Identification and Characterization of Androgen-Responsive Genes in

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Identification and characterisation of androgen– responsive genes in zebrafish embryos Eva Fetter, Sona Smetanova, Lisa Baldauf, Annegret Lidzba, Rolf Altenburger, Andreas Schüttler, and Stefan Scholz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01034 • Publication Date (Web): 26 Aug 2015 Downloaded from http://pubs.acs.org on August 27, 2015

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Environmental Science & Technology

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Identification and characterisation of androgen–responsive genes in zebrafish embryos

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Eva Fetter1*, Soňa Smetanová1,2, Lisa Baldauf1, Annegret Lidzba1, Rolf Altenburger1, Andreas

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Schüttler1, Stefan Scholz1

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1

5

Permoserstraße 15, 04318 Leipzig, Germany

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2

7

Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic

UFZ – Helmholtz Centre for Environmental Research, Department Bioanalytical Ecotoxicology,

RECETOX – Research Centre for Toxic Compounds in the Environment, Masaryk University,

1 Environment ACS Paragon Plus


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Abstract

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Responsive genes for fish embryos have been identified so far for some endocrine pathways but not

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for androgens. Using transcriptome analysis and a multiple concentration–response modelling, we

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identified putative androgen–responsive genes in zebrafish embryos exposed to 0.05–5000 nM 11–

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ketotestosterone for 24 hours. Four selected genes with sigmoidal concentration–dependent

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expression profiles (EC50=6.5–30.0nM) were characterised in detail. The expression of cyp2k22 and

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slco1f4 was demonstrated in the pronephros, lipca was detected in the liver and sult2st3 was found

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in the olfactory organs and choroid plexus. Their expression domains, the function of human

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orthologs and a pathway analysis suggested a role of these genes in the metabolism of hormones.

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Hence, it was hypothesised that they were induced to compensate for elevated hormone levels. The

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induction of sult2st3 and cyp2k22 by 11–ketotestosterone was repressed by co–exposure to the

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androgen receptor antagonist nilutamide supporting a potential androgen receptor mediated

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regulation. Sensitivity (expressed as EC50 values) of sult2st3 and cyp2k22 gene expression

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induction

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ketotestosterone~testosterone>progesterone>cortisol>ethinylestradiol) correlated with their known

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binding affinities to zebrafish androgen receptor. Hence, these genes might represent potential

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markers for screening of androgenic compounds in the zebrafish embryo.

after

exposure

to

other

steroidal

hormones

(11–

25 26

Keywords: endocrine disruptors, microarray, animal alternatives, Danio rerio, gene expression,

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transcriptome, chemical hazard assessment, toxicogenomics

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1. Introduction

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Chemicals that have the potential to disrupt the endocrine system of humans and wildlife are of high

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concern given the possible long–term impact on health and population development. The focus has

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been so far on estrogen–, androgen– and thyroid hormone regulation and an array of different in

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vitro and in vivo test systems has been developed, targeting e.g. the binding to hormone receptors,

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receptor transcriptional activation, enzymatic activities and protein– or hormone production [1-3].

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Particularly for the screening of many compounds or environmental samples in vitro cellular

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systems provide advantages such as specificity, simplicity and ability to automate the analysis.

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Alternatively, fish embryos have been suggested as a promising approach to screen for endocrine

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disruption [4]. In contrast to cellular assays, zebrafish embryos represent a complex system closely

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related to adult animals [5]. Further advantages of fish embryos in contrast to cellular assays are

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that they allow testing of hormonal effects in the context of other relevant endpoints such as

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neurotoxicity [6] or teratogenicity [7].

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Given the small dimension, transparency, and rapid ex utero development, the ease of production

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and the fact that many hormone signalling pathways are at least partially established already in

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embryonic stages [8], the zebrafish embryo as model organism is attractive and increasingly used in

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toxicological studies. This includes endocrine disruption using endpoints such as gene expression of

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target genes [9, 10], fluorescence of reporter genes/proteins in transgenic lines [11-14] or in in vivo

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immunoassays [15]. According to animal welfare regulations embryonic stages of fish are not

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protected and are therefore considered as alternatives to the testing of adult animals. [16].

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To date several assays using fish embryos are available for the screening of (anti)estrogens or

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goitrogens [1]. An appropriate assay for androgens is available only using a construct of the

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stickleback spiggin gene promoter with a reporter gene in medaka embryos [17]. Other androgen

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signalling–related sensitive genes have not yet been discovered for embryonic stages.

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Transcriptome analyses revealed responsive genes for androgen receptor agonists [18] and

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antagonists [19] in adult zebrafish liver and gonads, such as hydroxysteroid dehydrogenases 3 Environment ACS Paragon Plus


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(hsd17β3, hsd3β7, and hsd11β2) and cytochrome P450s (cyp2k7 and cyp17a1). These genes are

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involved in androgen biosynthesis and metabolism. For fish embryos potential anti–androgen

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responsive genes were identified for early stages (2 days post fertilisation) [20], but a detailed

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concentration–response and ligand specificity analysis has not been conducted. Therefore, in this

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study we aimed at identifying native androgen responsive genes with a concentration–dependent

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expression in post–hatched embryonic stages of zebrafish using microarray analysis. We selected

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the late embryonic period since previous studies with zebrafish have shown a higher sensitivity

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against exposure to endocrine disruptors for late embryonic stages [11, 15] presumably due to the

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onset of hormone synthesis [21] or the expression of nuclear receptors [22, 23]. Transcriptome data

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were obtained from exposure experiments with a series of concentrations of 11–ketotestosterone

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(0.05 – 5000 nM). We aimed at identifying genes directly regulated through the androgen receptor

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(AR). Therefore, the time course of the expression profile and the effect of a co–exposure to the

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androgen antagonist nilutamide and agonist 11–ketotestosterone on the gene expression were

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investigated in more detail for the two most responsive candidate genes (sult2st3 and cyp2k22).

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Furthermore, we assessed their specificity by comparing the response to various hormones. Since

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little was known about the identified target genes, we also performed in situ hybridisations to

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localise the expression domain and to obtain further information on the potential function of the

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genes.

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2. Experimental section

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2.1

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2.1.1 Chemicals

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For the exposure experiments, the following chemicals were used (CAS registry number, purity and

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manufacturer are given in parentheses): 11–ketotestosterone (53187–98–7, ≥98% Sigma Aldrich);

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17α–ethinylestradiol (57–63–6, ≥98% Sigma Aldrich), cortisol (50–23–7, ≥98% Sigma Aldrich),

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dimethylsulfoxide–DMSO (67–68–5, ROTIDRY ≥99.5%; ≤200 ppm H2O), nilutamide (63612–50–

Exposure experiments


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0, ≥98% Sigma Aldrich), progesterone (57–83–0, ≥99% Sigma Aldrich), testosterone (58–22–0,

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≥98% Sigma Aldrich).

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2.1.2 Zebrafish maintenance and exposure of embryos

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We used the UFZ–OBI wild type strain, which has been established from a stock of a local breeder

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and kept for several generations at the UFZ. Fish were cultured at 26±1°C at a 14:10 h light: dark

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cycle in a recirculating tank as described by Westerfield [24]. Husbandry and experimental

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procedures were performed in accordance with the German animal protection standards and were

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approved by the Government of Saxony, Landesdirektion Leipzig, Germany (Aktenzeichen 75–

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9185.64).

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Zebrafish embryos were exposed under static conditions for 24 h (96–120 hpf in the qPCR

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experiments, 72–96 and 96–120 hpf in the in situ hybridisation experiments) with the exception of

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the gene expression studies on different exposure durations where the exposure length was varied

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between 1-96 h (see details in chapter 3.5). The short exposure period was chosen in order to

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primarily detect genes directly regulated by hormones and to minimize potential secondary effects

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(e.g. autoregulatory feedback mechanisms). For the microarray analysis exposure to the non–

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aromatizable fish androgen 11–ketotestosterone was conducted in the range of 0.05–5000 nM in a

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single replicate but with three groups of control embryos (incubated in embryonic medium). For

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each sample 40–50 embryos were exposed in a volume of 100 ml. At the end of the exposure

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embryos were pooled in order to obtain enough material for microarray analysis and to avoid the

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necessity of further amplification steps and to obtain candidate genes with robust expression

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patterns. The 11–ketotestosterone concentration in the exposure media were analysed using an 11–

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ketotestosterone ELISA kit (Biomol GmbH, Hamburg, DE). About 80% of the nominal

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concentrations were detected after 24 h of exposure (Table S1). Validation experiments using qPCR

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were performed in three independent experiments pooling 40–50 embryos for each sample. For the

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combined exposure with 11–ketotestosterone and nilutamide two independent experiments were

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performed pooling 40–50 embryos for each sample. 5 Environment ACS Paragon Plus


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Penetration of RNA probes may be limited in later embryonic stages. Therefore, the exposure

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window of 72–96 hpf was used in addition to the 96–120 hpf exposure to 500 nM 11–

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ketotestosterone for part of the in situ hybridisation experiments. At least 25 individuals were

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analysed for each hybridisation probe and treatment, and a hybridisation has been repeated at least

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twice with the same probe in independent experiment.

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To prepare 11–ketotestosterone solutions no solvents were used. For all other compounds, embryos

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were exposed to dilutions of DMSO stock solutions (1 mg/mL for AR agonists and 7 mg/mL for

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nilutamide) in embryonic medium prepared according to the OECD guideline 236 [25]. An

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exposure of embryos to 0.1% DMSO served as solvent control. Dilutions of the stocks were

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prepared freshly before starting the exposure. Embryos were kept in an incubator at 26°C and a

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light cycle of 14:10 hours light: dark. Only embryos showing no malformations were subjected to

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gene expression analysis.

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2.2

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2.2.1 Microarray experiment

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Total RNA was isolated from homogenates of embryos using phase–lock tubes (5Prime GmbH,

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Hamburg) and Trizol (Invitrogen, Darmstadt) according to the manufacturer’s instructions. RNA

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was further purified using RNeasy Mini kit (Qiagen, Hilden, Germany). RNA concentrations were

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measured

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Spectrophotometer (Peqlab, Erlangen, Germany) and the RNA quality was checked using an

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Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA integrity values

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ranged from 9.6 to 10. RNA was labelled with the one–colour low input labelling and hybridization

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kit (Agilent Technologies) according to the manufacturer instructions. Scanning of the arrays was

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performed at Genovia GmbH (Zwenkau, Germany) using an Agilent DNA Microarray scanner.

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Microarray studies were performed using a commercial zebrafish oligonucleotide array

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(representing the whole zebrafish genome) based on the Ensembl zebrafish genome version 3 but

Transcriptome analysis using microarrays

by

spectrophotometric

absorption

at

260

nm


on

a

NanoDrop

ND–1000

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adapted to a custom 8x60k design (Amadid G4102A, Agilent). The annotation of the probes has

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been updated to the version 9 mapping the oligonucleotide probes to the zebrafish genome

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according to the method described by Arnold, et al. [26]. In brief, Agilent probe sequences were

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each aligned to the Zebrafish Genome (Zv9/danRer7, Jul. 2010) using BLAT [27]. Only hits with a

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minimum overlap of 95% were considered. All probes mapped more than once in the genome were

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disregarded. After mapping, the sequences were annotated using R version 3.0.1 and the biomaRt

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package [28, 29].

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2.2.2 Statistical analysis of microarray data

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Fluorescent intensities of individual microarray spots were extracted using the Agilent Feature

140

Extraction software (version 10.7.3.1). Raw microarray data were converted to log2 values and

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were quantile–normalised. For further statistical analysis, fold changes in relation to the mean of the

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controls were calculated for each treatment. In order to identify genes potentially regulated via the

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AR, a multiple concentration–response modelling using a logistic regression was performed. The

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four parametric log–logistic Hill concentration–response model commonly used in toxicology for

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concentration–response analysis was applied for the fitting of fold change data.

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y = E0 + (Emax - E0) × (C^ δ / (EC50 ^ δ +C^ δ))

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E0 represents the minimum effect level, Emax the maximum effect level, EC50 the half maximum

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effect concentration, δ the slope of the curve and C the exposure concentration.

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Probes showing a concentration–dependent response were identified and ranked by calculating the

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Akaike's Information Criterion (AIC) for each fit. The AIC was used as an indicator for the

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goodness of fit and is based on a maximized value of likelihood function and the number of

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parameters of the fitted model. Given the complexity and computational requirements of such a

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modelling it was restricted to genes showing at least a ratio of four between the maximum and

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minimum gene expression values across the treatments. Modelling was conducted for individual

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probes (all oligos were handled separately) after rescaling of the fold change data between 0 and 1

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(minimum–maximum normalisation). The rescaling of data prior to the concentration response 7 Environment ACS Paragon Plus

(1)


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modelling was necessary to be able to use the AIC as an indicator for the general goodness of fit

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among all tested responses of probes, since this criterion is dependent on the scale of the data. The

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curve fitting and the estimation of AIC were conducted using an R script based on the ‘drc’and

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‘epicalc’ R packages [30, 31].

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For further analyses, we selected probes based on the EC50 values and the AIC. The selected probes

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were clustered using the TMEV software package (version 4.9) and HCL clustering function with

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Pearson correlation [32]. In contrast to the concentration response–modelling, the cluster analysis

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was conducted with original (not rescaled) data.

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For the identification of biological functions and pathways associated with the changes of gene

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expression,

167

[https://analysis.ingenuity.com]. As input data, we used here the probes selected for the cluster

168

analysis as quantile normalised log2 ratios with respect to the mean of the controls. We used the

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“core analysis” function with a cut–off of 0.5 log2 ratio to identify over represented

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canonical pathways. As in the core analysis treatments are handled separately, a subsequent

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comparative analysis of the single core analyses was done.

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2.3

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2.3.1 Gene expression measurements with real–time quantitative PCR (qPCR)

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For the qPCR experiments total RNA was extracted from control or exposed zebrafish embryos at

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120 hpf (extraction was performed as specified at the microarray experiment). 2 µg of total RNA

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was reversely transcribed with RevertAidTM H Minus Reverse Transcriptase (Fermentas, Leon–

177

Roth, Germany) in 20 µl reaction mix according to the manufacturer instructions. qPCR was carried

178

out using a Step–One–Plus PCR System (Applied Biosystems, Darmstadt, Germany) and

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SensiMixTM SYBR with ROX as passive reference dye (Bioline, Luckenwalde, Germany) in 12.5 µl

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reaction volume in three technical replicates for each experimental replicate. gapdh was used as

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housekeeping gene. Full name of target genes and of the housekeeping gene, their NCBI reference

Ingenuity

Pathway

Analysis

(version

18030641)

was

performed

Validation of microarray experiment and characterisation of the candidate genes


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sequence identification code and literature references are listed in Table S3. Primer sequences,

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amplicon lengths and PCR protocol are listed in Table S4. The validation of gapdh as housekeeping

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gene is described in the supporting information (Figure S3 and Table S5). Melting curve analysis

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was conducted to ensure the gene specificity of the primers. Relative expression levels were

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determined by using the ∆∆Ct method [33]. In order to minimize variation across experiments, data

187

were mean–centred. Modelling of concentration response curves was performed with the Hill model

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(see equation 1) using GraphPad Prism v 5.01 (GraphPad Software Inc., La Jolla, CA, USA) by

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fixing E0 to 1.

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2.3.2 Toxicokinetics–toxicodynamic modelling of the time course of gene expression changes

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The time course of changes in gene expression in embryos exposed to 500 nM 11–ketotestosterone

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was described by a toxicokinetics–toxicodynamic model using an indirect effect model. The

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description of the indirect effect model and details on the modelling are given in the supporting

194

information. In brief, for the description of time course of internal concentration we used a TK

195

model for zebrafish embryos proposed by Kühnert, et al. [34]. The TD model was built under the

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assumption that gene expression is regulated by the binding of 11–ketotestosterone (ligand) to the

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AR resulting in an activation of the receptor via conformational changes of the AR. These activated

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hormone receptor complexes are transferred into the nucleus where they bind to the androgen

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response elements of the target gene promoter and trigger transcription (Figure S6).

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Here we applied two types of indirect effect models (direct stimulation and direct stimulation &

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indirect inhibition) used commonly in pharmacological studies [35] to describe the time course of

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gene expression profiles. We solved the differential equations numerically using a fourth–order

203

Runge–Kutta method in Berkeley Madonna (version 8.3.18). Using Madonna's “curve fitter”

204

option, we estimated kinetic parameters and took these to analyse the plausibility for describing the

205

observations.



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2.3.3 Localisation of gene expression with in situ hybridisation

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The expression domain of the target genes was investigated by in situ hybridisation in treated (500

208

nM 11–ketotestosterone) and untreated zebrafish embryos in order to reveal first evidence on the

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possible function of the genes. However, the design of the in situ hybridisations did not aim at

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demonstrating an increase in tissue–specific expression in 11–ketotestosterone exposed embryos but

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to obtain information on the potential function of the genes. To localise the expression domain of

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sult2st3, a control experiment was conducted with the clu gene, a marker of the zebrafish choroid

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plexus (Figure S4).

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A fragment of 600–900b of the cyp2k22, sult2st3, clu, slco1f4, and lipca genes were amplified

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using RT–PCR. The PCR reaction was conducted with GoTaq® Polymerase (Promega GmbH,

216

Mannheim, Germany) according to the manufacturer instruction. Fragments of cyp2k22, slco1f4,

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clu and lipca were cloned in a PCRII vector (Invitrogen, Darmstadt, Germany), linearised and

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purified. Fragments of sult2st3 were amplified with primers including a T7 promoter added to the 5’

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end of the reverse primer (see PCR protocol in the supporting information). Primers and amplicon

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length are given in Table S4. Synthesis of digoxigenin labelled RNA probes and whole mount in

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situ hybridizations were performed according to Thisse and Thisse [36] using linearised plasmids

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(cyp2k22, clu, slco1f4 and lipca) or the PCR fragment (sult2st3). For cyp2k22, clu, slco1f4 and lipca

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a control in situ hybridisation with sense RNA probes was performed.

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A Leica stereo microscope (MZ16F) equipped with a Leica colour digital camera (DFC 350FX)

225

(Leica, Wetzlar, Germany) was used to image embryos from in situ hybridisation mounted in

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glycerol.

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3. Results

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3.1

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The multiple concentration–response modelling of transcriptome data was restricted to 6877 probes

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of the microarray that showed at least a ratio of four between the maximum and minimum gene

Microarray experiment


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expression values across treatments. Since 11–ketotestosterone is known to transactivate AR

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already in low concentrations (EC50=~1 nM) [37], we selected probes showing an EC50

Identification and Characterization of Androgen-Responsive Genes in

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