Transcriptomic and physiological changes in Eastern Mosquitofish (Gambusia holbrooki) after exposure to progestins and anti-progestagens

Aquatic Toxicology 179 (2016) 8–17

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Transcriptomic and physiological changes in Eastern Mosquitofish (Gambusia holbrooki) after exposure to progestins and anti-progestagens Erica K. Brockmeier a,∗ , Philip D. Scott b , Nancy D. Denslow a , Frederic D.L. Leusch b a b

Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, PO Box 110885, Gainesville, FL 32611, USA Smart Water Research Centre, Australian Rivers Institute, Griffith School of Environment, Griffith University, Southport, Qld 4222, Australia

a r t i c l e

i n f o

Article history: Received 24 April 2016 Received in revised form 2 August 2016 Accepted 4 August 2016 Available online 10 August 2016 Keywords: Endocrine active chemicals Gonopodium Levonorgestrel Progestin Secondary sexual characteristics Transcriptomics Progestagens

a b s t r a c t Endocrine active compounds (EACs) remain an important group of chemicals that require additional evaluation to determine their environmental impacts. While estrogens and androgens were previously demonstrated to impact organisms during environmental exposures, progestagens have recently been shown to have strong impacts on aquatic organisms. To gain an understanding of the impacts of these types of chemicals on aquatic species, experiments evaluating the mechanisms of action of progestagen exposure were conducted with the Eastern Mosquitofish (Gambusia holbrooki). The objective of this study was to conduct hepatic microarray analysis of male and female G. holbrooki exposed to progestins and anti-progestagens. In addition, we evaluated the ability of levonorgestrel, a synthetic progesterone (progestin), to induce anal fin elongation and to determine how anal fin growth is modulated during co-exposures with progesterone and androgen receptor antagonists. Gene expression analyses were conducted on male and female G. holbrooki exposed for 48 h to the agonist levonorgestrel, the antagonist mifepristone, or a mixture of the two chemicals. Microarray analysis revealed that mifepristone does not act as an anti-progestagen in G. holbrooki in liver tissues, and that levonorgestrel elicits strong effects on the processes of embryo development and lipid transport. Levonorgestrel was also demonstrated to induce male secondary sexual characteristic formation in females, and co-exposure of either an androgen or levonorgestrel in the presence of the anti-androgen flutamide prevented anal fin elongation. These results provide indications as to the potential impacts of progestins, including non-target effects such as secondary sexual characteristic formation, and demonstrate the importance of this class of chemicals on aquatic organisms. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Even after many years of research, endocrine active chemicals (EACs) continue to be a class of pollutants for which crucial information is still needed in terms of biological and ecological impacts. Major classes of hormone mimics that have been extensively studied include environmental estrogens and androgens (Chen et al., 2010; Parks et al., 2001; Soto et al., 2004) but recent studies have also demonstrated significant impacts of natural and synthetic progestagens (i.e., progestins) on aquatic organisms (Zeilinger et al., 2009). Natural progestagens can enter the environment from operations such as paper production (Carson et al., 2008) and large

∗ Corresponding author. Present address: Institute of Integrative Biology University of Liverpool, Crown Street Liverpool L69 7ZB, United Kingdom. E-mail address: [email protected] (E.K. Brockmeier). http://dx.doi.org/10.1016/j.aquatox.2016.08.002 0166-445X/© 2016 Elsevier B.V. All rights reserved.

amounts of progestins are present in the form of excreted birth control products (King et al., 2016; Runnalls et al., 2013; Viglino et al., 2008; Vulliet et al., 2007). As the aquatic ecosystem is a large sink for the deposition of contaminants in the environment, where they become bioavailable and concentrated in sediment and water (Suedel et al., 1994), understanding the effects of these chemicals on aquatic organisms is of importance for providing a thorough basis of biological understanding for ecological risk assessments. In studies on aquatic organisms, progestins such as levonorgestrel have been demonstrated to act not as progestagens but as androgens in terms of the physiological endpoints that are produced upon exposure. In female fathead minnows (Pimephales promelas) exposed to levonorgestrel, the formation of significant male secondary sexual characteristics was apparent at pharmacological doses (Zeilinger et al., 2009). In female three-spined stickleback (Gasterosteus aculeatus), exposure to levonorgestrel induced the expression of spiggin mRNA, a protein normally

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only expressed in male kidneys and which serves as a potential biomarker of exposure to androgens (Svensson et al., 2013). In human cell lines, levonorgestrel is known to be a strong progesterone receptor (PR) agonist and a moderate androgen receptor (AR) agonist (Leusch et al., 2014). While it seems that levonorgestrel can elicit responses via an AR- mediated mechanism, it is not currently known by which specific mode of action (MOA) this chemical is able to induce the formation of male secondary sexual characteristics. In addition to the use of the fathead minnow and three-spined stickleback species as model organisms for the study of EACs in the lab, the Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis, respectively) are aquatic organisms that exhibit hormoneinduced sexual dimorphism. The male anal fin elongates into the gonopodium structure under the influence of increased androgen levels (Turner, 1941). Mosquitofish have been used to study the effects of androgens (Angus et al., 2001; Sone et al., 2005; Stanko and Angus, 2007) and estrogens (Angus et al., 2005) and have been used as a bioindicator organism in EAC exposure studies (Game et al., 2006; Orlando et al., 2007). In a recent article, levonorgestrel was also shown to induce anal fin elongation in females at exposures of 10 ng/L, providing additional support of this chemical acting as an androgen receptor agonist in fish (Frankel et al., 2016). In addition to their sexual dimorphism, molecular tools are also available for these species, including a method for in situ hybridization analysis of spatial gene expression patterns (Brockmeier et al., 2013a; Ogino et al., 2004) as well as a custom G. holbrooki microarray (Brockmeier et al., 2013b). These tools can be utilized to help understand the MOA of chemical exposure and how physiological masculinization manifests itself during exposure to hormone mimics. Because some progestagens appear to elicit androgenic effects, the objective of this study was to evaluate the molecular and physiological impacts of a progestin, an anti-progestagen, and mixture exposures in the Eastern Mosquitofish (G. holbrooki) using microarray technology. To address this objective, the two specific aims were: 1) conduct hepatic microarray analysis of male and female G. holbrooki exposed to the progestin levonorgestrel, the anti-progestagen mifepristone, and a mixture of the two chemicals, and 2) determine how anal fin growth is modulated during co-exposures of fish with levonorgestrel and either PR or AR antagonists. We hypothesized that effects on gene expression and changes in physiology would be indicative of an androgenic type of exposure in fish. Our results demonstrate that gene expression patterns exhibit overlap with profiles of AR ligands, as well as gene pathways which may be PR-related effects. In addition, co-exposure with the anti-androgen flutamide prevented anal fin elongation in exposure scenarios using either androgens or progestins.

2. Materials and methods 2.1. Animals and chemical treatments Exposures for specific aim 1 took place at the University of Technology (UTS) in Sydney, Australia. All procedures were conducted to minimize animal suffering and complied with animal ethics permit UTS ACEC 2008-022A. Four sexually mature fish per tank were acclimatized in randomized tanks, with two replicate tanks per treatment for a total of eight fish per treatment. Sexual maturity in males was determined by visual inspection of the gonopodium, a modified anal fin in males used for reproduction, while females were identified as sexually mature by presence of black gravid spots on either side of the abdomen. All tanks contained 2 L of dechlorinated Sydney tap water and fish were acclimatized in these tanks for 24 h prior to the 48 h exposure. A light cycle of 16:8 h light: dark

9

was utilized and fish loading did not exceed 1 g/L. G. holbrooki were fed 4% (w/w) body weight of Nutrafin Max Colour Enhancing Flakes (Hagen, Montreal, Canada) once daily. After acclimation, half of the water was exchanged and an additional 1 L was added. Tanks were dosed once at the beginning of the experiment with the exposure solutions made up in ethanol, and tank ethanol concentration did not exceed 10 ␮L/L (0.001%). After 30 min, 1 L grab samples were taken for solid-phase extraction (SPE) and subsequent chemical analysis. The following exposure concentrations were utilized for both male and female G. holbrooki exposures for specific aim 1: solvent control (ethanol), 0.3 ␮g/L (0.96 nM) levonorgestrel (13ethyl-17-ethynyl-17-hydroxy1,2,6,7,8,9,10,11,12,13,14,15,16, 17- tetradecahydrocyclopenta[a] phenanthren-3-one; CAS 79763-7) (Sigma-Aldrich, St. Louis, USA), 0.05 ␮g/L (0.166 nM) mifepristone (11␤-(4-dimethyl-amino)-phenyl-17␤-hydroxy17-(1-propynyl)--estra-4,9-dien-3-one; CAS 84371-65-3) (Sigma Aldrich, St. Louis, USA), and a mixture of levonorgestrel and mifepristone (0.3 ␮g/L (0.96 nM) and 0.05 ␮g/L (0.166 nM), respectively). These does were selected to represent a pharmacological exposure to the PR agonist, PR antagonist, and mixture experiments. For specific aim 2, a 21-day exposure was conducted on adult female G. holbrooki; this experiment was separately conducted from the experiment in specific aim 1 (for microarray data generation). As the focus of this experiment was on chemically-induced anal fin elongation and targeted anal fin gene expression, we used female G. holbrooki only. Sexually mature female G. holbrooki (defined as fish being greater than 15 cm standard length and with the presence of the gravid spot near the vent) were maintained as laboratory stocks at the University of Florida Aquatic Toxicology Facility (ATF) following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. Sexually mature female G. holbrooki were transferred to aerated 7 L glass tanks and exposed via semi-static renewal to either the vehicle control (ethanol, 0.0014% final concentration), 0.5 ␮g/L (1.85 nM) of 17␤-trenbolone (17␤-Hydroxyestra-4,9,11-trien-3one; CAS 10161-33-8) (Steraloids, Newport, USA); abbreviated as TB, or 0.1 ␮g/L (0.32 nM) of levonorgestrel (Steraloids, Newport, USA). For this exposure there were three replicate tanks per treatment and two fish per tank. The levonorgestrel exposure concentration was chosen as a high concentration known to induce male secondary sexual characteristic formation in fathead minnow (Zeilinger et al., 2009) and TB was selected at a concentration known to induce significant anal fin elongation as compared to controls in female mosquitofish after 21 days of exposure (Fig. 3). A follow-up experiment was conducted to determine if coexposures of levonorgestrel and TB with either PR or AR antagonists could reduce anal fin elongation. G. holbrooki from laboratory stocks at the ATF were transferred to aerated 4 L glass tanks and exposed via semi-static renewal (∼75% water change daily) to either the vehicle control (ethanol, 0.0374% final concentration), 0.1 ␮g/L (0.32 nM) of levonorgestrel, 0.1 ␮g/L (0.32 nM) of levonorgestrel with 10 ␮g/L (23.27 nM) of the anti-progestagen mifepristone (Sigma Aldrich, St. Louis, USA), 1 ␮g/L (3.70 nM) of TB with 100 ␮g/L (362 nM) of mifepristone, 0.1 ␮g/L (0.32 nM) of levonorgestrel with 100 ␮g/L (362 nM) of the anti-androgen flutamide (2-Methyl-N(4-nitro-3-[trifluoromethyl]phenyl)propanamide, CAS 1311-84-7) (Sigma Aldrich, St. Louis, USA), and 1 ␮g/L (3.70 nM) of TB with 1000 ␮g/L (3.62 ␮M) of flutamide. This ratio of the AR antagonist flutamide was chosen based on an observed decrease in AR signaling during co-exposures of the AR agonist TB with flutamide and a ratio of levonorgestrel to mifepristone as mifepristone was able to block PR signaling in vitro using the PR Gene BLAzer assay (Invitrogen, Carlsbad, USA). In this experiment we utilized TB as a positive control for anal fin elongation. This exposure was also conducted for

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2.2. Sample preparation and physiological measurements For the experiment conducted at UTS to address specific aim 1, fish were euthanized at the end of the 48 h exposure using 100 mg/L benzocaine (ethyl 4-aminobenzoate, CAS 94-09-7) (Sigma Aldrich, New South Wales, Australia) and livers were immediately excised and placed into vials with 2 mL RNAlater (Sigma Aldrich, New South Wales, Australia). Samples were held at 4◦ C overnight and stored at −80◦ C until RNA extraction. In experiments conducted at the ATF for specific aim 2, mosquitofish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Body length and anal fin ray 4 and ray 6 lengths were assessed upon dissection. For anal fin in situ analysis of sonic hedgehog (shh) expression in the 21-day levonorgestrel exposed G. holbrooki, the posterior half of the fish was cut away and fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight at 4◦ C. Samples were then washed with PBS/0.1% tween (PBT) and dehydrated by methanol in PBT washes (25/50/75/100%) before being stored at −20◦ C. 2.3. Solid-phase extraction (SPE) and chemical analysis of the water SPE was performed using a previously published method (Scott et al., 2014a; Scott et al., 2014b) with adjustments for using 1 L grab samples instead of 2 L, and pH adjustment was not necessary as samples were processed immediately. Levonorgestrel was analyzed by Gas Chromatography–Tandem Mass Spectrometry (GC–MS/MS) as previously described (Trinh et al., 2011). Mifepristone was quantified using Liquid Chromatography Quadrupole Time-of-Flight (LC-qTOF) using an Agilent 1290 high-pressure liquid chromatography (HPLC) attached to an Agilent 6530 qTOF (Agilent, Victoria, Australia). Separation was performed using an Agilent Zorbax Eclipse plus C18 column (2.1 × 100 mm with 1.8 ␮m particle size). Mobile phase A consisted of water with 0.1% formic acid. Mobile phase B was made of 95% acetonitrile and 5% water with 0.1% formic acid. Flow rate was 0.7 mL/min with an injection volume of 2 ␮L under the following optimized elution conditions: 10% B linearly programmed to 70% B over 10 min, followed by 70% B increased linearly to 95% B over 1 min and held for 3 min. 95% B was reduced to 10% B over 0.1 min and held for 2.9 min. The ionization source was an Agilent Jetstream ESI source in positive mode with a drying temperature of 300◦ C, a sheath gas temperature of 300◦ C, nozzle voltage of 500 V, capillary voltage of 3000 V and fragmenter (cone) voltage of 115 V. Data were acquired at three acquisitions per second. Data analysis was performed using the Agilent MassHunter Qualification and Quantification software packages. All analyses were based on the calculated monoisotopic exact mass for mifepristone (m = 429.2668 m/z; M + H = 430.2741 m/z), which eluted at 5.57 min, and all quantification was performed on the protonated precursor ion for mifepristone. For quantification, a 14-point standard curve was generated from 0.01 to 2000 ng/mL. The limit of quantification (LOQ) by direct injection for mifepristone was 3 ng/mL. 2.4. RNA extraction and quality control analysis For microarray sample analysis, RNA was extracted from RNAlater-preserved liver tissues by TRIzol (Invitrogen, Carlsbad, USA) at the University of Florida. After thawing on ice and drying

3

levo mife mix

2

Fold change over control

21 days. For the mixture exposures there were three replicate tanks per treatment and two fish per tank. All exposures were conducted so that each tank (vehicle control and treatment tanks) contained the same volume of ethanol.

A

Females

1 0 1

5

9

13

17

21

25

29

33

-1 -2 -3 -4

Gene number

3

Levo Mife Mix

2

Fold change over control

10

B

Males

1 0 1

5

9

13

17

21

25

29

33

37

41

-1 -2 -3

Gene number

Fig. 1. Hepatic differential gene expression that was statistically significant over the controls (one-way ANOVA, p < 0.05) in all treatment groups in the female (A) and male (B) data sets. Genes with reciprocal regulation between treatment groups in the female exposures are indicated in Fig. S1, with no reciprocal genes in the male data set.

off excess RNAlater, tissue homogenization was conducted in 1 mL TRIzol, followed by a 5 min room temperature incubation. Chloroform (200 ␮L) was added to the sample and following a 10-min room temperature incubation was put through high-speed centrifugation (15 min at 4◦ C at 20,800g). Isopropanol (500 ␮L) was used to precipitate RNA and all RNA pellets were washed twice with ethanol (80%). Samples were rehydrated with RNAsecure (Ambion, Grand Island, USA) and a NanoDrop spectrophotometer (ThermoScientific, Waltham, USA) was used to evaluate RNA quality and quantity. All samples were DNase treated (Ambion, Grand Island, USA) to reduce the presence of genomic DNA contamination. Additional quality analysis was conducted by obtaining an RNA Integrity Number (RIN) using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of A260 /A280 values was 1.88-1.99 and RINs ranged from 8.2-9.6. Four RNA samples from each treatment were used for microarray analysis. 2.5. Microarray analysis We selected the liver as the target organ for microarray analysis since the liver is responsible for both xenobiotic detoxification and steroid metabolism, so any changes seen in the this organ are indicative of both toxicologically and mechanistically relevant endpoints during EAC exposure. The low RNA input fluorescent linear amplification kit (Agilent, Santa Clara, USA) was used for conversion of RNA into labeled cRNA for microarray analysis. T7 primers were annealed to 200 ng of RNA, along with RNA spike-in controls that were added to each sample (One color RNA spikein mix; Agilent, Santa Clara, USA), via 10 min incubation at 65◦ C. MMLV-RT was used for cDNA synthesis, which took 2 h at 40◦ C immediately followed by 15 min at 70◦ C. T7 RNA polymerase (in

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were conducted using tRNA-free hybridization/saline-sodium citrate buffer and blocked with 2 mg/mL bovine serum albumin and 2% fetal bovine serum before incubation with antibodies against digoxigenin alkaline phosphatase (Roche, Penzberg, Germany) overnight at 4◦ C. Samples were incubated for 24 h with PBT and then alkaline phosphate buffer immediately before color development by the BM purple AP substrate (Roche, Penzberg, Germany). Upon color formation, the reaction was stopped using PBT with ∼1% paraformaldehyde (PFA). Representatives photographs from analyzed fins were captured with the MZ FLIII microscope (Leica Camera, Solms, Germany) and Leica Application Suite software v 3.6 (Leica Microsystems, Wetzlar, Germany). 2.7. Statistical analysis

Fig. 2. Significant differential gene expression differences between the vehicle control, levonorgestrel (levo), mifepristone (mife), and mixture (mix) exposed female G. holbrooki (A) and male G. holbrooki (B) as determined by K-means clustering. Genes with at least one treatment group having a one-way ANOVA p-values <0.05 (false discovery rate ␣ = 0.05) over the controls were used for this analysis. Abbreviations used: C, control; M, mifepristone; L, levonorgestrel; M, mixture.

addition to Cy3 for incorporation into the cRNA) was utilized to reverse transcribe cDNA into cRNA via in vitro transcription (2 h at 40◦ C) followed by column purification (RNEasy Kit, QIAGEN, Hilden, Germany). The NanoDrop spectrophotometer (ThermoScientific, Waltham, USA) was used to determine concentrations and Cy3 incorporation of the resulting cRNA. Using the Gene Expression hybridization kit (Agilent, Santa Clara, USA), 600 ng of cRNA was hybridized to the 8 × 15,000 G. holbrooki slides following the manufacturer’s recommended protocol. All slides were incubated for 17 h at 65◦ C while rotating at 10 rpm. Slides were washed and scanned using the Sure Scan microarray scanner (Agilent, Santa Clara, USA) at the University of Florida Gene Expression core facility. All reporting of data was conducted according to the Minimum Information About a Microarray Experiment (MIAME) guidelines and all data sets were deposited onto the Gene Expression Ontology (GEO) database (GSE55518). 2.6. In situ hybridization analysis Methods previously published (Brockmeier et al., 2013a) were utilized for qualitative gene expression analysis of shh based on modifications of a previously published protocol (Thisse et al., 1993). Samples kept in 100% methanol were rehydrated and washed in phosphate buffered saline with Tween20 (PBT) before 50 ␮g/mL proteinase-K treatment lasting for 45 min at room temperature. After stopping the enzymatic reaction by washing with 4% paraformaldehyde (PFA), PBT, and pre-hybridization buffer, the tissues were incubated with 0.5 ␮g/mL of the anti-sense RNA probe specific for G. holbrooki shh which was labeled with digoxigenin. Incubation occurred over three nights at 60◦ C with mixing. Washes

To determine anal fin elongation, the ratio of the length of the 4th anal fin ray (the longest part of the gonopodium in males) was divided by the length of the 6th anal fin ray (the termination of the fin rays that make up the gonopodium). These data were analyzed by one-way analysis of variance (ANOVA), with a cut-off for statistical significance set at p < 0.05. For significant ANOVA models, a Bonferroni post-hoc test was used to determine which treatment groups were significantly different from controls using SigmaPlot 11 (Systat Software Inc., San Jose, USA). For microarray data analysis, methods previously published (Brockmeier et al., 2013b) were utilized. In brief, all samples were subjected to quality control analysis; two of the male samples did not meet quality control requirements (one mixture-exposed fish and one control-exposed fish) and were removed from further analyses. Analysis of the remaining data was conducted on background subtracted signals after removal of non-uniform spots. JMP Genomics 6.0 (SAS Institute, Cary, USA) was used for data analysis unless otherwise noted. All data presented are log2 transformed and were subjected to loess normalization (based on the distributions of the parallel and box plots) before data analysis. Data analysis was conducted on male and female transcriptomes separately. A one-way ANOVA was used to determine genes that were differentially expressed by levonorgestrel, mifepristone, and mixture treatment groups as compared to the gender’s vehicle control. A false discovery rate (FDR) ␣=0.05 was utilized for this analysis and a p-value of <0.05 was considered statistically significant. Hierarchical cluster analysis was performed using significant genes using Cluster 3.0. Loess-transformed data were clustered via k-means clustering into four centers using the ‘stats’ package within R (v. 3.0.1). In addition to one-way ANOVA and cluster analyses, a Fisher’s Exact test was utilized as part of gene set enrichment analysis to determine significant sets of genes based on their Gene Ontology (GO) “Biological Process” category that were up or down-regulated as a group. An FDR ␣ = 0.05 was utilized for this analysis and a Fisher raw p-value of <0.05 was considered statistically significant. Significant pathways were compared to GO Biological Processes which were significantly enriched or under-represented in other data sets, including G. holbrooki females exposed to TB (Brockmeier et al., 2013b) and masculinized G. holbrooki females residing downstream of a paper mill impacted site (Brockmeier et al., 2014). 3. Results 3.1. Experimental parameters For the exposure conducted at UTS in specific aim 1, there was one mortality during the two-day exposure (one male G. holbrooki exposed to 0.3 ␮g/L (0.96 nM) levonorgestrel). In specific aim 2, we saw a higher number of mortalities in the

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Table 1 Chemical concentrations for levonorgestrel and mifepristone (experiment 1). Exposure

Levonorgestrel (ng/L) (LOQ: 5 ng/L)

Mifepristone (ng/L) (LOQ: 3 ng/L)

Levonorgestrel, males Levonorgestrel, females Mifepristone, males Mifepristone, females Levonorgestrel and mifepristone mixture, males Levonorgestrel and mifepristone mixture, females

208 ± 63 357 ± 23

<3 <3

<5 <5 270 ± 13

53 ± 1 46 ± 0 49 ± 4

280 ± 12

50 ± 2

LOQ: Limit of Quantitation.

levonorgestrel/mifepristone mixture and the TB/mifeprestone mixure. This may be due to mifepristone’s ability to serve as a non-selective antagonist for the GR in fish (Mazon et al., 2004). As the GR is known to regulate metabolism and immune responses, it is possible that reductions in these activities due to higher exposure/off-target effects could be inducing these higher mortality rates, although the LC50 for any fish species is not currently known for direct comparisons to the concentrations used in our study. Table 1 presents the measured concentrations of levonorgestrel and mifepristone for the experiment conducted in specific aim 1, with all concentrations from this experiment reported as measured throughout the manuscript. 3.2. Microarray analysis In both the male and female microarray data sets, a large number of genes were significantly differentially regulated compared to vehicle controls (Fig. 1). The numbers of genes with significant differential regulation over the female controls (oneway ANOVA, p < 0.05, FDR ␣ = 0.05) are as follows: levonorgestrel, 226 genes upregulated and 265 downregulated; mifepristone, 269 genes upregulated and 185 downregulated; and mixture, 341 genes upregulated and 282 downregulated. In general, there were more genes differentially regulated in the male G. holbrooki: levonorgestrel, 456 genes upregulated and 342 downregulated; mifepristone, 166 genes upregulated and 171 downregulated; and mixture, 292 genes upregulated and 271 downregulated versus control samples. In the female data set, 34 genes were significantly different from the controls in all of the treatment groups (Fig. 1a). Two of these genes were regulated differently in the treatment scenarios: an unknown gene product was upregulated by mifepristone and downregulated by both levonorgestrel and the mixture. Topoisomerase (DNA) II alpha was downregulated by both the levonorgestrel and mifepristone treatments but upregulated following the mixture exposure (Fig. 1a). When focusing on genes that were significantly differentially regulated as compared to the control by levonorgestrel and mifepristone (not in the mixture group), only three out of the list of 75 genes were reciprocally regulated, with two of these genes (ATPase family, AAA domain containing 2 B and an unknown gene product) upregulated by mifepristone and downregulated by levonorgestrel and one gene (kallikrein-related peptidase 2) upregulated by levonorgestrel and downregulated by mifepristone (Fig. S1). In the male data set, 41 genes were significantly different from the controls in all three of the treatment groups. However, none of these genes were reciprocally regulated in any of the treatments (Fig. 1b). This was also the case for the 128 genes which were significantly different from the controls in the levonorgestrel and mifepristone treatments only.

Table 2 Significantly enriched (↑) and under-represented (↓) Gene Ontology Biological Processes in adult female G. holbrooki exposed to levonorgestrel as compared to the vehicle control. GO Category (Biological Process)

Fisher raw P-value

Direction of change

go:0009790; embryo developmenta go:0008610; lipid biosynthetic processb go:0006511; ubiquitin-dependent protein catabolic processc go:0032355; response to estradiol stimulusb go:0051568; histone h3-k4 methylation go:0000398; nuclear mrna splicing, via spliceosome go:0006003; fructose 2,6-bisphosphate metabolic process go:0031124; mrna 3’-end processingd go:0050769; positive regulation of neurogenesis go:0060013; righting reflex go:0008380; rna splicinge go:0051297; centrosome organization go:0030168; platelet activatione go:0045941; positive regulation of transcription go:0006000; fructose metabolic process go:0006465; signal peptide processing go:0006621; protein retention in er lumend go:0015937; coenzyme a biosynthetic process go:0016579; protein deubiquitination go:0007409; axonogenesis go:0060041; retina development in camera-type eyed go:0046847; filopodium assembly go:0006368; transcription elongation from rna polymerase ii prom go:0006890; retrograde vesicle-mediated transport, golgi to er go:0007160; cell-matrix adhesion go:0006098; pentose-phosphate shunt go:0006476; protein deacetylation go:0006493; protein o-linked glycosylation go:0006364; rrna processing

0.0001 0.0028

↑ ↑

0.0041

↓

0.0047

↑

0.0064

↓

0.0119

↓

0.0131

↓

0.0131 0.0131

↓ ↓

0.0131 0.0150 0.0183 0.0199 0.0265

↓ ↑ ↓ ↑ ↑

0.0270

↓

0.0270 0.0270

↓ ↓

0.0270

↓

0.0270 0.0312 0.0322

↓ ↑ ↓

0.0339 0.0342

↓ ↓

0.0342

↓

0.0361 0.0397 0.0397 0.0397

↑ ↑ ↑ ↑

0.0493

↑

a

Pathway similarly expressed between females of all exposure groups. Pathway expressed in opposite directions when comparing levonorgestrel and mifepristone groups. c Pathway expressed in opposite directions when comparing levonorgestrel and mixture groups. d Pathway similarly expressed when comparing levonorgestrel and mixture groups. e Pathway similarly expressed when comparing levonorgestrel and TB-exposed groups. b

Fig. 2 is a principal components analysis (PCA) demonstrating the relationship between gene expression profiles between groups in which the genes utilized for clustering were statistically significant over the control in at least one treatment group. For the male data set, samples clustered into groups corresponding with the chemical treatment (Fig. 2a), whereas for females, one of the mixture samples was classified into the mifepristone group (Fig. 2b), albeit still clustering closely to the other mixture samples. There were several GO pathways with similar enrichment or under-representation patterns between treatment groups within both sexes. While there were overlapping pathways between the levonorgestrel and mixture groups, there were no similar pathways between the mifepristone and mixture groups, indicating that the effects seen in the mixture may be primarily driven

E.K. Brockmeier et al. / Aquatic Toxicology 179 (2016) 8–17 Table 3 Significantly enriched (↑) and under-represented (↓) Gene Ontology Biological Processes in adult female G. holbrooki exposed to mifepristone as compared to the vehicle control. GO Category (Biological Process)

Fisher raw P-value

Direction of change

go:0006414; translational elongation go:0009790; embryo developmenta go:0001756; somitogenesis go:0016126; sterol biosynthetic process go:0006917; induction of apoptosis go:0006869; lipid transport go:0006259; dna metabolic process go:0051603; proteolysis involved in cellular protein catabolism go:0043065; positive regulation of apoptosis go:0008299; isoprenoid biosynthetic process go:0032355; response to estradiol stimulusb go:0008610; lipid biosynthetic processb go:0044267; cellular protein metabolic process go:0030216; keratinocyte differentiation go:0042221; response to chemical stimulus go:0003007; heart morphogenesis go:0045597; positive regulation of cell differentiation go:0048545; response to steroid hormone stimulus go:0051384; response to glucocorticoid stimulus go:0006415; translational termination go:0046777; protein autophosphorylation go:0006694; steroid biosynthetic process go:0007017; microtubule-based process go:0006366; transcription from rna polymerase ii promoter go:0006633; fatty acid biosynthetic process go:0007283; spermatogenesis go:0030574; collagen catabolic process go:0007601; visual perception go:0009966; regulation of signal transduction go:0035050; embryonic heart tube development go:0051726; regulation of cell cycle go:0006935; chemotaxis

< 0.0001 < 0.0001 0.0043 0.0068

↑ ↑ ↑ ↓

0.0073 0.0082 0.0103 0.0125

↑ ↑ ↓ ↓

0.0139

↑

0.0193

↓

0.0195

↓

0.0205

↓

0.0206

↑

0.0216

↑

0.0234

↑

0.0270 0.0283

↑ ↓

0.0283

↓

0.0283

↓

0.0313 0.0313

↑ ↑

0.0321

↓

0.0321

↓

0.0329

↑

0.0329

↓

0.0344 0.0357 0.0380 0.0393

↑ ↓ ↓ ↑

0.0393

↑

0.0419 0.0453

↑ ↓

13

Tables 4 and 5 display the data from the male data set. Mixture data tables can be found in the Supplementary materials (Tables S1 and S2). Pathways with overlapping regulation within the same gender include embryo development (enriched in all female treatment groups), immune response (enriched in levonorgestrel and mifepristone males), and lipid transport (enriched in all male treatment groups). Pathways of regulation in the opposite direction between exposure groups of the same gender include response to estradiol stimulus (enriched in females exposed to levonorgestrel and under-represented in mifepristone), and lipid biosynthetic process (enriched in females exposed to levonorgestrel and under-represented in mifepristone). Between the two data sets, pathways of interest include isoprenoid biosynthetic process (under-represented in females exposed to mifepristone and enriched in males exposed to mifepristone), steroid biosynthetic process (under-represented in females exposed to mifepristone and males exposed to mixture), and regulation of protein metabolic process (under-represented in females exposed to mixture and enriched in males exposed to mixture). 3.3. Anal fin elongation and sonic hedgehog gene expression during levonorgestrel exposure Fig. 3 demonstrates the ability of the progestin levonorgestrel to induce anal fin elongation after a 21-day exposure to 0.1 ␮g/L (0.32 nM) levonorgestrel (one-way ANOVA with Bonferroni correction, p < 0.001). Elongation was also present in female G. holbrooki exposed to 0.5 ␮g/L of TB (one-way ANOVA with Bonferroni correction, p < 0.05) with no statistical significance between TB or levonorgestrel exposed G. holbrooki (p = 0.069). Fig. 4 depicts a representative in situ hybridization for shh in the levonorgestrelexposed G. holbrooki anal fins. Dark coloration at the distal portion of the elongated anal fin is indicative of shh expression. 3.4. Anal fin elongation during mifepristone and flutamide co-exposures Fig. 5 demonstrates anal fin elongation in adult female G. holbrooki after a 21-day exposure to 0.1 ␮g/L (0.32 nM) of the positive control levonorgestrel. When combined with the anti-androgen

a

Pathway similarly expressed between females of all exposure groups. Pathway expressed in opposite directions when comparing levonorgestrel and mifepristone groups. b

by levonorgestrel and not a mixture effect. There is also some overlap between the levonorgestrel and mifepristone exposures, which may be due to mifepristone’s ability to act as a partial antiestrogen and weak AR agonist (Kemppainen et al., 1992; Kettel et al., 1994). These results are consistent with other studies, which demonstrated that progesterone exposure resulted in a similarity of transcriptional responses when compared to mifepristone, and activation of the PR was also seen in zebrafish (Bluthgen et al., 2013b). Tables 2–5 depict the significantly enriched and underrepresented GO Biological Process categories as determined by gene set enrichment analysis. Tables 2 and 3 provide the pathways of females exposed to levonorgestrel and mifepristone that were significantly different from the vehicle control whereas

Fig. 3. Results of 21-day exposure of adult female G. holbrooki to a vehicle control, 100 ng/L levonorgestrel, or 500 ng/L 17␤-trenbolone. An asterisk indicates statistical significance (p < 0.05) from the control samples and the number inside each bar represents the number of female mosquitofish sampled. Error bars represent the standard deviation of the mean values.

14

E.K. Brockmeier et al. / Aquatic Toxicology 179 (2016) 8–17 Table 4 Significantly enriched (↑) and under-represented (↓) Gene Ontology Biological Processes in adult male G. holbrooki exposed to levonorgestrel as compared to the vehicle control.

Fig. 4. Sonic hedgehog (shh) gene expression during 21-day levonorgestrel exposure. Female G. holbrooki were exposed to 100 ng/L levonorgestrel or the vehicle control (ethanol) after 21 days. In situ hybridization by an anti-sense shh probe was used to evaluate spatial gene expression patterns; increased dark stain (indicated by black arrows) indicates probe binding and thus localized gene expression. Photographs are representative of all samples analyzed (N = 4 per treatment) with the control fin shown at a 2.5× objective and the levonorgestrel fin at a 4× objective.

Fig. 5. Results of 21-day exposure of adult female G. holbrooki to a vehicle control, 100 ng/L levonorgestrel, and mixtures of 100 ng/L levonorgestrel or 1000 ng/L 17␤trenbolone with either flutamide or mifepristone at a ratio of 1:100 (for mifepristone co-exposures) or 1:1000 (for flutamide co-exposures). An asterisk indicates statistical significance from the control samples (p < 0.05) and the number inside each bar represents the number of female mosquitofish sampled. Error bars represent the standard deviation of the mean values.

flutamide, there was no evident anal fin elongation compared to the control during both TB and levonorgestrel exposure (one-way ANOVA with Bonferroni correction, p = 1.000 levonorgestrel and p = 0.982 TB). However, during co-exposures of TB and mifepristone, anal fin elongation was significantly greater than the control (one-way ANOVA with Bonferroni correction, p = 0.003) (Fig. 5). While similar results were seen with the levonorgestrel and mifeprestone co-exposure as with the TB and mifepristone coexposure, a reduced sample size due to mortalities in this treatment group may have resulted in a lack of statistical significance (oneway ANOVA with Bonferroni correction, p = 0.420). 4. Discussion Results presented in this paper corroborate previous findings which suggested that mifepristone elicits effects other than PR

GO Category (Biological Process)

Fisher raw P-value

Direction of change

go:0051603; proteolysis involved in cellular protein catabolic processa go:0060041; retina development in camera-type eye go:0006260; dna replicationb go:0046686; response to cadmium ion go:0007067; mitosis go:0006278; rna-dependent dna replication go:0060271; cilium morphogenesis go:0006955; immune responsea go:0006270; dna-dependent dna replication initiation go:0060021; palate development go:0007596; blood coagulationa go:0009117; nucleotide metabolic process go:0001756; somitogenesisa go:0009615; response to virus go:0008277; regulation of g-protein coupled receptor protein sig go:0030593; neutrophil chemotaxis go:0019835; cytolysis go:0006888; er to golgi vesicle-mediated transport go:0032355; response to estradiol stimulusa go:0046777; protein autophosphorylation go:0006807; nitrogen compound metabolic processc go:0000084; s phase of mitotic cell cycle go:0000910; cytokinesis go:0030163; protein catabolic process go:0006869; lipid transportd go:0008360; regulation of cell shape go:0030199; collagen fibril organization

<0.0001

↓

0.0002

↓

0.0011 0.0057 0.0061 0.0069

↑ ↓ ↑ ↑

0.0089 0.0115 0.0118

↓ ↑ ↓

0.0118 0.0216 0.0227

↓ ↑ ↑

0.0232 0.0232 0.0248

↑ ↑ ↓

0.0248 0.0319 0.0319

↓ ↓ ↓

0.0337

↑

0.0377

↑

0.0407

↑

0.0422

↓

0.0422 0.0422 0.0437 0.0492 0.0492

↓ ↓ ↑ ↓ ↓

a Pathway similarly expressed between males exposed to levonorgestrel and mifepristone. b Pathway similarly expressed between males exposed to levonorgestrel and mixture. c Pathway expressed in opposite directions when comparing levonorgestrel and mixture groups. d Pathway similarly expressed between males in all treatment groups.

antagonism in G. holbrooki. While levonorgestrel exhibits androgenic responses at the physiological level, gene expression results were indicative of a general EAC effect that could not be directly linked to a specific type of EAC. In the microarray results presented here, a lack of antagonism during co-exposures between levonorgestrel and mifepristone was observed. Exposures of adult zebrafish (Danio rerio) to mifepristone resulted in an increase in cumulative egg output at exposure concentrations of 5 ng/L and 77 ng/L mifepristone; exposure of zebrafish to mifepristone also resulted in an increased number of vitellogenic oocytes, as well as the formation of morphological changes within the ovaries of females, but with no impacts on fertility or hatching success (Bluthgen et al., 2013a). Another study demonstrated that both natural progesterone and mifepristone were acting both as anti-estrogens and weak AR and PR agonists when using a recombinant yeast assay with human steroid receptors (Bluthgen et al., 2013b). Gene expression patterns were also evaluated in adults and embryos exposed to these chemicals, with downregulation of the AR, as well as the PR and glucocorticoid receptor (GR). Other

E.K. Brockmeier et al. / Aquatic Toxicology 179 (2016) 8–17 Table 5 Significantly enriched (↑) and under-represented (↓) Gene Ontology Biological Processes in adult male G. holbrooki exposed to mifepristone as compared to the vehicle control. GO Category (Biological Process)

Fisher raw P-value

Direction of changes

go:0044267; cellular protein metabolic processa go:0006955; immune responseb go:0006950; response to stress go:0006414; translational elongationc go:0001756; somitogenesisb go:0051436; negative regulation of ubiquitin-protein ligase actin go:0032355; response to estradiol stimulusb go:0008299; isoprenoid biosynthetic process go:0019221; cytokine-mediated signaling pathway go:0007596; blood coagulationb go:0008015; blood circulation go:0014070; response to organic cyclic compound go:0042254; ribosome biogenesis go:0045087; innate immune response go:0007399; nervous system development go:0007612; learning go:0007018; microtubule-based movement go:0051452; intracellular ph reduction go:0055086; nucleobase, nucleoside and nucleotide metabolism go:0001889; liver development go:0022900; electron transport chain go:0051603; proteolysis involved in cellular protein catabolismb go:0006979; response to oxidative stress go:0045727; positive regulation of translationd go:0006334; nucleosome assemblyc go:0001501; skeletal system development go:0006869; lipid transporte go:0008016; regulation of heart contraction

0.0021

↓

0.0023 0.0029 0.0034 0.0042 0.0071

↑ ↑ ↑ ↑ ↓

0.0076

↑

0.0107

↓

0.0107

↓

0.0118 0.0144 0.0144

↑ ↓ ↓

0.0162 0.0185 0.0204

↑ ↓ ↑

0.0225 0.0273

↑ ↑

0.0291 0.0291

↓ ↓

0.0310 0.0361 0.0385

↑ ↓ ↓

0.0388

↓

0.0391

↑

0.0412 0.0439

↑ ↑

0.0448 0.0461

↑ ↓

a Pathway expressed opposite between males exposed to mifepristone and mixture. b Pathway similarly expressed between males exposed to levonorgestrel and mifepristone. c Pathway similarly expressed between males exposed to mifepristone and mixture. d Pathway similarly expressed between males exposed to mifepristone and TB. e Pathway similarly expressed between males in all treatment groups.

genes for steroidogenic enzymes, including several types of steroid dehydrogenases and P450 enzymes, were also downregulated by mifepristone exposure. The authors concluded that mifepristone was not acting as an anti-progestagen in zebrafish (Bluthgen et al., 2013b). In our study, mifepristone did not change gene expression patterns during a co-exposure scenario with levonorgestrel (Fig. 1a and b) and it was unable to prevent levonorgestrel-induced anal fin elongation. While this is not a progestagenic effect, the lack of blocking of this effect during a co-exposure seems to indicate that levonorgestrel and mifepristone are interacting with different receptors in vivo or that they are interacting with the same receptor as agonists (Fig. 5). Gene set enrichment analysis was used to evaluate changes at the level of biological processes using gene expression data from the microarray data sets. This analysis was conducted in order to evaluate the types of pathways that are modulated by levonorgestrel and mifepristone exposure (Tables 2–5). When looking

15

at processes that are similar between the male and female data sets, several pathways of interest emerge. Both female and male G. holbrooki exposed to mifepristone had an under-representation of genes related to the isoprenoid biosynthetic process (go:0008299), a pathway that is important for the formation of steroid molecule precursors in the liver (Costigan et al., 2012). This same pathway was enriched in female G. holbrooki residing downstream of the pulp and paper mill on the Fenholloway River which is impacted by a mixture of EACs (Brockmeier et al., 2014). This pathway was also enriched in fathead minnows impacted by other paper mill effluent-impacted sites (Costigan et al., 2012). These findings suggest that impacts to isoprenoid biosynthesis are a potential target of mifepristone exposure, and the overlap between findings from two sites contaminated with EACs may indicate an overlap in the mode of action of these types of chemicals. In addition, the process of lipid transport (go:0006869) was enriched in all treatment groups from the male data set. This is an interesting parallel to the human literature, as ‘androgenic progestins’ are known to stimulate lipid movement including increased hepatic lipase activity (Tikkanen, 1996). In addition to the transport of lipids in males, embryo development (go:0009790) was an enriched biological process in all of the female treatment groups. This corroborates what is currently understood about fish oogenesis, as progression through early stages of egg cell development involves both estrogens and the fish progestagen 17␣,20␤-dihydroxy-4-pregnen-3-one (DHP). DHP serves to stimulate oogenesis via initiating meiotic divisions in the developing egg cells (Miura et al., 2007), so it is not surprising that exogenous progestagen exposure to female G. holbrooki stimulates the expression of genes related to embryo development. We provide further support of levonorgestrel’s ability to induce the formation of male secondary sexual characteristic (Fig. 3), findings that overlap with a recent publication on G. holbrooki (Frankel et al., 2016) as well as to studies in other fish species (Svensson et al., 2013; Zeilinger et al., 2009). In our study, levonorgestrel is able to induce anal fin elongation after 21 days at a ∼10-fold lower concentration than the potent androgen receptor agonist TB (0.32 nM versus 3.70 nM, respectively). This is supported by in vitro results that demonstrate that levonorgestrel and TB are both very potent androgens (with an EC50 of 0.87 and 0.48 nM, respectively, in a human androgen reporter gene assay, compared with an EC50 of 0.24 nM for the reference androgen dihydrotestosterone) (Leusch et al., 2014). Studies in the closely related Western mosquitofish (G. affinis) confirmed the presence of two androgen receptor subtypes, AR␣ and AR␤, with AR␣ more strongly expressed and present in the fin tissues of both male and female G. affinis (Sone et al., 2005). The secondary structures found in G. affinis ARs reflect those found in other teleost fish, and while TB exposure induces both AR␣ and AR␤ in the anal fin (Sone et al., 2005), other pathways such as wnt/␤catenin and shh/ptc1 are essential for androgen-induced secondary sexual characteristics (Ogino et al., 2011). One potential explanation for levonorgestrel’s potency could be via increased hormone load via tight binding of levonorgestrel to sex steroid globulin binding proteins. In rainbow trout (Oncorhynchus mykiss), measured bioaccumulation loads of levonorgestrel were nearly 1000-fold higher than the predicted values based on the octanol/water partition coefficient of this chemical (Svensson et al., 2013). Since levonorgestrel is known to bind tightly to the sex steroid binding globulins in the gills and liver (Miguel-Queralt and Hammond, 2008), this may serve as a means of significant uptake and subsequent bioaccumulation of this chemical, thus a lower concentration was required to elicit the masculinization response in mosquitofish (Zeilinger et al., 2009). In addition to anal fin elongation during levonorgestrel exposure, expression of the potential androgen biomarker gene shh was

16

E.K. Brockmeier et al. / Aquatic Toxicology 179 (2016) 8–17

also visualized using in situ hybridization after 21 days of exposure (Fig. 4). Shh is a growth factor gene found in many tissues and has a wide range of functions during development (Varjosalo and Taipale, 2007). Specifically in fin tissues, shh is known to induce early bone growth factors that facilitate the outgrowth of fins in both development (Ingham and McMahon, 2001) and regeneration (Poss et al., 2000). This gene is also expressed in the anal fins of members of the Poeciliidae family, including mosquitofish, during androgen exposure (Offen et al., 2008; Ogino et al., 2004; Zauner et al., 2003). While it is not known what its role is in androgen-induced anal fin elongation, it is hypothesized that this gene is co-regulated with AR during androgen exposures and its promoter region may include steroidlike binding elements (Ogino et al., 2004; Varjosalo et al., 2008). In chondrichthyes, it was recently discovered that shh expression in the male clasper structure, a penile-like structure found in early vertebrate species, was regulated by an androgen response element (ARE) in the hand2 gene (O’Shaughnessy et al., 2015), for which there could be a parallel mechanism in the gonopodia of G. holbrooki. We focused on the application of in situ methods for this paper to confirm that the spatial gene expression was in line with previous studies in mosquitofish and other Poeciliidae species. The expression of this gene at the distal tip of the gonopodial structure during levonorgestrel-induced anal fin elongation in this study provides additional support for levonorgestrel acting as an androgen receptor agonist in G. holbrooki. Results of AR and PR inhibitor co-exposures indicate that anal fin elongation is driven by interactions of chemicals with the AR instead of the PR (Fig. 5). Blocking the function of the AR by cotreatment of the anti-androgen flutamide resulted in an inhibition of anal fin elongation. This was true for exposures to an androgen, TB, and the progestin levonorgestrel. Results implicate the AR and not the PR as the crucial element in androgen and progestininduced anal fin elongation. This also correlates with previous studies on the importance of the AR on anal fin elongation as well as the expression of androgen-related genes such as shh (Brockmeier et al., 2013a; Ogino et al., 2004). Previous studies showing that progestins (such as levonorgestrel) have strong affinities for the AR of fish species also support this finding (Zeilinger et al., 2009). These results, in addition to the supporting evidence of shh expression, indicate that anal fin elongation is caused by the activation of the androgen receptor and not the progesterone receptor, even during progestin exposures.

5. Conclusions Using microarray analysis, we determined the hepatic transcriptome impacts of levonorgestrel, mifepristone, and mixture exposures on G. holbrooki and determined that there were significant modulations in gene expression categories such as isoprenoid biosynthesis, lipid transport, and embryo development. Based on gene expression profile comparisons, levonorgestrel may act as both an AR and PR agonist in this fish species. Similar to what was previously shown, we demonstrate that exposure to levonorgestrel induces the formation of male secondary sexual characteristics in adult female G. holbrooki, in addition to the expression of the androgen biomarker gene shh. During a co-exposure of levonorgestrel and the androgen TB with both flutamide and mifepristone, only the anti-androgen was able to prevent anal fin elongation, further demonstrating the importance of AR on inducing anal fin growth during exposure to either type of chemical. These results provide insights into how progestins may impact species in aquatic ecosystems and demonstrate the importance of studying the effects of this class of chemicals in a non-mammalian model organism.

Acknowledgements The authors gratefully acknowledge the assistance of A. Colville and R. Lim at the University of Technology in Sydney for support with the in vivo exposure. This study was funded in part by an Australian Research Council (ARC) Linkage grant (LP100100163) in collaboration with Water Research Australia. Additional support was provided by the U.S. Environmental Protection Agency Science to Achieve Results Fellowship 91728101-0 (EB). P.S. was supported by an Australian Postgraduate Award (Industry) scholarship and a Water Research Australia PhD top-up scholarship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2016.08. 002. References Angus, R.A., McNatt, H.B., Howell, W.M., Peoples, S.D., 2001. Gonopodium development in normal male and 11-ketotestosterone-treated female mosquitofish (Gambusia affinis): a quantitative study using computer image analysis. Gen. Comp. Endocr. 123, 222–234. Angus, R.A., Stanko, J., Jenkins, R.L., Watson, R.D., 2005. Effects of 17 alpha-ethynylestradiol on sexual development of male Western mosquitofish (Gambusia affinis). Comp. Biochem. Phys. C 140, 330–339. Bluthgen, N., Castiglioni, S., Sumpter, J.P., Fent, K., 2013a. Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 1. Reproductive and early developmental effects. Aquat. Toxicol. 144–145, 83–95. Bluthgen, N., Sumpter, J.P., Odermatt, A., Fent, K., 2013b. Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 2. Gene expression analysis and in vitro activity. Aquat. Toxicol. 144–145, 96–104. Brockmeier, E.K., Ogino, Y., Iguchi, T., Barber, D.S., Denslow, N.D., 2013a. Effects of 17beta-trenbolone on Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) anal fin growth and gene expression patterns. Aquat. Toxicol. 128–129, 163–170. Brockmeier, E.K., Yu, F., Moraga, D., Bargar, T.A., Denslow, N.D., 2013b. Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki). BMC Genomics 14, 660. Brockmeier, E.K., Jayasinghe, B.S., Pine, W.E., Wilkinson, K.A., Denslow, N.D., 2014. Exposure to paper mill effluent at a site in north central Florida elicits molecular-level changes in gene expression indicative of progesterone and androgen exposure. PLoS One 9, e106644. Carson, J.D., Jenkins, R.L., Wilson, E.M., Howell, W.M., Moore, R., 2008. Naturally occurring progesterone in loblolly pine (Pinus taeda L.): a major steroid precursor of environmental androgens. Environ. Toxicol. Chem. 27, 1273–1278. Chen, T.S., Chen, T.C., Yeh, K.J., Chao, H.R., Liaw, E.T., Hsieh, C.Y., Chen, K.C., Hsieh, L.T., Yeh, Y.L., 2010. High estrogen concentrations in receiving river discharge from a concentrated livestock feedlot. Sci. Total Environ. 408, 3223–3230. Costigan, S.L., Werner, J., Ouellet, J.D., Hill, L.G., Law, R.D., 2012. Expression profiling and gene ontology analysis in fathead minnow (Pimephales promelas) liver following exposure to pulp and paper mill effluents. Aquat. Toxicol. 122, 44–55. Frankel, T.E., Meyer, M.T., Orlando, E.F., 2016. Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki). Gen. Comp. Endocrinol., http://dx. doi.org/10.1016/j.ygcen.2016.01.007. Game, C., Gagnon, M.M., Webb, D., Lim, R., 2006. Endocrine disruption in male mosquitofish (Gambusia holbrooki) inhabiting wetlands in Western Australia. Ecotoxicology 15, 665–672. Ingham, P.W., McMahon, A.P., 2001. Hedgehog signaling in animal development: paradigms and principles. Gene Dev. 15, 3059–3087. Kemppainen, J.A., Lane, M.V., Sar, M., Wison, E.M., 1992. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J. Biol. Chem. 267 (2), 968–974. Kettel, L.M., Murphy, A.A., Morales, A.J., Yen, S.S., 1994. Clinical efficacy of the antiprogesterone RU486 in the treatment of endometriosis and uterine fibroids. Hum. Reprod., 116–120. King, O.C., van de Merwe, J.P., McDonald, J.A., Leusch, F.D., 2016. Concentrations of levonorgestrel and ethinylestradiol in wastewater effluents: is the progestin also cause for concern? Environ. Toxicol. Chem., http://dx.doi.org/10.1002/etc. 3304. Leusch, F.D.L., Khan, S.J., Laingam, S., Prochazka, E., Froscio, S., Trinh, T., Chapman, H.F., Humpage, A., 2014. Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water. Water Res. 49, 300–315.

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