

(KEs) and key event relationships (KERs), which are intended to inhibitors, which was ﬁrst described in mammals (Browne et al.,

facilitate regulatory decision making (Kramer et al., 2011; Segner, 1991). Fadrozole therefore exhibits a speciﬁc aromatase-inhibiting

2011, 2009). To date several AOPs have already been deﬁned for MoA with fewer side effects than e.g. prochloraz, which inhibits

endocrine disruption (aopkb.org/aopwiki). However, the AOP for steroidogenesis as well as androgen receptor signaling (Ankley

aromatase inhibition is only applicable to sexually mature female et al., 2005; Laier et al., 2006).

ﬁsh, as the AO at the organism level is reduced fecundity (Ankley In this study, a set of endocrine-related genes was measured in

et al., 2010, 2009; aopkb.org/aopwiki/index.php/Aop:25), and no early life stages, and in juvenile and pre-adult ﬁsh at the end of

measureable KE is described at the molecular level. the test, in addition to the default endpoints of the FSDT (hatch,

Zebraﬁsh are particularly sensitive to aromatase inhibitors dur- juvenile growth, sex ratio, plasma vitellogenin, and histopathol-

ing gonadal sex differentiation and maturation, the phase between ogy). The selection of potential marker genes was based on previous

the early life stage and adulthood (Baumann et al., 2015, 2013; results of our own and published studies (Schiller et al., 2013a,

Fenske and Segner, 2004; Kinnberg et al., 2007). As undifferentiated 2013b; Villeneuve et al., 2009), which indicated their sensitivity

gonochorists, zebraﬁsh’ gonads develop via a stage described as to endocrine disruption and especially aromatase inhibition. The

non-functional protogyne gonad (Maack and Segner, 2003). Testis group of selected genes covers also several critical steps in steroid

differentiation and male maturation are triggered by increasing lev- biosynthesis.

els of androgens, while maturation of ovaries requires higher levels Taken together, this study demonstrated that the existing

of estrogens. Aromatase inhibitors block the synthesis of estrogens Adverse Outcome Pathway (AOP) for aromatase inhibition in ﬁsh

by inhibition of the conversion of C19-androgens to C18-estrogens can be translated to the life stage of sexual differentiation. Fur-

(Callard et al., 1978). The surplus of androgens consequently favors ther, we were able to identify marker gene transcription involved

masculinization in the population (Baumann et al., 2015, 2013; in steroid biosynthesis which can be applied as measures of key

Fenske and Segner, 2004; Morthorst et al., 2010; Trant et al., 2001). events (KE) at the molecular level.

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 2. Material and methods

extension of the FELS test. It was originally developed as a “Fish Par-

tial Life Cycle Test” and considered an alternative to speciﬁcally test 2.1. Test species

endocrine disruptors in ﬁsh without the requirement of a whole

life span exposure. It covers the endocrine disruption prone devel- Wild-type zebraﬁsh (Danio rerio) originally obtained from West

opmental period of sexual differentiation of particularly zebraﬁsh Aquarium GmbH (Bad Lauterberg, Germany) and continuously bred

and allows the assessment of gonadal differentiation and sex ratio for several generations in the Fraunhofer IME laboratories, were

as endocrine disruption-associated endpoints (Thorpe et al., 2011). used for testing. Zebraﬁsh of this line correspond in their juve-

However, the information on the endocrine system functions which nile/sexual development to the line described by Takahashi (1977)

and Maack and Segner (2003).

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

Fish of the broodstock were maintained under ﬂow-through ﬁsh except those dedicated to gene expression analysis. Gonads

◦

conditions in 150 L tanks at 25 ± 2 C on a 12:12-h photoperiod in a were incubated in modiﬁed Davidsons’s ﬁxative 20% formalde-

temperature-controlled room. They were fed daily with TetraMin hyde (37%), 10% glycerol, 30% ethanol, and 10% acetic acid (100%)

main feed ad libitum and nauplii of Artemia salina. overnight, then transferred to 10% neutral buffered formalin

For egg collection, stainless steel spawning trays were inserted (according to the OECD Histopathology guidance document (OECD,

into the tanks, and spawning was induced at the beginning of the 2010)). Embedding was performed with parafﬁn and trunks orien-

light period (light intensity: 1000 lux). The eggs were microscopi- tated ventrally to the cutting surface. Sections of 4–5 ␮m were cut

cally examined for fertilization success. Only healthy eggs in at least with a microtome, mounted on glass slides and then stained with

blastula stage were inserted into the test system. hematoxylin-eosin.

Microscopic evaluation of the tissue sections was performed

2.2. Test substance according to the OECD Histopathology Guidance Document (OECD

2010). Each ﬁsh was either sexed or recorded as undifferenti-

Fadrozole (IUPAC-Name 4-(5,6,7,8-Tetrahydroimidazo[1,5- ated and the maturation stage of the gonads were categorized.

a]pyridine-5-yl)-benzonitrile), an aromatase inhibitor, was Endocrine-related effects were determined according to the pri-

purchased from Adooq Bioscience LLC. (Irvine, Canada). The sub- mary and secondary diagnostic evaluation criteria.

stance was directly dissolved in water for the preparation of the For determination of the VTG levels, an enzyme-linked

dosing solutions. immunosorbent assay (ELISA) for zebraﬁsh VTG (homologous ELISA

kit, Biosense, Bergen, Norway) was used according to the manu-

2.3. General exposure conditions facturers’ instructions. Plasma samples were measured in 96-well

plates by a microplate reader (iEMS Reader, Labsystems, Helsinki,

Purified tap water was used according to the OECD-Guideline Finland). The assay was calibrated against purified zebrafish VTG

210 (OECD, 2013). The puriﬁcation included ﬁltration with acti- (provided with the kit) as a standard. A blank control was run in

vated charcoal. The water was aerated to the level of oxygen each assay. The VTG concentrations were normalized to the total

saturation. The water quality was monitored in the testing facility. plasma protein content, and data expressed as ng VTG/␮g pro-

◦

Water temperature in the test vessels was adjusted to 25 2 C. tein (Fenske et al., 2001). Total protein was quantiﬁed using a BCA

Oxygen saturation of the test solutions was between 99.0% and Protein Assay Reagent Kit (Pierce, Rockford, USA).

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 2.5. Quantitative RT- PCR (QPCR)

Total RNA was isolated from homogenized frozen trunk tis-

The Fish Sexual Development Test (FSDT) was performed

sue (63 dpf) or pooled embryos/larvae/juveniles using TRIreagent

according to the OECD Test Guideline 234 (OECD, 2011) in a ﬂow-

(Sigma-Aldrich, T9424) method followed by a clean-up with

through exposure system. Minor modiﬁcations of the protocol were

RNeasy Mini Spin Columns (QIAGEN, Hilden, Germany) according

necessary to include gene expression analysis at different time

points. to the manufacturers’ protocols. Prior to cDNA synthesis, RNA was

DNAse (Sigma-Aldrich, AMP-D1) treated and subsequently mea-

The test was performed with three test concentrations (10 ␮g/L,

sured using a NanoDrop 1000 spectrophotometer (Thermo Fisher

32 ␮g/L, and 100 ␮g/L.) under ﬂow through conditions, with four

Scientiﬁc, Schwerte, Germany). Reverse transcription was car-

replicate tanks per concentration run in parallel. A number of 85

ried out using SuperScript III Reverse Transcriptase (Invitrogen,

eggs per replicate tank (12 L total volume) were introduced at test

Carlsbad, USA). Negative controls without reverse transcriptase

start divided into two stainless steel fry cages. One fry cage of 50

were prepared for each sample.

eggs provided samples for the gene expression analysis, the other

QPCR analysis was performed on three of four replicates of

fry cage of 35 eggs remained undisturbed.

each treatment (fadrozole exposures and controls) at each sam-

The eggs were kept in the fry cages until hatching and the

pling time point, using a BioRad iQ5 real-time PCR detection

number of hatched larvae was estimated at 4 and 6 days post fer-

system (BioRad , Hercules, USA). The fourth replicate samples

tilization (dpf). At 21 dpf, larvae were released from both fry cages

were excluded from the analysis due to RNA quality issues. For each

into the main tank. After the early life stage at 28 dpf, the juvenile

reaction, 40 ng of cDNA were added to Sybr Green qPCR Super Mix

ﬁsh were photographed for image-based evaluation of survival and

(ThermoFisher Scientiﬁc/Invitrogen, Waltham, USA) in white 96-

growth (ImageTool of the University of Texas Health Science Center

well PCR plates. Two technical replicates of each target gene and

at San Antonio, Version 3.0). Fish remained in the test system until

the respective reference genes were run for each cDNA sample.

test termination at 63 dpf. At test termination, survival rate, size in

Minus template controls were included on each plate; minus RT

terms of length and weight, and the sex of all ﬁsh were determined.

controls were included repeatedly, at least once for each sample,

Twenty ﬁsh per replicate were analyzed for their vitellogenin (VTG)

however not in every run. The reference genes 18s and rpl8 were

content, and were histopathologically evaluated.

used to normalize the expression values of the target genes.

For gene expression analysis, 20 embryos of 48 and 96 h post

The PCR protocol was as follows: After incubating for 2 min

fertilization (hpf) were sampled from each replicate, pooled in a

◦ ◦

at 50 C and for 10 min at 95 C (for DNA polymerase activation),

1.5 mL Eppendorf tube and snap frozen. At 28 dpf, 4 randomly cho-

◦

targets were ampliﬁed using the following cycle: 95 C for 15 s,

sen larvae per replicate were pooled and snap frozen in a 2.0 mL

◦

then 54, 54.5 or 60 C for 30 s (annealing temperatures for par-

Eppendorf tube; at test termination, the trunks of 5 ﬁsh per repli-

◦

ticular genes) and 60 C for 30 s (for acquisition; 40 cycles); the

cate (heads removed) were randomly chosen for gene expression

◦

run was ﬁnalized by a melting curve analysis: 95 C for 60 s, then

analysis and snap frozen individually. The heads were removed in

◦ ◦

55 C for 60 s (80 cycles, with +0.5 C increment each cycle and a

order to focus on endocrine effects related to gonad development.

◦

10 s acquisition). Relative gene expression ratios were calculated

All samples were stored at −80 C until further use.

by the comparative delta Ct method (ddCt) (Livak and Schmittgen,

For histopathology and VTG measurements, blood samples were

2001; Pfafﬂ, 2001).

collected (by cardiac puncture) and gonads dissected from all

E. Muth-Köhne et al. / Aquatic Toxicology 176 (2016) 116–127 119

2.6. Chemical water analysis 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-

Water samples of 10 mL volume were taken from each tank from zole treatment, with controls meeting the validity criterion of 75%

the mid water body, at test start, and thereafter once a week. The post-hatch survival recommended by the TG 234 (OECD, 2011). The

samples were taken alternating from one of the two test vessels 28 dpf post-hatch survival rates of the 32 ␮g/L and 100 ␮g/L treat-

served by one dosing pump. Water samples were stabilized by addi- ment decreased signiﬁcantly to 70.6% and 67.8% (Fig. 1A; *p < 0.05,

tion of acetonitrile (1:1; v + v) containing 0.2% formic acid prior to Williams t-test, one-sided smaller). The NOEC for post-hatch sur-

storage. vival was determined to be at 10 ␮g/L fadrozole. Survival was not

Fadrozole analysis was performed by high performance liq- further affected by fadrozole at 63 dpf.

uid chromatography tandem mass spectrometry (LC–MS/MS) Growth in terms of mean total body length at 28 dpf and in terms

with negative ionization. Data were collected on a Waters 2695 of mean standard length and wiped wet weight at 63 dpf was found

separations module coupled to a Quattro-Micro tandem mass spec- not to be affected by fadrozole. At 28 dpf, the average length of

trometer (Waters). Aliquots of 50 ␮L were injected into a Gemini the control ﬁsh was 10.1 mm whereas the treatment lengths varied

C18 high-performance LC column (150 mm × 3 mm, 5 ␮m parti- between 10.6 mm (100 ␮g/L) and 10.7 mm (at 10 ␮g/L and 32 ␮g/L).

cle size; Phenomenex) at a ﬂow rate of 0.5 mL/min and a column Thus, no signiﬁcant difference between the control and exposed

◦

temperature of 30 C. Matrix-free procedural blanks were run each 28 dpf ﬁsh was observed. The mean body length of 63 dpf control

working day to exclude possible cross-contaminations during lab- and exposed ﬁsh was 28.0 mm. The mean weight increased slightly

oratory work. Separation was performed on a binary gradient of with increasing exposure concentration, from 0.183 g for the con-

20 mmol ammonium acetate solution in (A) methanol and (B) a trols to 0.196 g in the highest treatment condition. However, the

90:10 water:methanol mixture. The gradient started with 60% A, differences in the mean values were not signiﬁcant.

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 Calibra-

3.3. Histopathology and sex ratio

tion was performed in a concentration range of 0 ␮g/L to 250 ␮g/L

fadrozole. Coefﬁcient of correlation (r ) of the calibration function

Gonadal sex ratios were determined during the histopatho-

was estimated ≥ 0.99. The substance-speciﬁc limit of quantitation

logical evaluation. The controls displayed a mean proportional

(LOQ) was deﬁned as 5 ␮g/L fadrozole.

ratio of males to females of 49.9–50.1% across all replicates. In

all fadrozole treatments, the sex ratios were signiﬁcantly differ-

2.7. Statistical analysis

ent from the controls (Fig. 1B; p < 0.05, Williams t-test, two-sided,

data arcsine-transformed prior to statistical analysis). At the low-

All physiological endpoints as well as the sex ratio were ana-

® est concentration, all ﬁsh were males, except for one female in one

lyzed using ToxRat Professional 3. Normal distribution of data

replicate. At the higher concentrations, only males were identiﬁed.

was conﬁrmed by the Shapiro-Wilks test, followed by Levene’s

As no females were present in the treatment groups,

test for variance homogeneity. A one-way ANOVA was performed,

histopathology of ovaries was limited to controls. The ovarian

with the Williams t-test as post-hoc test, applying a signiﬁcance

development of the control ﬁsh was characterized by gonads con-

level of p ≤ 0.05. Data on gonad histopathology were evaluated

sisting of early stage oocytes. Only one gonad had reached a

with GraphPad Prism (GraphPad Software, La Jolla, USA) including

maturation stage of 2, all other ovaries were at stage 0 or 1. Accord-

one-way ANOVA followed by Dunnett’s multiple t-test procedure,

ing to the maturity index of Baumann et al. (2013), this early ovary

≤

p 0.05. For the biological results, No Observed Effect Concentra-

maturation stage corresponded to a maturity index of 1.8. The ovar-

tions (NOEC) and Lowest Observed Effect Concentrations (LOEC)

ian maturation of the remaining single female at 10 ␮g/L fadrozole

were determined.

corresponded to a maturity stage of 0 (maturity index of 1). The

For gene expression, the statistical determination of signif-

delay in ovarian maturation was not associated with any patholog-

icant differences of the fadrozole treatments compared to the

ical alterations of the gonads.

controls, one-way ANOVAs followed by Dunnett’s multiple com-

Males of the control groups had an average testis maturity index

parison test (*p < 0.05; **p < 0.01; ***p < 0.01), or Students t-test

of 2.1. The average gonad maturity of males increased with increas-

(*p < 0.05; **p < 0.01; ***p < 0.01), were performed on relative tran-

ing fadrozole concentrations (Fig. 1C) and became statistically

script expression values of the three replicates. Control-normalized

signiﬁcant already from 10 ␮g fadrozole/L (**p < 0.01, ***p < 0.01;

data were log2-transformed for presentation.

one-way ANOVA followed by Dunnett’s multiple comparison test).

At 100 ␮g fadrozole/L the highest average maturity index reached

3. Results

2.7.

The gonads of control males were found to be in an undeveloped

3.1. Chemical analysis

protogynous state at 38.2 %. This state marks the transition phase

from an ovary-like structure to the male testis, which is a typical

The mean measured concentrations of the test solutions of

phase in zebraﬁsh sexual development (Maack and Segner, 2003).

fadrozole between day 1 and day 61 of the study ranged between

The incidence of the undeveloped gonad stage remained below

94.4% and 114.8% of nominal concentration per treatment (Table

20% in all fadrozole exposed ﬁsh (statistically no difference to the

S2). Test concentrations during the time course of the study did

control) (Fig. 1D), consistent with the increasing gonad maturity

not vary by more than 20% from nominal concentrations for most

indices of the treatment groups.

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. 3.4. Vitellogenin measurement

3.2. Effects of fadrozole on the FSDT standard endpoints VTG plasma concentration analysis was performed in pre-

adult development (i.e., 63 dpf). The plasma concentrations of

The hatching success of larvae was not inﬂuenced by fadrozole. the control females displayed high variation but were on average

␮

Complete hatch was observed at 6 dpf in controls as well as in 12.18 ng VTG/ g protein. Females with low amounts in the range

≤ ␮

treatments. Survival during early life, however, was diminished in 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 signiﬁcantly

␮

reduced 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 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 ␮g 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 biosyn-

Fig. 2. Signiﬁcantly changed genes in embryo (48 hpf) and larval stage (96 hpf)

thesis and signaling (Table S1) were analyzed by qPCR. MRNA levels

zebraﬁsh, after exposure to fadrozole at nominal 10 ␮g/L (light grey bars) 32 ␮g/L

of these genes were assessed for all sampling time points, calcu-

(medium grey bars) and 100 ␮g/L (dark grey bars). Bars depict log2-fold-changes

lating the log2-fold changes compared to the respective controls. versus the control mRNA levels. (A) At 48 hpf, mvd and igf1 were signiﬁcantly up-

Results are only shown for the most signiﬁcantly changed genes 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-

(Fig. 2 and Fig. 3).

ences of reference-normalized mean transcription values of treatments to control

At the end of embryogenesis (48 hpf), 21 of the 28 selected target

values (Livak and Schmittgen, 2001; Pfafﬂ, 2001) were determined by one-way

genes were found to be expressed. No expression could be detected

ANOVA with Dunnett’s multiple comparison test, and by Students t-test; *p < 0.05;

for neuropeptide Y1 receptor (npy1r), gonadotropin-releasing hor- **p < 0.01; ***p < 0.001; n = 3 replicates/treatment. Control-normalized data were

log2-transformed prior to presentation.

mone 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 at 32 ␮g/L fadrozole for igf1, and highly signiﬁcant (**p < 0.01) at

between controls and fadrozole treatments were observed for 32 ␮g/L fadrozole for mvd.

mevalonate (diphospho) decarboxylase (mvd), vitellogenin 1 (vtg1), At the larval stage of 96 hpf, 26 genes were found to be expressed

and insulin-like growth factor 1 (igf1). MRNA levels of mvd and and signiﬁcant differences compared to the control values were

igf1 was found to be up-regulated up to log2-fold changes of 2, observed for eight genes. No expression was observed for gnrhr1,

while vtg1 was found to be down-regulated with log2-fold changes and follicle stimulating hormone receptor (fshr) (Fig. 4). The genes

between 0 and −1 (Fig. 2A). These differences were statistically igf1, SRY (sex determining region Y)-box 9b (sox9b), and zgc:64022

signiﬁcant (*p < 0.05) at 10 ␮g/L fadrozole for mvd and vtg1, and were 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 displayed

high 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) Significant

differences in gene expression between low and high vtg1 expressers, depicted as log2-fold change. The gene esr2a, and cyp19a1b showed the same discriminating expression

levels 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:

dark grey bars) compared to (C) low vtg1 expressers (control) and (D) high vtg1 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 prior to presentation.

log2-fold changes between 0.5 and up to 2. Differences for sox9b tion was detected for kiss1 receptor a (kiss1ra), gnrhr1, and gnrhr2.

and zgc:64022 were signiﬁcant (*p < 0.05) at 32 ␮g/L and for igf1 The log2-fold change differences of low vtg1 compared to high vtg1

highly signiﬁcant (**p < 0.01) at 10 ␮g/L and most highly signiﬁ- expressers (Fig. 3 B) were highest for vtg1 (−9.0; most highly sig-

cant (***p < 0.001) at 32 ␮g/L and 100 ␮g/L (Figure 2 B). Lanostyryl niﬁcant, ***p < 0.001), at −0.8 for esr2a, and at −3.1 for the brain

synthase (lss), vtg1, estrogen receptor 2a (esr2a), gnrhr2, and gnrhr3 aromatase (cyp19a1b; both signiﬁcant, *p < 0.05). Large but non-

were down-regulated by log2-fold changes in the range of −1. signiﬁcant log2-fold change differences were additionally observed

These changes were statistically signiﬁcant (*p < 0.05) for vtg1 and for gnrhr4 (−5.8) and cyp19a1a (approx. 5.0) (Fig. 4).

gnrhr2 at 10 ␮g/L of fadrozole, for esr2a at 32 ␮g/L, and for gnrhr3 Gene expression measured in the fadrozole treated ﬁsh was

at 100 ␮g/L fadrozole. Highly signiﬁcant (**p < 0.01) was the differ- compared to low as well as high vtg1 expressing controls. According

ence for lss at 32 ␮g/L (Fig. 2B). to the results of the histopathological examinations, which identi-

The developmental stage at 28 dpf showed the lowest overall ﬁed 100% of fadrozole-treated ﬁsh at 63 dpf as males (compare

gene response. All target transcripts were found but none expressed skewed sex ratio described in Section 3.3), no statistically diver-

at signiﬁcantly different fold-change levels. Particularly high vari- gent vtg1 mRNA expression in individual treatments was observed.

ations were observed between replicates. Compared to the low vtg1 controls, a signiﬁcant induction was

The pre-adult 63 dpf ﬁsh which were analyzed for gene expres- observed for lss at 32 ␮g/L and 100 ␮g/L of fadrozole, and for star

sion, had not been subjected to gonadal sex determination or and prox1 at 100 ␮g/L (all **p < 0.01; Fig. 3C).

histopathology. Thus, the morphological sex of these ﬁsh was Compared to the high vtg1 expressers of the controls, more

unknown. However, these ﬁsh showed divergence in the relative genes were found to be differentially expressed in fadrozole treated

mRNA amounts of vtg1 in the control samples (after normaliza- 63 dpf ﬁsh. Statistically signiﬁcant differences were observed for

tion to the reference genes). A group of control ﬁsh exhibiting lss, vtg1, star, esr2a, igfbp5a, prox1, cyp19a1b, and zgc:64022. Highly

relative vtg1 mRNA amounts of 0.00005–0.0017, separated clearly signiﬁcantly (**p < 0.01) up-regulated was lss at fadrozole concen-

(by a factor of 500) from a high level vtg1 group with mRNA lev- trations of 32 ␮g/L and 100 ␮g/L, and still signiﬁcant (*p < 0.05) was

els of 0.13–0.46 (Fig. 3A). A sex-dependent expression of the vtg1 star, igfbp5a, and prox1 at 100 ␮g/L. Down-regulation was most

mRNA was assumed and consequently, the control ﬁsh divided into highly signiﬁcant (***p < 0.001) for vtg1 at all fadrozole concentra-

a low and a high level vtg1 group for analysis. Accordingly, the tions, and for esr2a at 32 ␮g/L. Highly signiﬁcant down-regulation

mRNA levels of the other target genes were compared between was observed for esr2a at 10 ␮g/L and 100 ␮g/L; still signiﬁcant

the “low” and “high” vtg1 expresser control groups. Twenty-ﬁve of was cyp19a1b and zgc:64022 at all concentrations (*p < 0.05). The

the 28 target genes were expressed in the controls; no transcrip- log2-fold changes ranged from −9.0 for vtg1 to 2 for lss (Fig. 3D).

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

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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 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 showed a steady increase from 48 hpf to 63 dpf (Fig. 5A) whereas

28 target genes showed signiﬁcant regulation for at least one treat- star was down-regulated at 96 hpf and 28 dpf compared to 48 hpf,

ment level and time point, seven genes were found to be regulated before returning to the 48 hpf level at 63 dpf. Expression of vtg1

in more than one exposure at a given time-point and ﬁve genes (lss, showed a decrease below the 48 hpf level at the juvenile stage

vtg1, esr2a, igf1, zgc:64022) were regulated at more than one time (28 dpf) before it increased sharply in the female ﬁsh with sexual

point. No gene was signiﬁcantly regulated at all developmental maturation at 63 dpf. In male ﬁsh, the vtg1 mRNA was on average

stages investigated. Based on the mRNA expression patterns (Fig. 4), slightly above the 48 hpf level. Sex related differences in mRNA lev-

potential marker genes for aromatase inhibition were identiﬁed, els of control ﬁsh at 63 dpf were, apart from vtg1, also displayed by

which responded signiﬁcantly at least to two fadrozole exposure cyp19a1b.

concentrations, or at two developmental stages. Accordingly, igf1, Fadrozole exposure affected the ﬁve marker genes differently

star, lss, cyp19a1b, and vtg1 were selected as potential markers and in the course of development (Fig. 5B–F). During early life, igf1

analyzed in more detail in terms of their transcription over time (Fig. 5B) was up-regulated at 48 hpf (signiﬁcantly at 32 ␮g/L fadro-

at control conditions as well as in response to fadrozole treatment zole) and at 96 hpf (signiﬁcant at all treatment levels), lss (Fig. 5D)

(Fig. 5). At control conditions, the mRNA of lss, igf1 and cyp19a1b was down-regulated at 96 hpf at a concentration of 32 ␮g/L, and

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

vtg1 (Fig. 5F) responded by up-regulation at 48 hpf (200 ␮g/L) and fadrozole decreases the estrogen level and consequently results

96 hpf (10 ␮g/L). On the other hand, a response to fadrozole of star in an increased male-to-female ratio (Fenske and Segner, 2004;

(Fig. 5C) and cyp19a1b (Fig. 5E) only occurred at 63 dpf, suggest- Takatsu et al., 2013). Additionally, steroid biosynthesis may be

ing that the reduced levels of estrogen affected the transcription stimulated by positive feedback regulation due to low levels of

of these genes only at a later developmental stage. Most notably, estrogens via a loop induced. This would lead to even higher testos-

cyp19a1b was down-regulated in the 63 dpf exposed ﬁsh only in terone levels with aromatase conversion being inhibited (Ankley

relation to the high vtg1 control group. Most importantly, vtg1 and Villeneuve, 2015; Villeneuve et al., 2013, 2009). The under-

was strongly down-regulated in relation to the high vtg1 puta- lying regulatory molecular mechanisms are not straightforward

tive female controls at 63 d. This result is consistent with results of as gene expression regulation can occur directly (e.g. via genes

histopathology showing that almost all fadrozole treated ﬁsh were possessing an estrogen receptor-responsive element (ERE) in their

morphologically identiﬁed as males. 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

4. Discussion 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.

Alternative concepts are currently being pursued in regulatory The average maturity index of the male gonads increased from

ecotoxicology to discriminate, prioritize, and assess endocrine dis- approximately 2.0 in the control groups to approximately 2.7 at

rupting chemicals (EDCs). These concepts integrate in silico and the highest fadrozole concentration (Fig. 1C). A promoting effect

molecular data to better explain adverse outcomes of regulatory on the maturity index of zebraﬁsh was described by Baumann

concern (Ankley et al., 2010; Bradbury et al., 2004; Meek et al., 2014; et al. (2013) also for other masculinizing substances. This observa-

NRC, 2007; Perkins et al., 2015; Villeneuve and Garcia-Reyero, tion underpins the assumption that aromatase inhibition elevates

2011). The knowledge of the responses at the molecular level and levels of androgen and in turn, stimulates testis maturation. Fur-

how these responses relate to a particular adverse outcome is ther strengthening evidence is provided e.g., by Seki et al. (2006),

instrumental in predicting endocrine disruption more reliably and who reported elevated gonadosomatic indices, and Morthorst et al.

possibly earlier than traditionally, where only apical effect end- (2010), who reported a stimulating effect on testis maturation in

points and few physiological biomarkers are used. The aim of the zebraﬁsh after exposure to 17ß-trenbolone (a strong androgen).

study was thus to investigate the value of additional molecular However, particular attention of the study was directed to the

endpoints, like gene expression, for the identiﬁcation of EDCs and gene expression endpoints. Even though mRNA synthesis does

potentially also the mode of endocrine action, prior to the manifes- not unequivocally result in protein expression (i.e. translation of

tation of adverse effects. transcribed genes, compare Nikinmaa and Rytkönen, 2011), tran-

Classiﬁed as a level 4 study, the FSDT covers early developmen- scription level changes can serve as measurable markers in the

tal stages as well as the phase of sexual maturation of zebraﬁsh cascade form the initial event to effect manifestation at organism

(Maack and Segner, 2003). It therefore delivers apical and physio- level. Thus, transcription of selected genes involved in regula-

logical data, as well as the possibility to obtain early molecular data tory events of hypothalamus-pituitary-gonadal (HPG) signaling,

from treated embryos or larvae. steroidogenesis, steroid signaling, and signaling responses was

During the FSDT, we observed a weak, but signiﬁcant effect on analyzed at key developmental stages during early life (48 hpf, 96

post-hatch survival at 28 dpf (Fig. 1A). This observation suggested hpf) and early (28 dpf) and late puberty (63 dpf). Five of the 28

low systemic toxicity, which could be explained by an elevated selected genes showed signiﬁcant regulation by fadrozole at more

stress susceptibility known for larval stages of ﬁsh, especially than one concentration and time point and were therefore consid-

after chemical treatment (Belanger et al., 2010; Diekmann and ered promising candidates for biomarkers of aromatase inhibition.

Nagel, 2004; Woltering, 1984). During the larval stage, the energy These genes are igf1, star, lss, cyp19a1b and vtg1. The correspond-

metabolism changes from intrinsic nutrient absorption from the ing proteins are involved in the regulation of transcription factors,

yolk to external feeding, which often results in increased mor- steroid/terpenoid synthesis, cholesterol transport, and direct reg-

tality also under control conditions. Supporting this assumption ulation of expression by estradiol (see Fig. 6).

of increased sensitivity of larval fish, zebrafish embryos exposed Significant responses to fadrozole were not restricted to these

to even higher fadrozole concentrations (0.1–1.0 mg fadrozole/L; genes but the discussion primarily focuses on these genes in terms

unpublished results) did not display increased mortality. Generally, of their function as biomarkers for the identiﬁcation of endocrine

factors such as systemic toxicity were proposed to inﬂuence end- disruptive properties of tested substances. In order to put the tran-

points known as endocrine indicators in a non-endocrine manner scriptional changes caused by aromatase inhibition into a broader

(Wheeler et al., 2013). However, it was deemed very unlikely that perspective, also the role of the corresponding proteins in steroido-

the observed effects of fadrozole on sex ratio and gonad maturation genesis was contemplated. Alterations at the transcription level

(Fig. 1B–D) in the present study were a result of systemic toxicity. often results in changes at the translational level, even though post-

Fadrozole has a very speciﬁc aromatase inhibition MoA, and the transcriptional processing and regulation disallow a one-to-one

same effects on zebraﬁsh sex ratio have already been described transfer from transcription to translation (Nikinmaa and Rytkönen,

elsewhere (Fenske and Segner, 2004; Takatsu et al., 2013). Accord- 2011).

ingly, we concluded that the observed effects on gene expression The vtg1 gene is a well-established estrogenic biomarker

were also caused by aromatase inhibition and not by systemic tox- (Muncke and Eggen, 2006) as it contains an ERE in its promoter

icity. region. In addition, vtg1 is expressed at reliably measurable mRNA

During development, zebraﬁsh gonads respond particularly and protein levels in adult zebraﬁsh only in females (Wang et al.,

sensitive to aromatase modulations during the phase of gonadal 2005). The differential transcription of vtg1 in control ﬁsh at 63

sex differentiation. They are described as undifferentiated gono- dpf was interpreted as sexually dimorphic analogue to the VTG

chorists, which develop via a stage described as non-functional protein. Therefore, the division into high and low vtg1 mRNA lev-

protogyne gonad (Maack and Segner, 2003). This undifferentiated els of controls at 63 dpf, allowed us to perform a differential

gonadal stage is inﬂuenced by the estrogen-to-androgen ratio dur- sex-related analysis of the other genes without the knowledge of

ing steroid biosynthesis. An imbalanced ratio easily results in a the histopathological sex of these ﬁsh. Thus, the results obtained

skewed sex ratio. Thus, treatment with an aromatase inhibitor like for fadrozole-treated ﬁsh discerned genes that were signiﬁcantly

E. Muth-Köhne et al. / Aquatic Toxicology 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 be

considered a direct marker of aromatase inhibition in pubescent

zebraﬁsh.

The proposed marker gene igf1 showed a concentration-

dependent increase in mRNA expression at all investigated time

points (compare Figs. 2–4), although the up-regulation was sig-

niﬁcant 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 develop-

ment in vertebrates. For ﬁsh, the expression of igf genes, including

igf1, 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). Further-

more, it was shown that Igf1 protein stimulated aromatase activity

and also cyp19a1 gene expression in red sea bream (Kagawa et al.,

2003). In turn, the observed up-regulation of igf1 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 igf1 mRNA upon

treatment with prochloraz, an imidazole fungicide known to act as

aromatase inhibitor.

In contrast to igf1, transcription of star was exposure

concentration-dependently up-regulated only at 63 dpf in both

high and low vtg1 expressers. A differential expression of star upon

treatment with EDCs was already evidenced for early life stage fat-

head minnow and adult goldﬁsh (Johns et al., 2011; Sharpe et al.,

Fig. 6. Role of the selected biomarker genes within the steroid biosynthesis path- 2007). Since StAR protein coordinates cholesterol transport to the

way. Fadrozole exposure (pink star) directly inhibited the activity of the aromatase

inner mitochondrial membrane, which is a rate-limiting step dur-

and subsequently also of cyp19a1b (and the other isoform cyp19a1a; not shown).

ing steroidogenesis (Johns et al., 2011), we assumed a functional

The conversion of testosterone to estradiol (E2) is inhibited and the decrease in E2

role of star in maintenance of steroid concentrations in adulthood

directly lowers the transcription of the ERE-responsive genes vtg1, and cyp19a1b.

Aromatase inhibition also has a stimulating effect on the steroid biosynthesis affect- rather than during development. The up-regulation of star mRNA

ing key steps, e.g. IGF-R signaling, the synthesis of fatty acids to cholesterol or the would indicate an increased demand in cholesterol shuttling due to

cholesterol transport to the inner membrane of the mitochondrion. The genes igf1,

the stimulation of steroidogenesis caused by the aromatase inhibi-

lss, and star are involved and respond either already early (igf1) or later during

tion. The up-regulation of star mRNA was equally strong in putative

puberty (lss and star) with up-regulation. In contrast to the ERE-responsive genes,

genetic male and female zebraﬁsh (low and high vtg1 expressers)

regulation of these genes is subject to mostly indirect control mechanisms, which

complicates the prediction of expression changes. and thus, suggested an upstream regulation of star transcription

rather than through the downstream imbalance in estrogen syn-

thesis.

changed compared to putative females as differentially expressed The functional role of the protein lanostyryl synthase dur-

due to sex. Genes that were signiﬁcantly changed compared to ing steroid biosynthesis is the synthesis of cholesterol from fatty

both control groups, suggested their involvement in a fadrozole acids. Together with sc4mol (see Fig. 4), which is involved in the

related but sex independent steroid biosynthesis dysregulation. same key process, the lss gene was found down-regulated early in

Expression of vtg1 mRNA in the fadrozole exposed ﬁsh showed development (signiﬁcant at 96 hpf), and up-regulated at 63 dpf.

down-regulation already in the early developmental stages (48/96 Positive regulation of lss was already described for estrogenic and

hpf, Fig. 2), which was not surprising since the estrogen respon- anti-androgenic substances in 48 hpf zebraﬁsh and 7 dpf medaka

siveness of vtg1 in zebraﬁsh embryos as early as 48 hpf was known (Schiller et al., 2014). An anti-estrogenic effect of aromatase inhi-

from previous estrogenic exposures (Schiller et al., 2014, 2013b). bition in the present study could therefore explain the suppression

At 63 dpf, vtg1 showed a strong down-regulation versus the high of lss transcription at 96 hpf. The up-regulation of lss mRNA at 63

vtg1 control group, and no regulation compared to the low vtg1 dpf rather signiﬁed a higher demand for cholesterol due to the

control group, likely due to the difference in estrogen levels and stimulation of steroid biosynthesis (in compensation of aromatase

consequently, in VTG concentrations in females and males. This inhibition), which would be expected to be higher in the putative

result is consistent with results of histopathology showing that females.

almost all fadrozole-treated fish were morphologically identified Four of the five genes which we selected as potential biomark-

as males. Moreover, these results are in line with the in AOP of ers, based on their regulation upon fadrozole treatment at different

aromatase inhibition established in adult females in which vtg1 developmental stages, were considered potential markers of estro-

down-regulation at mRNA and protein level was identiﬁed as KE. genic disruption already earlier. Johns et al. (2011) demonstrated

Similar to vtg1, transcription of cyp19a1b is controlled by an regulation of igf1, star, vtg1 in embryo and larval fathead minnow

ERE in its promoter region. Accordingly, its mRNA level was signiﬁ- after exposure to estrogenic and anti-estrogenic EDCs. Further-

cantly down-regulated, as it was anticipated for genes requiring the more, Hao et al. (2013) performed a study with larval zebraﬁsh and

binding of estrogen-bound estrogen receptors for activation. Aro- E2 and found regulation of vtg1 and cyp19a1b starting from 4 dpf.

matase inhibition leads to reduced estrogen levels, thus inhibiting We postulate that the transcription of these genes is applicable as

the expression of ERE-possessing genes. Transcriptional regulation additional marker endpoint to facilitate the interpretation of FSDT

of these genes by aromatase inhibitor treatment or other estro- results. The veriﬁcation of this hypothesis, however, will require

genic disruptors has been described for ﬁsh in several studies (Filby further investigations involving different EDCs.

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

The results of the present study did not only provide valu- Ankley, G.T., Villeneuve, D.L., 2015. Temporal changes in biological responses and

uncertainty in assessing risks of endocrine- disrupting chemicals: insights

able ﬁndings on potential gene biomarkers for the FSDT, they also

from intensive time-course studies with ﬁsh. Toxicol. Sci. 144, 259–275.

allowed us to extend the scope of the current AOP of aromatase

Ankley, G.T., Kahl, M.D., Jensen, K.M., Hornung, M.W., Korte, J.J., Makynen, E.a.,

inhibition. The existing AOP for aromatase inhibition (Ankley et al., Leino, R.L., 2002. Evaluation of the aromatase inhibitor fadrozole in a

short-term reproduction assay with the fathead minnow (Pimephales

2010, 2009; aopkb.org/aopwiki/index.php/Aop:25) was developed

promelas). Toxicol. Sci. 67, 121–130.

based on data obtained by Fish Short Term Reproduction Assays

Ankley, G.T., Jensen, K.M., Durhan, E.J., Makynen, E.a., Butterworth, B.C., Kahl, M.D.,

(FSTRAs) with fathead minnow. It describes the gonadal aromatase Villeneuve, D.L., Linnum, a., Gray, L.E., Cardon, M., Wilson, V.S., 2005. Effects of

two fungicides with multiple modes of action on reproductive endocrine

inhibition in adult females, which results in reduced female fecun-

function in the fathead minnow (Pimephales promelas). Toxicol. Sci. 86,

dity due to reduced VTG synthesis. When transferring this AOP of

300–308.

aromatase inhibition to the sensitive life stage of ﬁsh sexual devel- Ankley, G., Daston, G.P., Degitz, S.J., Denslow, N.D., Hoke, R.A., Kennedy, S.W.,

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Toxicogenomic in regulatory ecotoxicology. Environ. Sci. Technol. 40,

to an altered sex ratio. The MIE of fadrozole exposure during ﬁsh

4055–4065.

sexual development is the effective inhibition of the P450 aro-

Ankley, G.T., Bencic, D.C., Breen, M.S., Collette, T.W., Conolly, R.B., Denslow, N.D.,

matase (CYP19), which regulates the conversion of C19-androgens Edwards, S.W., Ekman, D.R., Garcia-Reyero, N., Jensen, K.M., Lazorchak, J.M.,

Martinovic,´ D., Miller, D.H., Perkins, E.J., Orlando, E.F., Villeneuve, D.L., Wang,

to C18-estrogens (Callard et al., 1978). This conversion is a key event

R.-L., Watanabe, K.H., 2009. Endocrine disrupting chemicals in ﬁsh: developing

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reduction of estrogen synthesis and subsequently lowers E2 plasma action. Aquat. Toxicol. 92, 168–178.

Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D.,

levels. In developing zebraﬁsh, this estradiol imbalance promotes

Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.a., Tietge,

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for Fish and Wildlife Health, University of Bern, for performance

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for performing the chemical analysis and Maike Lutter und Tom

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