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Transcriptional effects of androstenedione and 17α-hydroxyprogesterone in zebrafish embryos
Accepted Manuscript Title: Transcriptional effects of androstenedione and 17␣-hydroxyprogesterone in zebrafish embryos Authors: Karl Fent, Patricia Franziska Siegenthaler, Andreas Alexandre Schmid PII: DOI: Reference:
S0166-445X(18)30344-8 https://doi.org/10.1016/j.aquatox.2018.06.012 AQTOX 4967
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
9-4-2018 19-6-2018 21-6-2018
Please cite this article as: Fent K, Siegenthaler PF, Schmid AA, Transcriptional effects of androstenedione and 17␣-hydroxyprogesterone in zebrafish embryos, Aquatic Toxicology (2018), https://doi.org/10.1016/j.aquatox.2018.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptional effects of androstenedione hydroxyprogesterone in zebrafish embryos
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Karl Fent a,b,+, Patricia Franziska Siegenthaler a, Andreas Alexandre Schmid b
a University
of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences,
b Swiss
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Gründenstrasse 40, CH–4132 Muttenz, Switzerland
Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and
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Pollution Dynamics, Department of Environmental System Sciences, CH–8092 Zürich, Switzerland +
Corresponding author: Karl Fent
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Tel.: +41 61 228 55 87, E-mail: [email protected]; [email protected]
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ORCID
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Karl Fent: 0000-0002-3916-7196
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Highlights
A4 and testosterone induced the formation of sult2st3, cyp19b and cyp2k7 transcripts
Induction of sult2st3, cyp19b and cyp2k7 may serve as biomarkers for androgenic exposure
17-hydroxyprogesterone showed no physiological effects up to 10 g/L
17-hydroxyprogesterone induced little expressional changes
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Summary
Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of these steroids and transcriptional changes were determined
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at 96 hours post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 g/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120
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hpf embryos at 6.56 g/L A4 and 1 g/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10
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g/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 g/L. Our study highlights
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sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-
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OHP4 is not likely to pose a risk for fish at environmental concentrations.
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Different classes of steroid hormones and their metabolites occur in surface waters at concentrations in the range of ng/L, and rarely up to g/L (Chang et al., 2011, 2009; Fent 2015; Zhang et al., 2017; Zhang and Fent, 2018). They originate from natural excretion and medical use in humans and livestock. Maximal concentrations of androstenedione (or 4-androstenedione or androst-4-ene-3,17dione, A4) of up to 360 ng/L in wastewaters (Chang et al., 2011; 2009; Zhang et al., 2017), 480 ng/L in rivers (Chang et al., 2008; Liu et al., 2012), 130 ng/L in hospital wastewater (Zhang et al., 2017) and 40 ng/L in paper mill effluents (Durhan et al., 2002) have been reported. River water concentrations were
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generally in the range of few ng/L A4 or below 1 ng/L (Zhang et al., 2017; Zhang and Fent, 2018; Conley
et al., 2017). However, they represent major contributors to the measured androgenic activity (Thomas et al., 2002).
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A4 is a weak agonist of the androgen receptor (ar) and has a lower potency than the fish
androgen 11-ketotestosterone (11-KT). The potency of A4 was 15-24% relative to 11-KT in rainbow fish and was lower than that of testosterone (Bain et al., 2015). This androgen can cause sex reversal in
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zebrafish and induced masculinization in female fathead minnow (DeQuattro et al., 2015) and in mosquitofish (Hou et al., 2017; Stanko and Angus, 2007). Similarly, pulp mill effluent waters
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contaminated by A4 can cause masculinization (Jenkins et al., 2001; Parks et al., 2001). Induction of
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vitellogenin was also reported after exposure to androgens (Hornung et al., 2004; Pawlowski et al.,
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2004).
17-hydroxyprogesterone (17-OHP4) is an endogenous steroid hormone, which serves as an
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intermediate in the biosynthesis of hydrocortisone and gonadal steroids. It is also a metabolite of progesterone (P4), whicn reaches aquatic ecosystems from natural excretion by humans and livestock. Reported concentrations of 17-OHP4 were up to 66 ng/L in WWTP influents In Switzerland (Zhang et
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al., 2017; Zhang and Fent, 2018), 25 ng/L in WWTP discharge sites and 1.1 ng/L in rivers in China (Chang et al., 2009). High levels were found in runoff from cattle feedlots in the United States with maximum
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concentrations of 1250 ng/L (Bartelt-Hunt et al., 2012). 17-OHP4 binds to the mammalian progesterone receptor (pgr) but the relative binding affinity
was only 1% of that of P4 (Attardi et al., 2007), which suggests a low activity of this metabolite. In contrast, binding of 17-OHP4 to zebrafish pgr was similar to P4 (Chen et al., 2010), which suggests a
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different potency between humans and fish. Thus, it is interesting to test whether the currently unknown biological potency of 17-OHP4 in fish is similar to that of P4 (Zucchi et al., 2013, 2012). Despite their ubiquitous environmental occurrence little is known about ecotoxicological implications of A4 (Hou et al. 2017, 2018), and nothing about 17-OHP4. The frequent occurrence of A4 and 17-OHP4 prompted us to study potential effects in fish. As generally, androgen responsive genes in fish are poorly known, a further aim of this study was to explore a series of target genes and to assess the activity of these steroids in fish. 3
We used a similar experimental design of embryo exposures as previously (Zucchi et al. 2012; Siegenthaler et al., 2017; Rossier et al., 2016; Zhao et al. 2016; Willi et al., 2018). Details of materials and chemicals (Tables S1, S2, Figure S1), exposures, measured endpoints at 96 hpf and 120 hpf, RNA isolation, RT-qPCR, primers (Table S3) are given in the supplementary material (SM). In brief, the A4 and 17-OHP4 experiments consisted of four concentrations (nominal concentrations of 0.01, 0.1, 1, and 10 µg/L), the positive control 1 µg/L testosterone (T) in case of A4 experiments, and a solvent control (0.01% DMSO). The low A4 and 17-OHP4 concentrations are environmentally relevant
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reflecting surface and wastewater contamination, respectively, while the higher concentrations are
pharmacological ones. We conducted three independent experiments with 4-5 replicates each with A4, and four replicates in the 17-OHP4 experiment. Physiological effects were analyzed in the 17-OHP4
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experiment. Spontaneous muscle contractions were determined at 24 hpf, hatching success at 72 hpf
and at 120 hpf, swimming activity as performed previously (Zhao et al., 2016; Zhang et al., 2017; Willi et al., 2018). We quantified A4 concentrations in exposure water in one experiment. Details of the
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sampling and chemical analysis are given in the SM. Mean concentrations in the A4 experiment were 0.00413, 0.0456, 0.619, and 6.563 µg/L (Table S4). A4 concentrations were relatively stable during the
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24 h static-renewal exposure.
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The series of selected 18 genes in the A4 and 15 in the 17-OHP4 experiment included genes
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with the following types of products: hormone receptors, vitellogenin, proteins in the regulation of circadian rhythm and enzymes involved in steroidogenesis and metabolism. The selection of target
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genes was based on mechanistic considerations and previous studies with androgens for A4 (Fetter et al., 2015; Hoffmann et al., 2008) and P4 metabolites (Rossier et al., 2016; Zhao et al., 2015; Zucchi et al., 2012). Testosterone and 6.563 µg/L A4 led to significant transcriptional induction of the genes
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encoding aromatase, cyp19b, and sulfotransferase, sult2st3, at 120 hpf (Figure 1), while no significant changes occurred at 96 hpf (Figures 1 and Figures S2-S6). The magnitude and number of transcriptional
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responses showed some variability among the experiments (transcriptional alterations below log2 = 1, e.g. two-fold changes were not regarded as being significant) (Figures S2-S6). Additionally, a significant induction of cyp2k7 occurred at 6.563 µg/L A4 in two experiments. The heat map including hierarchical clustering of the mean of the three experiments shows that, in addition to induction of cyp19b and
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sult2st3 at 120 hpf, cyp2k7 induction occurred at both time points at 6.563 µg/L A4 (Figure 1b). 17-OHP4 exposure did not induce physiological effects (spontaneous muscle contractions,
heart rate, hatching success or swimming activity) (Figure S7). This contrasts to pronounced effects that were induced by other steroids like glucocorticoids (Willi et al., 2017; Zhao et al., 2016). Figure 2a illustrates that among the 15 selected genes, pgr, cyp19a, cyp19b, hsd17b3, cyp2k7 and nr1d1 transcripts showed significant induction at 10 g/L 17-OHP4, either at 96 or 120 hpf. The
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transcriptional changes were very minor and above two-fold for cyp2k7 only, which suggests a low potency of 17-OHP4 (Figure S8). The heat map (Figure 2b) indicates that transcriptional effects occurred mainly at the highest concentration of 17-OHP4. Our study shows that 6.563 µg/L A4 led to significant induction of the genes encoding aromatase cyp19b and sulfotransferase sult2st3, and to a minor extent of cyp2k7 at 120 hpf. Expression patterns of A4 and testosterone were similar and varied for different genes between different developmental stages (96 and 120 hpf), although effects at 120 hpf were often more
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pronounced. Induction of sult2st3 and cyp2k22 (cyp2k7 according to new nomenclature) but not of cyp19b was previously reported at extremely high concentrations of 11-ketotestosterone (11-KT) or
50 nM (14.4 µg/L) testosterone (Fetter et al., 2015). Cyp2k7 upregulation by 17alpha-
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methyldihydrotestosterone has also been reported in the liver of zebrafish (Hoffmann et al., 2008), and 1 µg/L testosterone led to induction of cyp19b and sult2st3 in the brain of adult zebrafish (Mouriec et al., 2009). Recently, Hou et al. (2018) reported transcriptional effects in zebrafish exposed to 4 to
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980 ng/L A4 exposed for up to 144 hpf. Induction of several transcripts including pgr, vtg1, and several steroidogenesis genes including cyp19a were found to be induced. In contrast, we did not find
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induction of pgr and vtg1 even at higher concentrations. However, here we assessed alteration of
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complementary to the data of Hou et al. (2018).
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cyp19b, sult2st3 and cyp2k, which have not been previously been assessed, thus our data are
Induction of cyp19b, cyp2k7 and sult2st3, which encode phase I and phase II enzymes and are regulated by the androgen receptor (Fetter et al., 2015), suggests alteration of metabolism of
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androgenic steroids by A4 and testosterone, in case the transcriptional alteration translates to the enzyme protein and activity. In contrast to A4 and testosterone, 11-ketotestosterone, the most potent
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androgen in fish, led to induction of vtg1 at 0.1 μg/L (Fuzzen and Bernier, 2011). Endogenous and synthetic progestins mediate their biological activities mainly by binding to
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the pgr but also bind to additional steroid hormone receptors, and thus, they may interfere with signaling of the endocrine system (Fent, 2015). We found little transcriptional changes in the pgr and genes encoding steroidogenesis enzymes, cyp19a, cyp19b, hsd17b3, and cyp2k7 and nr1d1 at 10 g/L, either at 96 or 120 hpf. Thus, the P4 metabolite 17-OHP4 altered similar gene transcripts as did P4
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(Rossier et al., 2016; Zhao et al., 2015; Zucchi et al., 2012), although at lower potency. This indicates a lower potency of the metabolite. A4 is ubiquitously found in surface waters contaminated by wastewater, runoff from livestock, or paper mill effluents. Highest environmental levels of A4 in WWTP effluents and river water were in the range of a few hundred ng/L in China and Japan (Chang et al., 2011, 2009, 2008), whereas in Switzerland, 2 and 1 ng/L, respectively, were found (Zhang et al., 2017). However, testosterone concentrations may reach 9.33 µg/L in untreated wastewater (Poltrock, 2010) and 6.1 ng/L in WWTP 5
effluents (Kolodziej et al., 2003). In our study, the lowest observed effect concentrations (LOECs) of A4 and testosterone for transcriptional changes were 0.62 and 1 µg/L, respectively, which are well above environmental levels in surface waters. Environmental levels of 17-OHP4 were 66 ng/L in WWTP influents in Switzerland (Zhang et al., 2017; Zhang and Fent, 2018), up to 25 ng/L in WWTP discharge sites and 1.1 ng/L in river water in China (Chang et al., 2009), while up to very high levels of 1250 ng/L were found in runoff from cattle
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feedlots in the United States (Bartelt-Hunt et al., 2012). In our study, the LOEC for low transcriptional alterations was 10 µg/L, thus well above environmental levels. Our data therefore suggest that single occurrence of A4, testosterone and 17-OHP4 may not pose a risk to fish embryos. However, steroids
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occur always as mixtures and they may sum up to effect concentrations in embryos (Zhang et al., 2017) or adult fish (Thrupp et al., 2018).
Together, our study leads to the conclusion that A4 and testosterone induce effects in developing zebrafish embryos at higher than environmental concentrations. Our findings with A4 and
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testosterone also suggests that sult2st3, cyp19b and cyp2k7 may serve as biomarkers of androgenic
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Acknowledgements
We thank Lilian Gremlich for contributions on effects of A4. The research was funded by the Swiss
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National Science Foundation (contract no. 310030_141040 to K.F.).
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Figure Legends Figure 1: (a) Relative transcript levels in zebrafish embryos after exposure to androstenedione (A4) and testosterone (T). Relative transcript levels of cyp19b, sult2st3 and cyp2k7 at 96 and 120 hpf. Significant transcriptional changes compared to solvent control are indicated with asterisks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001. Data are shown as mean ± SEM of three independent experiments.
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(b) Hierarchical clustering of transcriptional responses of zebrafish eleuthero-embryos exposed to different concentrations of A4 and testosterone at 96 hpf (-96) or 120 hpf (-120).
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Figure 2
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(a) Relative transcript levels in zebrafish embryos after exposure to 17-OHP4. Relative transcript levels of pgr, cyp2k7, cyp19a, cyp19b and nr1d1 at 96 and 120 hpf. Significant transcriptional changes compared to solvent control are indicated with asterisks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001. Data are shown as mean ± SEM of four biological replicates and 17-OHP4 concentrations are given in μg/L on the x-axis.
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(b) Hierarchical clustering of transcriptional responses of zebrafish eleuthero-embryos exposed to different concentrations of 17-OHP4 at 96 hpf (-96) or 120 hpf (-120).
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S0166-445X(18)30344-8 https://doi.org/10.1016/j.aquatox.2018.06.012 AQTOX 4967
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
9-4-2018 19-6-2018 21-6-2018
Please cite this article as: Fent K, Siegenthaler PF, Schmid AA, Transcriptional effects of androstenedione and 17␣-hydroxyprogesterone in zebrafish embryos, Aquatic Toxicology (2018), https://doi.org/10.1016/j.aquatox.2018.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptional effects of androstenedione hydroxyprogesterone in zebrafish embryos
and
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Karl Fent a,b,+, Patricia Franziska Siegenthaler a, Andreas Alexandre Schmid b
a University
of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences,
b Swiss
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Gründenstrasse 40, CH–4132 Muttenz, Switzerland
Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and
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Pollution Dynamics, Department of Environmental System Sciences, CH–8092 Zürich, Switzerland +
Corresponding author: Karl Fent
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Tel.: +41 61 228 55 87, E-mail: [email protected]; [email protected]
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ORCID
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Karl Fent: 0000-0002-3916-7196
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Highlights
A4 and testosterone induced the formation of sult2st3, cyp19b and cyp2k7 transcripts
Induction of sult2st3, cyp19b and cyp2k7 may serve as biomarkers for androgenic exposure
17-hydroxyprogesterone showed no physiological effects up to 10 g/L
17-hydroxyprogesterone induced little expressional changes
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Summary
Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of these steroids and transcriptional changes were determined
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at 96 hours post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 g/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120
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hpf embryos at 6.56 g/L A4 and 1 g/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10
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g/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 g/L. Our study highlights
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sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-
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OHP4 is not likely to pose a risk for fish at environmental concentrations.
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Different classes of steroid hormones and their metabolites occur in surface waters at concentrations in the range of ng/L, and rarely up to g/L (Chang et al., 2011, 2009; Fent 2015; Zhang et al., 2017; Zhang and Fent, 2018). They originate from natural excretion and medical use in humans and livestock. Maximal concentrations of androstenedione (or 4-androstenedione or androst-4-ene-3,17dione, A4) of up to 360 ng/L in wastewaters (Chang et al., 2011; 2009; Zhang et al., 2017), 480 ng/L in rivers (Chang et al., 2008; Liu et al., 2012), 130 ng/L in hospital wastewater (Zhang et al., 2017) and 40 ng/L in paper mill effluents (Durhan et al., 2002) have been reported. River water concentrations were
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generally in the range of few ng/L A4 or below 1 ng/L (Zhang et al., 2017; Zhang and Fent, 2018; Conley
et al., 2017). However, they represent major contributors to the measured androgenic activity (Thomas et al., 2002).
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A4 is a weak agonist of the androgen receptor (ar) and has a lower potency than the fish
androgen 11-ketotestosterone (11-KT). The potency of A4 was 15-24% relative to 11-KT in rainbow fish and was lower than that of testosterone (Bain et al., 2015). This androgen can cause sex reversal in
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zebrafish and induced masculinization in female fathead minnow (DeQuattro et al., 2015) and in mosquitofish (Hou et al., 2017; Stanko and Angus, 2007). Similarly, pulp mill effluent waters
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contaminated by A4 can cause masculinization (Jenkins et al., 2001; Parks et al., 2001). Induction of
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vitellogenin was also reported after exposure to androgens (Hornung et al., 2004; Pawlowski et al.,
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2004).
17-hydroxyprogesterone (17-OHP4) is an endogenous steroid hormone, which serves as an
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intermediate in the biosynthesis of hydrocortisone and gonadal steroids. It is also a metabolite of progesterone (P4), whicn reaches aquatic ecosystems from natural excretion by humans and livestock. Reported concentrations of 17-OHP4 were up to 66 ng/L in WWTP influents In Switzerland (Zhang et
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al., 2017; Zhang and Fent, 2018), 25 ng/L in WWTP discharge sites and 1.1 ng/L in rivers in China (Chang et al., 2009). High levels were found in runoff from cattle feedlots in the United States with maximum
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concentrations of 1250 ng/L (Bartelt-Hunt et al., 2012). 17-OHP4 binds to the mammalian progesterone receptor (pgr) but the relative binding affinity
was only 1% of that of P4 (Attardi et al., 2007), which suggests a low activity of this metabolite. In contrast, binding of 17-OHP4 to zebrafish pgr was similar to P4 (Chen et al., 2010), which suggests a
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different potency between humans and fish. Thus, it is interesting to test whether the currently unknown biological potency of 17-OHP4 in fish is similar to that of P4 (Zucchi et al., 2013, 2012). Despite their ubiquitous environmental occurrence little is known about ecotoxicological implications of A4 (Hou et al. 2017, 2018), and nothing about 17-OHP4. The frequent occurrence of A4 and 17-OHP4 prompted us to study potential effects in fish. As generally, androgen responsive genes in fish are poorly known, a further aim of this study was to explore a series of target genes and to assess the activity of these steroids in fish. 3
We used a similar experimental design of embryo exposures as previously (Zucchi et al. 2012; Siegenthaler et al., 2017; Rossier et al., 2016; Zhao et al. 2016; Willi et al., 2018). Details of materials and chemicals (Tables S1, S2, Figure S1), exposures, measured endpoints at 96 hpf and 120 hpf, RNA isolation, RT-qPCR, primers (Table S3) are given in the supplementary material (SM). In brief, the A4 and 17-OHP4 experiments consisted of four concentrations (nominal concentrations of 0.01, 0.1, 1, and 10 µg/L), the positive control 1 µg/L testosterone (T) in case of A4 experiments, and a solvent control (0.01% DMSO). The low A4 and 17-OHP4 concentrations are environmentally relevant
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reflecting surface and wastewater contamination, respectively, while the higher concentrations are
pharmacological ones. We conducted three independent experiments with 4-5 replicates each with A4, and four replicates in the 17-OHP4 experiment. Physiological effects were analyzed in the 17-OHP4
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experiment. Spontaneous muscle contractions were determined at 24 hpf, hatching success at 72 hpf
and at 120 hpf, swimming activity as performed previously (Zhao et al., 2016; Zhang et al., 2017; Willi et al., 2018). We quantified A4 concentrations in exposure water in one experiment. Details of the
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sampling and chemical analysis are given in the SM. Mean concentrations in the A4 experiment were 0.00413, 0.0456, 0.619, and 6.563 µg/L (Table S4). A4 concentrations were relatively stable during the
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24 h static-renewal exposure.
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The series of selected 18 genes in the A4 and 15 in the 17-OHP4 experiment included genes
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with the following types of products: hormone receptors, vitellogenin, proteins in the regulation of circadian rhythm and enzymes involved in steroidogenesis and metabolism. The selection of target
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genes was based on mechanistic considerations and previous studies with androgens for A4 (Fetter et al., 2015; Hoffmann et al., 2008) and P4 metabolites (Rossier et al., 2016; Zhao et al., 2015; Zucchi et al., 2012). Testosterone and 6.563 µg/L A4 led to significant transcriptional induction of the genes
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encoding aromatase, cyp19b, and sulfotransferase, sult2st3, at 120 hpf (Figure 1), while no significant changes occurred at 96 hpf (Figures 1 and Figures S2-S6). The magnitude and number of transcriptional
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responses showed some variability among the experiments (transcriptional alterations below log2 = 1, e.g. two-fold changes were not regarded as being significant) (Figures S2-S6). Additionally, a significant induction of cyp2k7 occurred at 6.563 µg/L A4 in two experiments. The heat map including hierarchical clustering of the mean of the three experiments shows that, in addition to induction of cyp19b and
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sult2st3 at 120 hpf, cyp2k7 induction occurred at both time points at 6.563 µg/L A4 (Figure 1b). 17-OHP4 exposure did not induce physiological effects (spontaneous muscle contractions,
heart rate, hatching success or swimming activity) (Figure S7). This contrasts to pronounced effects that were induced by other steroids like glucocorticoids (Willi et al., 2017; Zhao et al., 2016). Figure 2a illustrates that among the 15 selected genes, pgr, cyp19a, cyp19b, hsd17b3, cyp2k7 and nr1d1 transcripts showed significant induction at 10 g/L 17-OHP4, either at 96 or 120 hpf. The
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transcriptional changes were very minor and above two-fold for cyp2k7 only, which suggests a low potency of 17-OHP4 (Figure S8). The heat map (Figure 2b) indicates that transcriptional effects occurred mainly at the highest concentration of 17-OHP4. Our study shows that 6.563 µg/L A4 led to significant induction of the genes encoding aromatase cyp19b and sulfotransferase sult2st3, and to a minor extent of cyp2k7 at 120 hpf. Expression patterns of A4 and testosterone were similar and varied for different genes between different developmental stages (96 and 120 hpf), although effects at 120 hpf were often more
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pronounced. Induction of sult2st3 and cyp2k22 (cyp2k7 according to new nomenclature) but not of cyp19b was previously reported at extremely high concentrations of 11-ketotestosterone (11-KT) or
50 nM (14.4 µg/L) testosterone (Fetter et al., 2015). Cyp2k7 upregulation by 17alpha-
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methyldihydrotestosterone has also been reported in the liver of zebrafish (Hoffmann et al., 2008), and 1 µg/L testosterone led to induction of cyp19b and sult2st3 in the brain of adult zebrafish (Mouriec et al., 2009). Recently, Hou et al. (2018) reported transcriptional effects in zebrafish exposed to 4 to
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980 ng/L A4 exposed for up to 144 hpf. Induction of several transcripts including pgr, vtg1, and several steroidogenesis genes including cyp19a were found to be induced. In contrast, we did not find
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induction of pgr and vtg1 even at higher concentrations. However, here we assessed alteration of
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complementary to the data of Hou et al. (2018).
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cyp19b, sult2st3 and cyp2k, which have not been previously been assessed, thus our data are
Induction of cyp19b, cyp2k7 and sult2st3, which encode phase I and phase II enzymes and are regulated by the androgen receptor (Fetter et al., 2015), suggests alteration of metabolism of
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androgenic steroids by A4 and testosterone, in case the transcriptional alteration translates to the enzyme protein and activity. In contrast to A4 and testosterone, 11-ketotestosterone, the most potent
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androgen in fish, led to induction of vtg1 at 0.1 μg/L (Fuzzen and Bernier, 2011). Endogenous and synthetic progestins mediate their biological activities mainly by binding to
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the pgr but also bind to additional steroid hormone receptors, and thus, they may interfere with signaling of the endocrine system (Fent, 2015). We found little transcriptional changes in the pgr and genes encoding steroidogenesis enzymes, cyp19a, cyp19b, hsd17b3, and cyp2k7 and nr1d1 at 10 g/L, either at 96 or 120 hpf. Thus, the P4 metabolite 17-OHP4 altered similar gene transcripts as did P4
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(Rossier et al., 2016; Zhao et al., 2015; Zucchi et al., 2012), although at lower potency. This indicates a lower potency of the metabolite. A4 is ubiquitously found in surface waters contaminated by wastewater, runoff from livestock, or paper mill effluents. Highest environmental levels of A4 in WWTP effluents and river water were in the range of a few hundred ng/L in China and Japan (Chang et al., 2011, 2009, 2008), whereas in Switzerland, 2 and 1 ng/L, respectively, were found (Zhang et al., 2017). However, testosterone concentrations may reach 9.33 µg/L in untreated wastewater (Poltrock, 2010) and 6.1 ng/L in WWTP 5
effluents (Kolodziej et al., 2003). In our study, the lowest observed effect concentrations (LOECs) of A4 and testosterone for transcriptional changes were 0.62 and 1 µg/L, respectively, which are well above environmental levels in surface waters. Environmental levels of 17-OHP4 were 66 ng/L in WWTP influents in Switzerland (Zhang et al., 2017; Zhang and Fent, 2018), up to 25 ng/L in WWTP discharge sites and 1.1 ng/L in river water in China (Chang et al., 2009), while up to very high levels of 1250 ng/L were found in runoff from cattle
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feedlots in the United States (Bartelt-Hunt et al., 2012). In our study, the LOEC for low transcriptional alterations was 10 µg/L, thus well above environmental levels. Our data therefore suggest that single occurrence of A4, testosterone and 17-OHP4 may not pose a risk to fish embryos. However, steroids
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occur always as mixtures and they may sum up to effect concentrations in embryos (Zhang et al., 2017) or adult fish (Thrupp et al., 2018).
Together, our study leads to the conclusion that A4 and testosterone induce effects in developing zebrafish embryos at higher than environmental concentrations. Our findings with A4 and
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testosterone also suggests that sult2st3, cyp19b and cyp2k7 may serve as biomarkers of androgenic
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exposure in fish.
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Acknowledgements
We thank Lilian Gremlich for contributions on effects of A4. The research was funded by the Swiss
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National Science Foundation (contract no. 310030_141040 to K.F.).
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Figure Legends Figure 1: (a) Relative transcript levels in zebrafish embryos after exposure to androstenedione (A4) and testosterone (T). Relative transcript levels of cyp19b, sult2st3 and cyp2k7 at 96 and 120 hpf. Significant transcriptional changes compared to solvent control are indicated with asterisks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001. Data are shown as mean ± SEM of three independent experiments.
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(b) Hierarchical clustering of transcriptional responses of zebrafish eleuthero-embryos exposed to different concentrations of A4 and testosterone at 96 hpf (-96) or 120 hpf (-120).
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Figure 2
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(a) Relative transcript levels in zebrafish embryos after exposure to 17-OHP4. Relative transcript levels of pgr, cyp2k7, cyp19a, cyp19b and nr1d1 at 96 and 120 hpf. Significant transcriptional changes compared to solvent control are indicated with asterisks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001. Data are shown as mean ± SEM of four biological replicates and 17-OHP4 concentrations are given in μg/L on the x-axis.
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(b) Hierarchical clustering of transcriptional responses of zebrafish eleuthero-embryos exposed to different concentrations of 17-OHP4 at 96 hpf (-96) or 120 hpf (-120).
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