The test compound used in this study was Enzalutamide (purity 98%, CAS No.: 915087-33-1) purchased from Advanced Chemblocks Inc. (G-7963). Corn oil was purchased from Sigma-Aldrich (Copenhagen, Denmark) and used as control compound and vehicle (product number: C8267-2.5L).
The antagonistic effects of enzalutamide on AR were investigated using the Androgen Receptor Stably Transfected Transcriptional Activation Assay (AR STTA) described in OECD test guideline no. 458 (19). Experiments were run with three technical replicates and repeated thrice, with the mean value of technical replicates representing one biological replicate. AR-EcoScreen™ cells (JCRB1328, Japanese Collection of Research Bioresources) were grown in CellBIND® Surface cell culture flasks (Corning® Inc., Corning, New York, USA) in growth medium consisting of Gibco® DMEM/F-12 Nutrient Mixture with L-glutamine and HEPES and without phenol red supplemented with 5% FBS, 1% Penicillin-Streptomycin, 200 µg/ml Zeocin™ Selection Reagent, and 100 µg/ml Hygromycin B (all reagents from Invitrogen™, Life Technologies™, Carlsbad, California, USA). The cells were incubated at 37 °C with a humidified atmosphere of 5% CO2 and 95% air. Only cells of passage 3–15 were used for experiments. One day prior to the experiment cells were plated in white 96-well plates (Corning® Inc., Corning, New York, USA) at a density of 9,000 cells/well in assay medium consisting of Gibco® DMEM/F-12 Nutrient Mixture medium, 1% Penicillin-Streptomycin, with 5% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS) (all reagents from Invitrogen™, Life Technologies™, Carlsbad, California, USA). The following day the medium was changed to assay medium containing various concentrations of enzalutamide or the control compounds R1881 (known AR agonist, Perkin Elmer) or hydroxyflutamide (known AR antagonist, CAS No. 52806-53-8, Toronto Research Chemicals, Toronto, Ontario, Canada). Enzalutamide was added to the cells in 2-fold serial dilutions ranging from 0.05–12.5 µM. The DMSO vehicle concentrations were constant in all wells. R1881 was added at a concentration of 0.1 nM to all wells when testing for AR antagonism. After cells had been exposed to test compounds for ~ 20 hours firefly luminescence was measured in a luminometer (LUMIstar® Galaxy, BMG LABTECH, Offenburg, Germany) using Dual-Glo®lLuciferase Reagent from the Dual-Glo® Luciferase Assay System from Promega (Madison, Wisconsin, USA). In order to distinguish a decrease in luciferase activity caused by pure antagonism from that caused by cytotoxicity, we measured Renilla luminescence using Dual-Glo® Stop & Glo® Reagent from the Dual-Glo® Luciferase Assay System (Promega, Madison, Wisconsin, USA).
H295R steroidogenesis assay
The ability of enzalutamide to affect steroidogenesis was investigated using the H295R Steroidogenesis Assay described in OECD test guideline no.456 (20). The H295R human adrenocortical carcinoma cell line (CRL-2128™, ATCC®, Manassas, Virginia, USA) was cultured in CellBIND® Surface cell culture flasks (Corning® Inc., Corning, New York, USA) in DMEM/F-12 growth medium with L-glutamine and HEPES and without phenol red (Invitrogen™, Life Technologies™, Carlsbad, California, USA) supplemented with 1% ITS + premix media additive (Corning®Inc., Corning, New York, USA) and 2.5% Nu-SerumTM (Corning® Inc., Corning, New York, USA). The cells were incubated at 37 °C with a humidified atmosphere of 5% CO2 and 95% air. Only cells of passage 4–9 were used for experiments. One day prior to the experiment cells were plated in clear 24-well plates (Corning® Inc., Corning, New York, USA) at a final density of 300,000 cells/well. Cells were incubated with enzalutamide (2-fold serial dilutions ranging from 0.05–12.5 µM) for 48 hrs after which the supernatant was repositioned to new 24-well plates stored at -80 °C until quantification of hormone levels by HPLC-MS/MS. A quality control plate with the control compounds Forskolin (1 and 10 µM) (Sigma-Aldrich, Copenhagen, Denmark) and prochloraz (0.3 and 3 µM) (Ehrenstorfer, VWR & Bie & Berntsen, Denmark) were included in all experiments to monitor assay performance. Three independent experiments were performed each with technical triplicates.
After removal of the supernatant, cytotoxicity was evaluated using fluorescamine (CAS no. 38183-12-9, Sigma-Aldrich®, St. Louis, Missouri, USA). Cells were washed with 500 µl/well pre-warmed (37 °C) PBS. 150 µl/well lysis buffer were added and plates were shaked for 15 min. Hereafter, 400 µl of pre-warmed (37 °C) fluorescamine diluted in acetonitrile to a final concentration of 500 µg/ml was added to each well and placed dark in an incubator with a humidified atmosphere of 5% CO2 and 95% air for 20 min. 300 µl of the supernatant was transferred to white opaque 24-well plates (PerkinElmer, Waltham, Massachusetts, USA) and cell viability was evaluated by measuring fluorescence using an EnSpire® Multilabel Reader (PerkinElmer, Massachusetts, USA) with excitation and emission wavelengths of 390 nm and 460 nm, respectively.
Hormone analysis by HPLC–MS/MS
Steroid hormones were extracted and quantified as previously described (21, 22), with minor modifications. Before LC-MS/MS analysis, deuterated internal standards were added to the cell extracts and samples were centrifuged (15,000 g, 10 min). Steroid hormones were separated, detected, and quantified using on-line-SPE LC-MS/MS. For on-line SPE an Oasis HLB column (2.1 × 20 mm, 15 µm) was used. For 17β-estradiol and estrone analysis, a Kinetex C18 column (2.1 × 100 mm, 2.6 µm) was used with an injection volume of 100 µl, measuring in ESI- mode using methanol, and 1 mM ammonia in water as the mobile phases (gradient flow rate was 0.4 ml/min). For the other hormones an Ascentis Express C8 column (2.1 × 100 mm, 2.7 µm) was used with an injection volume of 100 µl, measuring in ESI-/ESI + mode with acetonitrile and 0.1% formic acid in water as the mobile phases (gradient flow rate was 0.25 ml/min). Ten hormones: testosterone, androstenedione, dehydroepiandrosterone (DHEA), corticosterone, cortisol, pregnenolone, progesterone, 17α-OH-progesterone, estradiol and estrone were quantified. The limit of quantification (LOQ) was 0.1 ng/ml for corticosterone, 1.0 ng/ml for DHEA and pregnenolone, 0.02 ng/ml for testosterone and androstenedione, 0.05 ng/ml for 17α-OH-progesterone, and 0.01 ng/ml for all the other hormones. For quantification, external calibration standards were run before and after the samples at levels of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 20 ng/ml, with 5.0 ng/ml internal standards: (testosterone-d2, methyltestosterone-d3, progesterone-c2, and estradiol-d3). The mass spectrometer was an EVOQ Elite Triple Quadropole Instrument from Bruker (Bremen, Germany) and the UPLC system was an Ultimate 3000 system with a DGP-3600RS dual-gradient pump.
Twelve time-mated nulliparous, young adult Sprague Dawley rats with a body weight of approximately 255 ± 25 g were supplied at gestational day (GD)3 (NTac:SD, SPF, Taconic Europe, Ejby, Denmark). The day of vaginal plug detection was designated GD1. On GD4, dams were randomized and distributed into 2 groups of 6 animals with similar body weight distributions. Animals were housed in pairs until GD17 and thereafter individually. The animals were housed under standard conditions in semi-transparent polysulfone (PSU) type III cages (PSU 80-1291HOOSU Type III, Tecniplast, Buguggiate, Italy) (15 × 27 × 43 cm) with Aspen wood chip bedding (Tapvei, Gentofte, Denmark), Enviro Dri nesting material (Brogaarden, Lynge, Denmark) and Tapvei Arcade 17 (Aspen wood) shelters (Brogaarden, Lynge, Denmark). They were placed in an animal room with controlled environmental conditions: 12 hr light-dark cycles with light starting at 9 pm, temperature 22 ± 1 °C, humidity 55 ± 5%, 10 air changes per hr.
All animals were fed a standard diet with Altromin 1314 (soy- and alfalfa-free, Altromin GmbH, Lage, Germany). Acidified tap water (to prevent microbial growth) in PSU bottles (84-ACBTO702SU Tecniplast) were provided ad libitum. The PSU bottles and cages as well as the aspenwood shelters (instead of plastic) were used to eliminate any risk of migration of bisphenol A that could potentially confound the study results. From GD7-21, dams were weighed daily and dosed by oral gavage by qualified animal technicians with a stainless steel probe 1.2 × 80 mm (Scanbur, Karlslunde, Denmark) with either vehicle control (corn oil) or enzalutamide (10 mg/kg bw/day) at a constant volume of 2 ml/kg bw per day. All animals were decapitated (guillotined) under CO2/O2-anesthesia at GD21.
Caesarean sections GD 21
Dams were decapitated (guillotined) under CO2/O2-anesthesia at GD21 and fetuses were collected by caesarean section. The dams were exposed 1h ± 15 min before decapitation in the same order as Caesarean sections were performed to adjust for the chemical analysis of maternal blood, fetal blood and amniotic fluid. Uteri were taken out and weighed, and the number of live fetuses, resorptions, and implantations were registered. Body weights of the fetuses were recorded prior to decapitation (by a scissor). Maternal trunk blood was collected and transferred to heparin-coated vials. Trunk blood from all fetuses was collected and transferred to heparin-coated vials and pooled for each gender within each litter. Blood samples were kept on ice and centrifuged at 4000 rpm, 4 ºC for 10 min. Plasma was transferred to new tubes and stored at -80 ºC. Amniotic fluid was collected from all fetuses, pooled within each litter, snap frozen in liquid nitrogen and subsequently stored at -80 ºC.
AGD was measured as the distance between the genital papilla and the anus by the same, blinded technician using a stereomicroscope with a micrometer eyepiece. The AGD index (AGDi) was calculated by dividing AGD by the cube root of the body weight. Fetal testes were isolated by dissection under a stereomicroscope and placed in liquid nitrogen (for steroid hormone analysis) or RNAlater (for RT-qPCR analysis) (Qiagen, Hilden, Germany) and stored at -80˚C. The animal experiments were carried out at DTU Food (Mørkhøj, Denmark) with ethical approval from the Danish Animal Experiments Inspectorate (license number 2015-15-0201-00553) and by the in-house Animal Welfare committee. All methods in this study were performed in accordance with relevant guidelines and regulations.
Enzalutamide concentrations in vivo
Acetonitrile, formic acid and 25% ammonium hydroxide, all of LC-MS grade, were obtained from Sigma Aldrich, Schneldorf, Germany. Evolute ABN express columns, 30 mg, were purchased from Biotage, Sweden. Water was purified on a Milli Q system, Millipore Corporation, US. First, 50 µl of plasma or amniotic fluid was transferred to an Eppendorf tube and 150 µl ice cold acetonitrile was added. The samples were centrifuged at 10000 x g, 4 ˚C for 10 minutes (Ole Dich Instrument makers, Denmark) and the supernatants were passed through an Evolute express ABN column. 20 µl eluate was transferred to an HPLC vial and 80 µl 50% acetonitrile was added prior to analysis by Liquid Chromatography – Mass Spectrometry (LC-MS).
LC was performed on a Dionex Ultimate 3000 RS (Thermo Scientific, CA) with a Poroshell SB C-18 (100 × 2.1 mm, 2.7 µm particle size) column held at 30 °C (Agilent technologies, Walbron, Germany). The solvent system consisted of A: 2.5 mM ammonium hydroxide + 0.1% formic acid in water and B: acetonitrile. Solvent programming were: 2% B from 0 to 1 min followed by a linear gradient to 95% B to 14 min, isocratic 95% B from 14 to 16 min followed by reversal to initial conditions to 16.1 min and re-equilibration of the column to 20 min. The flow rate was 0.3 ml/min from 0 to 1 min followed by a linear gradient to 0.4 ml/min to 14 min, which was held to 16 min followed by reversal to initial conditions.
The LC system was connected to a Bruker Daltonics, maXis qTOF mass spectrometer equipped with an electrospray ion source operated in positive ion mode (Bruker Daltonics, Bremen, Germany). The ion source settings were: nebulizer pressure 2 bars, drying gas flow 8 l/min, dry gas temperature 200 °C, capillary voltage 2500 V. The scan range was from 80 to 1000 m/z with an acquisition rate of 2 Hz. Sodium formate dissolved in 50% 2-propanol was introduced in the ion source in a 0.2–0.4 min time segment and used for internal calibration of the data files. Hexakisperflouroetoxyphophazene was used as lock mass calibrant. Matrix matched standard samples were prepared in blank plasma at 6 different levels in the concentration range of: 3 to 10,000 nM. Standards and blanks were analyzed in the beginning of a sequence and after each set of 20 samples. Data files were processed using QuantAnalysis (Bruker Daltonics, Bremen, Germany). Extracted ion chromatograms of m/z 465.1003 ± 0.002 Da were constructed and integrated. Plasma concentrations were calculated based on linear calibrations curves constructed using 1/x weighing.
One testicle was transferred to a beetBeater tube (Fischer Scientific, Waltham, MA, USA) and 1 mL of 80% acetonitrile (with internal standards, 1 ng/ml testosterone–d2 (Rikilt, Wageningen, Netherlands) was added. The sample was homogenised for 1 min and 50 mg of Que Z-Sep (Sigma-Aldrich, St. Louis, Missouri, USA) was added followed by whirlmix for 1 min. The sample was centrifuged at 3000 x g, 4 oC for 10 min and placed at -18 oC for 1 hr. The sample was then centrifuged at 10000 x g 4 oC for 10 min., followed by filtration through a SPE column (Waters Corporation, Milford, MA, USA) (HLB, 30 mg), conditioned with 2 mL 80% acetonitrile. The sample was evaporated to dryness (50 oC, N2) and dissolved in 500 µl 10% acetonitrile. Testosterone was detected and quantified using on-line-SPE LC-MS/MS. For on-line SPE, an Oasis HLB column (2.1 × 20 mm, 15 µm) was used and as analytical column an Ascentis Express C8 column (2.1 × 100 mm, 2.7 µm) was used with an injection volume of 100 µl in ES + mode with acetonitrile and 0,1% formic acid in water as the mobile phases (flow rate 0.25 ml/min, gradient method).
The MS was an EVOQ Elite Triple Quadropole Instrument from Bruker (Bremen, Germany) and the UPLC system was an Ultimate 3000 system with a DGP-3600RS dual-gradient pump. For quantification, external calibration standards were run before and after the samples at levels of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 20 ng/ml (with 5.0 ng/ml internal standards). The limit of quantification (LOQ) for testosterone was estimated as the concentrations corresponding to six times signal-to-noise, and was < 20 pg/testicle.
Synthesis of cDNA and RT-qPCR analysis
Protocols were essentially as previously described (23). Briefly, total RNA was extracted from GD21 testis (n = 12/group) using RNeasy mini kit and on-column DNaseI treatment, according to manufacturer’s instructions (Qiagen, Hilden, Germany). RNA was quantified by Nanodrop spectrophotometry (Nanodrop Technologies, Wilmington, DE) and 500 ng total RNA used to synthesize cDNA using a random primer mix (New England Biolabs, Ipswich, MA, USA) and the Omniscript kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Quantitative RT-PCR (RT-qPCR) reactions were run in technical duplicates on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Thermo Fischer Scientific) in 11 µl reactions containing: 3 µl diluted (1:20) cDNA, TaqMan Fast Universal Mastermix (2X) (Life Technologies, Carlsbad, CA, USA) and TaqMan Gene Expressions Assays (Life Technologies). TaqMan assays were: Hsd3b1 Rn01774741_m1, Hsd17b1 Rn00563388_g1, Nr5a1 Rn00584298_m1, Ddx4 Rn01489814_m1, Sox9 Rn01751069_mH, Rps18 Rn01428913_gH, and Sdha Rn00590475_m1. In addition, primers and probes Cyp17a1, Cyp11a1 and Star were designed in our lab (24). The following cycling conditions were used: an initial step of 95 °C for 20 sec followed by 45 two-step thermal cycles of 95 °C for 1 sec and 60 °C for 20 sec. The relative transcript abundance was calculated using the 2−ΔCT method using Rps18 and Sdha as normalizing genes.
Data from the AR-Eco Screen and H295R assay were analyzed by one-way ANOVA followed by Dunnett’s post hoc test in GraphPad Prism 5 (GraphPad Software, San Diego California, USA). Results are presented as mean ± SEM for the three independent experiments. One measurement of DHEA in the H295R assay was lost so the DHEA data presented are from two independent experiments.
In vivo data on maternal parameters, fetal body weight, AGD and AGDi, were analyzed by one-way ANOVA followed by Dunnett’s post hoc test, using SAS® (SAS Enterprise Guide 6.1, SAS Institute, Inc., Cary, NC, USA). AGD was analyzed using fetal weight as a covariate and fetal body weights were analyzed using the number of offspring per litter as covariate. For all analyses, the litter was the statistical unit. Statistical analyses were adjusted using litter as an independent, random and nested factor. For data presentation, group mean ± SEM was calculated from 6 litters/group based on litter means.
Analysis of enzalutamide concentrations in plasma and amniotic fluid as well as RT-qPCR data and intra-testicular testosterone levels was performed with student’s t-test in GraphPad Prism 8 (GraphPad Software, San Diego California, USA). In cases of non-normal distribution or non-equal variance between groups, data was log-transformed prior to analysis, while the graphs still represent the untransformed data. For data presentation, mean ± SEM was calculated from 6 litters/group (maternal plasma, amniotic fluid and fetal plasma), 1 testicle from 6 fetuses /group (fetal intra-testicular testosterone) and 1 testicle from 12 fetuses/group (RT-qPCR).