ELSEVIER
Biochemical Pharmacology
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Bjochemical P
Pharmacology
Profiling of anabolic androgenic steroids and selective androgen receptor modulators for interference with adrenal steroidogenesis
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Melanie Patta,b, Katharina R. Becka,b,1, Tobias Di Marcob, Marie-Christin Jägera,b, Victor González-Ruiza,c, Julien Boccarda,c, Serge Rudaza,c, Rolf W. Hartmannd,e, Mohamed Salah®, Chris J. van Koppene, Matthias Grillf, Alex Odermatta,b,*
a Swiss Centre for Applied Human Toxicology (SCAHT), University of Basel, Basel, Switzerland
b Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
” Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva 4, Switzerland
d Department Drug Design and Optimization (DDOP), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Universitätscampus E8 1, 66123 Saarbrücken, Germany
e Department of Pharmaceutical and Medicinal Chemistry, Universitätscampus C2.3, 66123 Saarbrücken, Germany ‘Lipomed AG, Fabrikmattenweg 4, 4144 Arlesheim, Switzerland
ARTICLE INFO
Keywords:
Adrenal gland Steroid biosynthesis Hypertension Cardiovascular disease H295R
ABSTRACT
Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treat- ment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain un- known. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spec- trometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone in- creased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell- free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3B-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxy- metholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, sug- gesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic informa- tion. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.
1. Introduction
Anabolic androgenic steroids (AAS) are synthetic derivatives of the male sex hormone testosterone, developed to increase bioavailability
and reduce adverse androgenic properties, while maximizing anabolic effects. Traditional indications for the use of AAS have been advanced breast cancer, osteoporosis and anemia associated with leukemia and kidney failure. Nowadays, AAS are clinically used in hormone
* Corresponding author at: Swiss Centre for Applied Human Toxicology (SCAHT), University of Basel, Basel, Switzerland. E-mail addresses: melanie.patt@unibas.ch (M. Patt), katharina.beck@unibas.ch (K.R. Beck), tobias.dimarco@outlook.com (T. Di Marco),
m.jaeger@unibas.ch (M .- C. Jäger), victor.gonzalez@unige.ch (V. González-Ruiz), julien.boccard@unige.ch (J. Boccard), serge.rudaz@unige.ch (S. Rudaz),
rolf.hartmann@helmholtz-hzi.de (R.W. Hartmann), mohamed.rezk87@hotmail.com (M. Salah), vankoppen@elexopharm.de (C.J. van Koppen),
grill78@web.de (M. Grill), alex.odermatt@unibas.ch (A. Odermatt).
1 Current address: Labormedizinisches Zentrum Dr. Risch, Lagerstrasse 30, 9470 Buchs, Switzerland.
https://doi.org/10.1016/j.bcp.2019.113781
ACTH
Cholesterol
StAR
CYP11A1
Pregnenolone
CYP17A1
17aOH-Pregnenolone
Dehydroepiandrosterone
3฿HSD2
CYP17A1
Progesterone
17aOH-Progesterone
-
Androstenedione
CYP21A2
Deoxycorticosterone
11-Deoxycortisol
CYP11B1
Corticosterone
Cortisol
CYP11B2
Aldosterone
replacement therapies, including hypogonadism and aging, and to treat muscle wasting due to cancer, AIDS, severe burns, chronic renal failure and pulmonary diseases [1-7]. Therapeutic doses of synthetic testos- terone aim to raise serum testosterone concentrations to the mid- normal range between 350 ng/dL (14 nmol/L) and 600 ng/dl (25 nmol/L) [8,9], and treatment with testosterone analogues should achieve equivalent activity corresponding to this range. Although im- provements in the chemical scaffold of testosterone were made, a clear dissociation of anabolic from androgenic effects in the respective tes- tosterone analogues has not yet been achieved [10]. The undesirable androgenic properties of AAS including acne, hirsutism and alopecia are responsible for their limited clinical use [8,9].
The beneficial effects of AAS on muscle mass and bone mineral density led to the development of tissue-selective alternatives with re- duced adverse effects. Selective androgen receptor modulators (SARMs) are supposed to act as full agonists in anabolic tissues such as muscle and bone with ideally no or minimal activation of the androgen re- ceptor (AR) in prostate, heart or liver [11,12]. Most SARMs are non- steroidal compounds, expected to exhibit less interactions with steroid metabolizing enzymes and possessing fewer adverse effects. Currently, SARMs are studied in phase I and II clinical trials to assess their efficacy in the treatment of cachexia, benign prostatic hyperplasia, prostate cancer, breast cancer and stress urinary incontinence in post- menopausal women [13].
Besides therapeutic applications, AAS are used as anabolic agents to enhance muscle mass and burn fat by athletes to enhance performance and by the general population to improve body shape. AAS are the most frequently detected doping agents with about 44% of adverse analytical findings in WADA-accredited laboratories in 2017 [14]. Due to frequent doping tests, AAS misuse cannot be considered a serious health risk among elite athletes; however, it has become a public health concern [15]. The majority of AAS users are individuals striving for a muscular body shape without competitive athletic ambitions. According to the
latest ‘Monitoring the Future’ statistics (2017), an annual survey on drug abuse in adolescents across the United States funded by the Na- tional Institute on Drug Abuse, the lifetime prevalence of AAS use is 1.4% for young adults (ages 19-28) [16]. Nevertheless, these data do not accurately reflect the population encountering serious adverse ef- fects, since most of those arise during long-term AAS use. Prolonged AAS use can be assumed to be more prevalent in fitness and strength training environments. Higher estimates of AAS misuse have been documented among gym-goers, bodybuilders and security personnel compared to the general population [15,17,18]. Whereas the medical use of AAS aims to achieve a physiologic replacement level on a con- tinuous basis, recreational users usually take supra-physiologic doses of AAS, reaching 10 to 100 times the physiological level. Furthermore, AAS are often applied in a sophisticated multidrug regimen involving varying doses, time courses and simultaneously using oral and in- tramuscular preparations [19-22]. High-dose AAS use is associated with a wide range of adverse health effects including liver toxicity, kidney diseases, psychological disorders, endocrine disturbances and dermatologic effects [23-25]. Additionally, AAS affect the cardiovas- cular system. Several studies described cardiovascular consequences occurring after abusive AAS use including hypertension, myocardial hypertrophy, cardiomyopathy, myocardial infarction and sudden car- diac death [26,27].
Mechanisms involving steroidogenesis have been suggested to contribute to the hypertensive effects of AAS. In bovine adrenal cells, testosterone hemisuccinate stimulated the membrane binding of an- giotensin as well as aldosterone biosynthesis [28]. Another mechanism causing hypertension includes the elevation of the mineralocorticoid 11-deoxycorticosterone, which may be caused by a testosterone-de- pendent decrease in cytochrome P450 11B1 (CYP11B1, 11ß-hydro- xylase) mRNA levels [29,30]. In addition, inhibition of cytochrome P450 17A1 (CYP17A1, 17a-hydroxylase-17, 20-lyase) is accompanied with mineralocorticoid excess, since the lack of CYP17A1 activity forces
steroid substrates to pass through the biosynthetic pathway of aldos- terone via corticosterone and 11-deoxycorticosterone [31-33]. The feedback regulation via adrenocorticotropic hormone (ACTH) upon inhibition of cortisol biosynthesis further stimulates adrenal ster- oidogenesis, thereby enhancing mineralocorticoid production.
Although the adverse cardiovascular effects of AAS misuse are well recognized, the underlying molecular mechanisms are still not fully understood. The current study examined the effects of 19 AAS and 3 SARMs on adrenal steroidogenesis, aiming to identify compounds that increase the production of mineralocorticoids and potentially con- tribute to the development of hypertension and cardiovascular diseases [34]. The use of a modified protocol of the OECD test guideline 456, based on human H295R adrenocortical carcinoma cells, and subsequent quantification of major adrenal steroids ([35], see Fig. 1 for an over- view of steroid biosynthesis), provided initial insight into the me- chanism of interference by different AAS. Finally, this study allowed a comparison of the potential steroidogenic-disrupting effects of AAS and SARMs as a first insight of chemical grouping in the context of system toxicology.
2. Materials and methods
2.1. Chemicals and reagents
Danazol (CAS 17230-88-5), fluoxymesterone (CAS 76-43-7), mes- tanolone (CAS 521-11-9), methandienone (CAS 72-63-9), nandrolone (CAS 434-22-0), oxandrolone (CAS 53-39-4), oxymesterone (CAS 145-12-0), oxymetholone (CAS 434-07-1), stanozolol (CAS 10418-03- 8), trenbolone (CAS 10161-33-8), and ostarin (CAS 841205-47-8) were obtained from Lipomed (Arlesheim, Switzerland) at the highest purity available. Boldenone (CAS 846-48-0), clostebol (CAS 1093-58-9), drostanolone (CAS 58-19-5), methasterone (CAS 3381-88-2), LDG- 2226 (CAS 328947-93-9), LDG-4033 (CAS 1165910-22-4), and oxy- stanolone (2-hydroxymethylene-androstan-17ß-ol-3-one or 4,5a-di- hydro-2-(hydroxymethylene)testosterone) were synthesized as de- scribed elsewhere [36]. Mesterolone (CAS 1424-00-6) and turinabol (clostebol acetate, CAS 855-19-6) were kindly provided by Dr. Daniela Schuster (Paracelsus Medical University, Salzburg, Austria). Metheno- lone (CAS 153-00-4) and norbolethone (CAS 1235-15-0) were pur- chased from Cerilliant Corporation (Round Rock, TX, USA), and the reference compounds forskolin (CAS 66575-29-9) and prochloraz (CAS 67747-09-5) from Sigma-Aldrich (Buchs, Switzerland) at the highest purity available. Stock solutions (10 mM or 5 mM) were prepared in dimethyl sulfoxide (DMSO, AppliChem, Darmstadt, Germany). UPLC- grade purity methanol, acetonitrile and formic acid were purchased from Biosolve (Dieuze, France) and ethyl acetate from Fisher Scientific (Reinach, Switzerland). Aldosterone, corticosterone, 11-deox- ycorticosterone, androstenedione, testosterone and [2,2,4,6,6,21,21-2H7]-aldosterone (98% isotopic purity) were obtained from Sigma-Aldrich. 11-Dehydrocorticosterone, dehydroepian- drosterone, progesterone, 17a-hydroxyprogesterone, 11-deoxycortisol, cortisol and cortisone were purchased from Steraloids (Newport, RI, USA). [2,2,4,6,6,16,16-2H7]-44-androstene-3,17-dione (98% isotopic purity) and [2,2,4,6,6,17a,21,21-2H8]-corticosterone (98% isotopic purity) were purchased from C/D/N Isotopes Inc. (Pointe-Claire, Ca- nada). Stock solutions (10 mM and/or 1 mg/mL) of above-mentioned steroids were prepared in methanol.
2.2. Culture and treatment of H295R cells
H295R human adrenocortical carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/ Ham’s nutrient mixture F-12 (1:1, v/v) (Life Technologies, Zug, Switzerland), containing 1% (v/v) ITS + Premix (BD Bioscience, Bedford, MA, USA), 2.5% (v/v) Nu-serum (Lot: 2342913, BD
Bioscience, Bedford, MA, USA), 15 mM HEPES buffer, pH 7.4, and 1% (v/v) penicillin-streptomycin (Sigma-Aldrich). The H295R steroid biosynthesis assay was carried out according to the OECD test guideline 456 [37], with several modifications [38]. H295R cells were used be- tween passages 5-10. They were grown in 24-well plates (200,000 cells/mL) and the medium was changed 24 h later to new medium supplemented with the reference or test compound (1 uM). The con- centration of AAS used was chosen because such a level may be reached in AAS abusers. Measurements included a vehicle control (DMSO, 0.01% (v/v)), a steroid biosynthesis inducer as positive control (for- skolin, 10 µM) and a negative control to inhibit adrenal steroidogenesis (prochloraz, 1 µM). Additionally, the compounds (1 µM) were tested in activated cells in the presence of 10 uM forskolin. Forskolin served as vehicle control for general stimulation of steroidogenesis. Complete medium without cells served as control at time zero. Following 48 h of exposure, culture media was collected and stored at -20 ℃ until steroid hormones were quantified. Experiments were conducted three times independently, each in duplicates.
2.3. Cell viability assay
Possible effects of tested compounds on cell viability were evaluated using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car- boxanilide (XTT, Sigma-Aldrich) and phenazine methosulfate (PMS, Sigma-Aldrich) as an electron-coupling reagent. Briefly, H295R cells were seeded in 96-well plates at a density of 30,000 cells/100 uL complete medium. After 24 h, the medium was replaced by fresh phenol red-free medium containing test compound at a concentration of 0.1 µM, 1 µM or 10 uM, with or without 10 uM forskolin. Digitoxin (10 µM) was used as a cytotoxicity control. After 48 h of incubation, cells were subjected to microscopic inspection for morphological changes, and 25 µL of XTT/PMS solution (1 mg/mL and 7.5 µg/mL) was added to each well, followed by incubation for another 2 h. Absorbance was then recorded at 450 and 650 nm (reference wavelength). Control wells containing phenol red-free medium were used as a value for background absorbance. Experiments were performed three times in- dependently with technical triplicates.
2.4. Targeted steroid quantification
The concentrations of nine steroid hormones in H295R culture su- pernatants were quantified using ultra-high performance liquid chro- matography tandem mass spectrometry (UHPLC-MS/MS) as described previously, with minor adaptations [35,38]. Briefly, 1 mL of cell su- pernatant was spiked with deuterium-labeled aldosterone, corticos- terone and androstenedione as internal standards (final concentrations: 0.11 ng/mL, 0.22 ng/ml and 0.18 ng/ml, respectively). The samples were then extracted using Oasis HLB 1 cc SPE cartridges (30 mg, 30 um particle size, Waters, Massachusetts, USA), preconditioned with 1 mL ethyl acetate and 1 mL Milli-Q water. After washing the columns three times with water and a mixture of methanol/water (10/90, v/v), ster- oids were eluted twice with 0.5 mL of ethyl acetate. The eluates were evaporated to dryness, and reconstituted with 50 uL of methanol. Steroids were separated on a reverse-phase column (Waters Acquity UPLC BEH C18, 1.7 um, 2.1 mm × 150 mm) using an Agilent 1290 UHPLC system. Mobile phases A and B consisted of water-acetonitrile- formic acid (95/5/0.1; v/v/v) and (5/95/0.1; v/v/v), respectively. Detection was performed using an Agilent 6490 triple quadrupole mass spectrometer equipped with a jet-stream electrospray ionization source. Mass Hunter software version B.07.01 (Agilent Technologies) was used to analyze the acquired data.
2.5. Gene expression analysis
Following the indicated treatment, RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) on a QIAcube extraction
Table 1
Effects of AAS and SARMs on steroid profile in H295R cells in the basal state. H295R cells were incubated for 48 h with vehicle (solvent control (SC); 0.01% DMSO) or the respective anabolic androgenic steroid (AAS) or selective androgen modulator (SARM) at a final concentration of 1 uM. Forskolin (10 uM) and prochloraz (1 µM) served as reference compound (RC). A complete medium control (MC; t = 0 h) taken at the start of the experiment was included for comparison. Steroid hormone levels were quantified by UHPLC-MS/MS. Data are expressed as fold changes relative to SC and are depicted as mean + SD from three independent experiments, each performed in duplicate. Steroids significantly downregulated compared to SC are represented in green (printed version: dark grey) and those significantly upregulated in red (printed version: light grey). Differences with p < 0.05 were considered significant. Absolute concentrations (ng/ml) are shown for the DMSO control.
| progestins | mineralocorticoids | glucocorticoids | adrenal androgens | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| steroid treatment | progesterone | 17a-hydroxy- progesterone | 11-deoxy- corticosterone | corticosterone | aldosterone | 11-deoxycortisol | cortisol | dehydro- epiandrosterone | androstenedione | ||||||||
| SC | 0.01% DMSO | 1.00 ± 0.02 | 1.00 ± 0.04 | 1.00 ± 0.03 | 1.00 | ± | 0.05 | 1.00 | ± 0.06 | 1.00 | ± 0.04 | 1.00 | ± 0.06 | 1.00 ± 0.04 | 1.00 | ± 0.05 | |
| MC | complete medium; t=0 | 1.00 ± 0.13 | n.d. | 0.01 ± 0.00 | n.d. | n.d. | 0.02 | ± 0.01 | 0.30 | ± 0.13 | n.d. | 0.03 | ± 0.00 | ||||
| RC | forskolin 10 uM | 1.20 ± 0.20 | 3.90 ± 0.32 | 1.30 ± 0.20 | 4.50 | ± | 0.33 | 2.50 | ± 0.28 | 2.60 | ± 0.36 | 3.90 ± 0.96 | 4.00 | ± 0.33 | |||
| prochloraz 1 uM | 16.0 ± 1.80 | 1.70 ± 0.23 | 1.70 | ± 0.24 | 0.35 | ± | 0.06 | 0.35 | ± 0.06 | ± 0.01 | 0.32 | ± 0.16 | n.d. | 0.08 | ± 0.01 | ||
| AAS | clostebol | 0.87 ± 0.05 | 1.40 ± 0.09 | 0.67 ± 0.06 | n.d. | 0.24 | ± 0.10 | 0.94 | ± 0.07 | 0.48 | ± 0.13 | 1.90 ± 0.23 | ± | ||||
| turinabol | 0.62 ± 0.06 | 1.30 ± 0.11 | 0.47 ± 0.07 | n.d. | 0.22 | ± 0.13 | 0.75 | ± 0.04 | 0.35 ± 0.11 | 2.80 ± 0.45 | 0.92 ± 0.06 | ||||||
| oxymetholone | 0.73 ± 0.12 | 1.10 ± 0.08 | 0.57 | ± 0.11 | 0.43 | ± | 0.06 | 0.46 | ± 0.11 | 0.81 | ± 0.11 | 0.92 ± 0.22 | 2.10 ± 0.54 | 0.95 ± 0.05 | |||
| mesterolone | 2.30 ± 0.08 | 0.57 ± 0.03 | 2.30 ± 0.04 | 2.50 | ± | 0.17 | 2.70 | ± 0.32 | 0.42 | ± 0.04 | 0.70 ± 0.29 | n.d. | 0.30 ± 0.02 | ||||
| mestanolone | 2.00 ± 0.09 | 0.77 ± 0.06 | 1.90 ± 0.11 | 1.90 | ± | 0.33 | 1.70 | ± 0.19 | 0.65 | ± 0.05 | 0.81 | ± 0.13 | n.d. | 0.50 | ± 0.03 | ||
| methenolone | 1.30 ± 0.17 | 0.98 ± 0.06 | 1.40 ± 0.16 | 1.80 | ± | 0.18 | 1.80 | ± 0.21 | 0.84 | ± 0.06 | 0.95 | ± 0.29 | n.d. | 0.74 | ± 0.06 | ||
| stanozolol | 3.60 ± 0.35 | 2.80 ± 0.44 | 1.40 ± 0.10 | 1.60 | ± | 0.10 | 1.80 | ± 0.41 | 0.62 | ± 0.06 | 0.86 | ± 0.18 | 0.65 ± 0.39 | 0.48 ± 0.03 | |||
| drostanolone | 2.20 ± 0.22 | 1.40 ± 0.16 | 1.70 ± 0.16 | 1.60 | ± | 0.21 | 1.90 | ± 0.31 | 0.85 | ± 0.10 | 1.10 | ± 0.27 | 0.82 ± 0.48 | 0.92 ± 0.10 | |||
| danazol | 0.68 ± 0.06 | 0.41 ± 0.04 | 0.90 ± 0.03 | 0.89 | ± | 0.12 | 0.87 | ± 0.16 | ± | 0.54 | ± 0.14 | 0.57 ± 0.18 | 0.20 ± 0.03 | ||||
| methandienone | 1.80 ± 0.35 | 1.80 ± 0.31 | 1.20 ± 0.13 | 0.61 | ± | 0.08 | 0.83 | ± 0.11 | 0.96 | ± 0.10 | 0.78 | ± 0.21 | 0.79 ± 0.34 | 1.20 ± 0.10 | |||
| boldenone | 3.10 ± 0.30 | 2.80 ± 0.28 | 1.50 | ± 0.09 | 0.95 | ± | 0.09 | 1.00 | ± 0.15 | 1.00 | ± 0.08 | 0.58 | ± 0.12 | 0.78 ± 0.12 | 1.10 ± 0.05 | ||
| methasterone | 1.20 ± 0.06 | 1.10 ± 0.09 | 1.30 ± 0.06 | 1.00 | ± | 0.10 | 0.99 | ± 0.17 | 0.91 | ± 0.05 | 0.88 | ± 0.19 | 0.93 ± 0.15 | 0.85 ± 0.05 | |||
| oxystanolone | 1.30 ± 0.04 | 1.00 ± 0.07 | 1.30 ± 0.05 | 1.20 | ± | 0.10 | 1.10 | ± 0.13 | 0.91 | ± 0.05 | 0.82 ± 0.17 | 0.82 ± 0.23 | 0.83 ± 0.05 | ||||
| fluoxymesterone | 1.00 ± 0.03 | 0.98 ± 0.04 | 0.98 ± 0.03 | 0.69 | ± | 0.15 | 1.20 | ± 0.11 | 1.00 | ± 0.13 | 1.20 | ± 0.20 | 0.91 ± 0.12 | 0.95 ± 0.05 | |||
| nandrolone | 1.20 ± 0.08 | 1.30 ± 0.07 | 1.20 ± 0.06 | 0.89 | ± | 0.07 | 0.75 | ± 0.13 | 1.00 | ± 0.08 | 0.88 | ± 0.17 | 0.85 ± 0.22 | 1.20 ± 0.05 | |||
| norbolethone | 1.00 ± 0.10 | 1.10 ± 0.11 | 1.00 ± 0.10 | 1.10 | ± | 0.08 | 1.20 | ± 0.17 | 1.00 | ± 0.09 | 1.20 ± 0.09 | 1.00 ± 0.11 | 1.00 ± 0.07 | ||||
| oxandrolone | 0.94 ± 0.03 | 1.10 ± 0.02 | 0.85 ± 0.05 | 0.75 | ± | 0.10 | 0.70 | ± 0.15 | 0.98 | ± 0.08 | 1.10 ± 0.20 | 1.40 ± 0.08 | 1.20 ± 0.03 | ||||
| oxymesterone | 1.10 ± 0.11 | 1.10 ± 0.07 | 0.97 ± 0.05 | 0.75 | ± | 0.07 | 0.77 | ± 0.14 | 0.98 | ± 0.14 | ± | 1.00 ± 0.17 | 1.00 ± 0.07 | ||||
| trenbolone | 1.00 ± 0.08 | 1.20 ± 0.09 | 0.97 ± 0.04 | 0.86 | ± | 0.05 | 0.76 | ± 0.14 | 1.00 | ± 0.09 | 1.10 | ± 0.25 | 1.20 ± 0.28 | 1.20 ± 0.06 | |||
| SARMs | LGD-2226 | 1.10 ± 0.03 | 0.96 ± 0.05 | 1.00 ± 0.03 | 1.20 | ± | 0.06 | 1.30 | ± 0.19 | 0.89 | ± 0.05 | 1.00 | ± 0.20 | 0.84 ± 0.11 | 0.94 ± 0.03 | ||
| LGD-4033 | 0.98 ± 0.08 | 1.00 ± 0.14 | 0.96 ± 0.08 | 1.20 | ± | 0.07 | 1.40 | ± 0.19 | 0.97 | ± 0.07 | ± | 0.98 ± 0.15 | 1.00 ± 0.14 | ||||
| ostarin | 0.90 ± 0.06 | 0.94 ± 0.07 | 0.95 ± 0.06 | 1.10 | ± | 0.07 | 1.30 | ± 0.18 | 0.93 | ± 0.09 | 1.10 | ± 0.19 | 0.86 ± 0.31 | 0.97 ± 0.08 | |||
| absolute value (DMSO control, ng/ml) | 0.49 ± 0.06 | 1.24 ± 0.23 | 21.7 ± 4.04 | 2.09 ± | 0.35 | 0.07 ± 0.02 | 62.3 ± 14.3 | 5.36 ± 1.93 | 1.15 ± 0.36 | 7.53 ± 1.43 | |||||||
robot (QIAGEN, Hilden, Germany) according to the manufacturer. Complementary DNA (cDNA) was synthesized and quantitative poly- merase chain reaction (qPCR) was performed as described previously [35].
2.6. Determination of CYP17A 17a-hydroxylase activity in cell lysates
Lysates of recombinant E. coli pJL17/OR co-expressing human CYP17A1 and rat NADPH-P450-reductase were prepared in phosphate buffer (50 mM sodium phosphate, pH 7.4, 1 mM MgCl2, 0.1 mM EDTA, and 0.1 mM dithiothreitol) with 20% glycerol, and enzyme activity measurements were performed as described in detail earlier [39,40]. Briefly, CYP17A1 17a-hydroxylase activity was determined by mea- suring the conversion of progesterone to the main product 17a-hy- droxyprogesterone and the byproduct 16a-hydroxyprogesterone. The corresponding AAS (5 uL, final concentration: 5 or 10 uM) or DMSO (vehicle control), and 50 µL NADPH regenerating system (10 mM NADP+, 100 mM glucose-6-phosphate, 2.5 U glucose-6-phosphate de- hydrogenase) were pre-incubated at 37 ℃ for 5 min in 145 µL of phosphate buffer containing 6.25 nmol progesterone (final substrate concentration 25 uM). Protein suspension (50 µL, 0.8-1 mg protein per mL) was added, followed by incubation for 30 min at 37 ℃. The re- action was terminated with 50 uL 1 M HCI, followed by steroid ex- traction using ice-cold ethyl acetate. The analysis of steroids was
performed by UV spectroscopy and data were normalized to the vehicle control and presented as mean ± SD from two independent experi- ments.
2.7. Molecular modeling
Based on the crystal structures of human CYP17A1 co-crystallized with abiraterone (PDB code 3RUK [41]) and human CYP21A2 in complex with 17a-hydroxyprogesterone (PDB code 5VBU [42]), docking calculations were performed using GOLD 5.2 (The Cambridge Crystallographic Data Centre, Cambridge, UK, [43]). In a first step, the co-crystallized ligands of each crystal structure were deleted and re- docked into the corresponding enzyme’s substrate binding pocket in order to determine whether the original binding orientation could be restored and thus the adapted docking settings could be validated. The root mean square deviation (RMSD) values obtained were 0.522 for CYP17A1 and 0.384 for CYP21A2 using the following settings: the li- gand binding pockets were defined as spheres with a 10 Å radius around the coordinates X = 28.53, Y = - 8.76, Z = 36.51 for CYP17A1 and X = - 35.87, Y = - 2.26, Z = 28.00 for CYP21A2. ChemPLP was used as scoring function for all docking calculations. The binding poses predicted by GOLD were then further analyzed using LigandScout 4.1 (Inte:Ligand GmbH, Vienna, Austria) [44].
2.8. Statistics
Statistical evaluation was performed in GraphPad Prism version 7.04. Shapiro-Wilk normality test was performed to verify the nor- mality of data (N = k x n) obtained from three independent steroid profiling experiments (k = 3) each performed in duplicate (n = 2). One-way analysis of variance (ANOVA) and Dunnett’s multiple-com- parison test were used to compare chemical treatments to the solvent control, followed by a Bonferroni correction to adjust the false dis- covery rate. Differences were considered significant at a p-value < 0.05.
3. Results
3.1. Effects of AAS and SARMs on cell viability
Cell viability was assessed after 48 h of incubation with the re- spective compound using cells in the basal state and upon forskolin stimulation by visual inspection under a microscope and by performing an XTT assay, reflecting mitochondrial activity. No alteration of the normal cell morphology nor any reduction of mitochondrial activity (values > 80% of vehicle control were considered non-significant) were observed at the tested concentrations up to 10 uM (data not shown).
3.2. Effects of AAS and SARMs on steroid profiles in H295R cells in the basal and stimulated state
H295R cells were incubated with the respective compound in the basal state, allowing easy detection of compounds inducing the pro- duction of steroids, and upon forskolin stimulation of steroidogenesis, to facilitate identification of inhibitory effects. The amounts of nine adrenal steroids were quantified by UHPLC-MS/MS to investigate ef- fects of the compounds in cells at the basal state (treated with vehicle: 0.01% DMSO, Table 1) and in stimulated cells (treated with 10 uM forskolin, Table 2). Since the Nu-serum already contained a significant amount of progesterone and cortisol, steroid measurements of the complete medium at the beginning of the experiment (t = 0 h) were assessed in order to distinguish between steroids produced by the cells and those contributed by the Nu-serum [35]. Following the OECD test guideline 456, forskolin (10 µM) and prochloraz (1 µM) were included in non-stimulated cells as reference compounds to verify the respon- siveness. As expected, forskolin, a known inducer of adenylyl cyclase that increases cAMP levels [45], significantly increased the production of the progestin 17a-hydroxyprogesterone, the mineralocorticoids 11- deoxycorticosterone, corticosterone and aldosterone as well as the glucocorticoids 11-deoxycortisol and cortisol and the adrenal andro- gens dehydroepiandrosterone and androstenedione, in accordance with an overall stimulation of adrenal steroidogenesis. Prochloraz, known to inhibit CYP17A1 [46], led to a pronounced accumulation of the adrenal precursor steroid progesterone and a more moderate increase in the intermediate 11-deoxycorticosterone, which precedes CYP17A1, whilst the levels of the adrenal androgens and glucocorticoids were sig- nificantly decreased. Levels of 17a-hydroxyprogesterone were slightly elevated, which may be due to the accumulation of progesterone and incomplete CYP17A1 inhibition at a concentration of 1 uM prochloraz.
Next, the effects of various AAS and SARMs were analyzed for their potential to interfere with adrenal steroidogenesis. The three AAS mesterolone, mestanolone and methenolone significantly enhanced the levels of mineralocorticoids but decreased adrenal androgens (Table 1 and 2). The steroid patterns obtained in unstimulated and forskolin- activated cells exhibited an important overlap. Furthermore, the pat- terns resembled the steroid profile observed for abiraterone [47], in- dicating decreased CYP17A1 activity as an underlying mechanism. Whereas treatment with mesterolone led to high levels of progesterone and decreased 17a-hydroxyprogesterone and 11-deoxycortisol,
suggesting potent inhibition of CYP17A1 activity, mestanolone resulted in high progesterone levels but had slightly weaker effects on the pro- duction of 17a-hydroxyprogesterone and 11-deoxycortisol. Metheno- lone exhibited the weakest CYP17A1 inhibition with only a trend to increase progesterone production and no change in 17a-hydro- xyprogesterone. The effect of mesterolone, mestanolone and metheno- lone on 17a-hydroxylase enzymatic activity was estimated as the ratio of the product 17a-hydroxyprogesterone and the substrate proges- terone. Similarly, CYP17A1 17/20-lyase activity (that is enhanced in H295R cells, which exhibit some properties of fetal adrenal cells com- pared to normal adult adrenal steroidogenesis [48,49]) and total ac- tivity were assessed under basal conditions as the ratio of androstene- dione/17a-hydroxyprogesterone and androstenedione/progesterone, respectively (Fig. 2A). Total CYP17A1 activity was decreased by a compound-dependent manner, with mesterolone showing the most potent inhibition and methenolone the weakest. Interestingly, these three AAS seemed to lower 17a-hydroxylase activity more efficiently than 17/20-lyase activity. A very similar pattern was found under for- skolin-stimulated conditions (data not shown). Furthermore, the steroid output in mineralocorticoid, glucocorticoid and adrenal androgen pathways was estimated by summing up the relative changes in 11- deoxycorticosterone, corticosterone and aldosterone (miner- alocorticoids), 11-deoxycortisol and cortisol (glucocorticoids), and de- hydroepiandrosterone and androstenedione (adrenal androgens), re- spectively (Fig. 2B). The treatment of unstimulated cells with mesterolone, mestanolone and methenolone resulted in markedly in- creased mineralocorticoids, while adrenal androgens were decreased and glucocorticoids unaffected or moderately decreased. Similar effects on mineralocorticoid and androgen pathways were obtained in cells stimulated with forskolin (data not presented).
A significantly increased biosynthesis of mineralocorticoids was si- milarly observed for stanozolol and drostanolone both in unstimulated and stimulated cells (Table 1 and 2, and Fig. 2B). In addition, both AAS significantly enhanced the production of progestins. However, whereas stanozolol significantly decreased the levels of androstenedione, dros- tanolone did not affect adrenal androgen biosynthesis. Stanozolol and drostanolone could be distinguished by the ratios of 17a-hydro- xyprogesterone to progesterone reflecting CYP17A1 17a-hydroxylase activity, and androstenedione to 17a-hydroxyprogesterone denoting 17/20-lyase activity. The data suggest that stanozolol lowers CYP17A1 activity mainly through inhibition of 17/20-lyase activity, whereas drostanolone equally affected 17/20-lyase and 17a-hydroxylase activ- ities, in the basal as well as forskolin-stimulated state (Fig. 2A, data under forskolin-stimulated conditions not shown).
A different steroid profile was observed upon treatment with clos- tebol, turinabol (clostebol acetate) and oxymetholone (Table 1 and 2, Fig. 2C). In cells in the basal state, these AAS led to significantly re- duced levels of mineralocorticoids, enhanced dehydroepiandrosterone and a trend increase in 17a-hydroxyprogesterone. The observed steroid profiles resembled those obtained from cells in the forskolin-stimulated state, indicating decreased mineralo- and glucocorticoids along with increased adrenal androgens, suggesting inhibition of CYP21A2 activity as mode-of-action. Since cortisol levels decreased or tended to decrease, an inhibition of CYP11B1/2 activity cannot be fully excluded.
All other AAS tested exerted very moderate effects (danazol, me- thandienone, boldenone, methasterone, oxystanolone and fluox- ymesterone) or did not affect the steroid profiles (nandrolone, norbo- lethone, oxandrolone, oxymesterone and trenbolone) in unstimulated and stimulated cells. In particular, none of the tested SARMs (LGD- 2226, LGD-4033 and ostarin) showed any effect on adrenal ster- oidogenesis.
3.3. Evaluation of the effects of selected AAS on the mRNA expression of steroidogenic enzymes
To test whether altered expression of steroidogenic enzymes
Table 2 Effects of AAS and SARMs on steroid profiles in forskolin-stimulated H295R cells. H295R cells were incubated for 48 h with 10 uM forskolin (reference stimulating control, FC) and the corresponding anabolic androgenic steroid (AAS) or selective androgen receptor modulator (SARM) at a concentration of 1 uM. A complete medium control (MC; t = 0 h) was taken at the start of the experiment. Quantitative analysis of steroid levels was performed by UHPLC-MS/MS. Data obtained from three independent experiments, each performed in duplicate, were normalized to the respective steroid concentration in forskolin treated cells. Fold changes are shown as means ± SD and depicted in a color code: green (printed version: dark grey) represents significant downregulation and red (printed version: light grey) significant upregulation compared to FC. Differences with p < 0.05 were considered significant. Absolute concentrations (ng/ml) are shown for the forskolin control.
| progestins | mineralocorticoids | glucocorticoids | adrenal androgens | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| steroid treatment | progesterone | 17a-hydroxy- progesterone | 11-deoxy- corticosterone | corticosterone | aldosterone | 11-deoxycortisol | cortisol | dehydro- epiandrosterone | androstenedione | ||||||||||||||
| FC | forskolin 10 uM | 1.00 | ± 0.03 | 1.00 ± 0.03 | 1.00 ± 0.03 | 1.00 | ± | 0.05 | 1.00 | ± 0.06 | 1.00 | ± | 0.05 | 1.00 | ± | 0.11 | 1.00 ± 0.05 | 1.00 ± 0.02 | |||||
| MC | complete medium; t=0 | 0.84 | ± 0.07 | n.d. | 0.01 | ± | 0.00 | n.d. | n.d. | 0.01 | ± | 0.01 | 0.07 | ± | 0.04 | n.d. | 0.01 ± 0.00 | ||||||
| AAS | clostebol | 0.74 | ± 0.06 | 1.10 | ± 0.04 | 0.60 ± 0.07 | 0.28 | ± | 0.03 | 0.21 21 | ± 0.03 | 0.98 | ± | 0.12 | 0.58 | ± | 0.21 | 2.60 ± 0.26 | 1.10 ± 0.10 | ||||
| turinabol | 0.59 | ± 0.09 | 0.96 | ± | 0.06 | 0.43 | ± 0.10 | 0.19 | ± | 0.03 | 0.19 | ± 0.04 | 0.72 | ± | 0.12 | 0.38 | ± | 0.12 | 3.10 ± 0.22 | 0.73 ± 0.12 | |||
| oxymetholone | 0.70 | ± 0.08 | 0.90 | ± | 0.03 | 0.52 | ± 0.12 | 0.50 | ± | 0.08 | 0.54 | ± 0.06 | 0.89 | ± | 0.05 | 1.00 | ± | 0.36 | 2.60 ± 0.74 | 0.81 ± 0.07 | |||
| mesterolone | 2.10 | ± 0.22 | 0.83 | ± | 0.09 | 2.20 | ± 0.25 | 1.80 | ± | 0.19 | 2.10 | ± 0.36 | 0.74 | ± | 0.02 | 0.73 | ± | 0.03 | 0.55 ± 0.11 | 0.52 ± 0.06 | |||
| mestanolone | 1.70 | ± 0.22 | 1.00 | ± | 0.08 | 1.70 | ± 0.19 | 1.40 | ± | 0.09 | 1.40 | ± 0.13 | 0.90 | ± | 0.05 | 0.73 | ± | 0.16 | 0.86 ± 0.13 | 0.68 ± 0.08 | |||
| methenolone | 1.20 | ± 0.12 | 0.97 | ± | 0.09 | 1.40 | ± 0.24 | 1.40 | ± | 0.24 | ± | 0.95 | ± | 0.17 | 0.94 | ± | 0.04 | 0.85 ± 0.12 | 0.74 ± 0.10 | ||||
| stanozolol | 3.30 30 | ± 0.31 | 3.00 | ± 0.29 | 1.40 | ± 0.12 | 1.30 | ± | 0.12 | 1.40 | ± 0.15 | 0.99 | ± | 0.04 | 1.00 | ± | 0.39 | 1.00 ± 0.12 | 0.61 ± 0.06 | ||||
| drostanolone | 1.40 | ± 0.14 | 1.10 | ± 0.07 | 1.30 ± 0.13 | 1.20 | ± | 0.12 | 1.60 | ± 0.13 | 1.10 | ± | 0.07 | 1.10 | ± | 0.46 | 0.94 ± 0.08 | 0.90 ± 0.09 | |||||
| danazol | 1.30 | ± 0.13 | 0.60 | ± | 0.03 | 1.40 | ± 0.21 | 0.90 | ± | 0.09 | 1.10 | ± 0.13 | 0.45 | ± | 0.06 | 0.31 | ± | 0.08 | 0.66 ± 0.07 | 0.20 ± 0.02 | |||
| methandienone | 1.20 | ± 0.15 | 1.30 | ± | 0.11 | 1.00 | ± 0.12 | 0.68 | ± | 0.06 | 0.68 | ± 0.09 | 1.10 | ± | 0.08 | 0.69 | ± | 0.11 | 1.30 ± 0.18 | 1.10 ± 0.10 | |||
| boldenone | 1.60 | ± 0.14 | 1.60 | ± | 0.07 | 1.30 | ± 0.12 | 1.00 | ± | 0.07 | 0.92 | ± 0.03 | 1.10 | ± | 0.10 | 0.84 | ± | 0.31 | 0.81 ± 0.12 | 0.97 ± 0.07 | |||
| methasterone | 1.00 | ± 0.03 | 0.93 | ± | 0.07 | 1.20 | ± 0.06 | 0.93 | ± | 0.10 | 1.20 | ± 0.13 | 1.20 | ± | 0.10 | 0.84 | ± | 0.22 | 0.96 ± 0.12 | 0.86 ± 0.11 | |||
| oxystanolone | 1.10 | ± 0.04 | 0.98 | ± | 0.09 | 1.10 | ± 0.04 | 1.20 | ± | 0.15 | 1.20 | ± 0.20 | 1.10 | ± | 0.08 | 1.10 | ± | 0.44 | 1.00 ± 0.25 | 0.92 ± 0.15 | |||
| fluoxymesterone | 0.98 | ± 0.10 | 1.00 | ± | 0.07 | 1.00 | ± 0.06 | 0.59 | ± | 0.08 | 1.10 | ± 0.10 | 1.00 | ± | 0.11 | 0.94 | ± | 0.15 | 1.00 ± 0.17 | 1.10 ± 0.14 | |||
| nandrolone | 1.10 | ± 0.12 | 1.10 | ± | 0.12 | 1.10 | ± 0.13 | 1.10 | ± | 0.07 | 0.71 | ± 0.08 | 1.10 | ± | 0.14 | 1.10 | ± | 0.29 | 1.10 ± 0.21 | 1.10 ± 0.12 | |||
| norbolethone | 0.92 | ± 0.09 | 0.99 | ± | 0.08 | 0.98 | ± 0.09 | 1.10 | ± | 0.04 | 1.10 | ± 0.22 | 1.10 | ± | 0.17 | 0.96 | ± | 0.23 | 1.00 ± 0.16 | 1.00 ± 0.11 | |||
| oxandrolone | 0.89 | ± 0.03 | 0.96 | ± | 0.08 | 0.79 | ± 0.04 | ± | 0.86 | ± 0.10 | 1.00 | ± | 0.05 | 1.10 | ± | 0.33 | 1.40 ± 0.35 | 0.99 ± 0.11 | |||||
| oxymesterone | 0.92 | ± 0.05 | 1.00 | ± | 0.07 | 0.91 | ± 0.08 | 0.86 | ± | 0.04 | 0.86 | ± 0.07 | 1.00 | ± | 0.09 | 0.96 | ± | 0.16 | 1.10 ± 0.10 | 1.00 ± 0.12 | |||
| trenbolone | 0.89 | ± 0.10 | 1.10 | ± | 0.07 | 0.82 | ± 0.08 | 0.95 | ± | 0.10 | 0.70 | ± 0.11 | 1.10 | ± | 0.07 | 1.00 | ± | 0.33 | 1.40 ± 0.30 | 0.98 ± 0.10 | |||
| SARMs | LGD-2226 | 1.00 | ± 0.05 | 1.00 | ± | 0.07 | 1.00 | ± 0.06 | 1.10 | ± | 0.13 | 1.30 | ± 0.05 | 1.00 | ± | 0.10 | 1.00 | ± | 0.16 | 1.00 ± 0.15 | 0.96 ± 0.05 | ||
| LGD-4033 | 0.84 | ± 0.08 | 0.94 | ± | 0.07 | 0.89 | ± 0.05 | 0.97 | ± | 0.05 | 1.10 | ± 0.10 | 0.91 | ± | 0.04 | 1.00 | ± | 0.14 | 1.10 ± 0.12 | 1.10 ± 0.13 | |||
| ostarin | 0.91 | ± 0.03 | 0.98 | ± | 0.05 | 0.97 | ± 0.06 | 0.97 | ± | 0.10 | 1.10 | ± 0.12 | 0.92 | ± | 0.07 | 0.97 | ± | 0.19 | 1.00 ± 0.13 | 1.10 ± 0.11 | |||
| absolute value (forskolin control, ng/mL) | 0.60 | ± 0.14 | 4.87 | ± 0.88 | 28.7 ± 8.20 | 9.33 | ± | 1.79 | 0.18 | ± 0.03 | 159 | ± | 25.8 | 30.1 | ± | 19.6 | 4.32 ± 1.11 | 30.0 ± 6.32 | |||||
A
17a OH-Prog/Prog
B
mineralocorticoids
C
mineralocorticoids
AD/17a OH-Prog
glucocorticoids
glucocorticoids
1.2
AD/Prog
3.0
adrenal androgens
2.5
adrenal androgens
(relative to DMSO control)
1.0
steroidogenic pathway (relative to DMSO control)
2.5
steroidogenic pathway (relative to DMSO control)
2.0
0.8
2.0
ratio
1.5
0.6
1.5
1.0
0.4
1.0
0.2
0.5
0.5
0.0
control
0.0
mesterolone
mestanolone
methenolone
stanozolol
drostanolone
control
mesterolone
mestanolone
methenolone
stanozolol
drostanolone
0.0
control
clostebol
turinabol
oxymetholone
| Compound | CYP17A1 inhibition (% of control) [5 [M] |
|---|---|
| mesterolone | 19.0 ± 4.1 |
| mestanolone | 2.9 ± 6.3 |
| methenolone | 3.0 ± 1.9 |
| stanozolol | 22.3 ± 0.1* |
| drostanolone | 3.0 ± 0.1 |
| abiraterone | IC50 = 15.6 nM |
*tested at 10 µM.
contributed to the observed effects on the steroid profiles by the iden- tified AAS (mesterolone, mestanolone, methenolone, clostebol, oxy- metholone, stanozolol, and drostanolone), H295R cells were incubated with these AAS for 48 h and mRNA levels of 30-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were determined by qPCR. At con- centrations of 1 µM, none of the selected AAS affected the mRNA ex- pression levels of the five key steroidogenic genes as compared to the DMSO vehicle control (data not shown).
3.4. Effects of selected AAS on CYP17A1 17a-hydroxylase activity
Next, the impact of mesterolone, mestanolone, methenolone, sta- nozolol and drostanolone on the conversion of progesterone to 17a- hydroxyprogesterone and the byproduct 16a-hydroxyprogesterone were tested in a cell-free CYP17A1 activity assay. At a high con- centration of 5 uM, the selected AAS showed very weak (mester- olone: < 20% inhibition) or no inhibition (mestanolone, methenolone, stanozolol and drostanolone) of CYP17A1 17a-hydroxylase activity (Table 3).
3.5. Predicted binding of the selected AAS to CYP17A1 and CYP21A2
The potent irreversible inhibition of CYP17A1 by abiraterone is characterized by two key interactions: first, the heme iron forming a covalent coordinating bond with the nitrogen in the heterocycle of abiraterone, and second, the hydrogen bond between the C3-hydroxyl of abiraterone and the side chain of Asn202 in helix F, at a distance of 2.6 Å [41,50] (Fig. 3A-C). Additionally, hydrophobic interactions of the heterocycle ring and the two methyl groups with hydrophobic side chains of several residues on CYP17A1 contribute to the stabilization of abiraterone binding (Fig. 3C). The docking calculations predicted a si- milar binding mode for mesterolone and mestanolone but with an in- verted orientation of the steroid backbone compared to abiraterone where the C3-carbonyl forms an interaction with the heme iron (Fig. 3A, B, D, E). Additionally, some stabilizing hydrophobic interac- tions by the methyl groups could be identified; however, the key sta- bilizing hydrogen bond interaction with Asn202 is absent and no hy- drogen bonds of the C17-hydroxyl with CYP17A1 residues at a distance closer than 5 Å could be found. Methenolone showed even weaker ef- fects on CYP17A1 activity in the H295R assay and docking calculations also did not reveal any stabilizing interactions with Asn202 (data not shown).
17a-hydroxyprogesterone binding to CYP21A2 is characterized by a hydrogen bond (2.5 Å) of the C3-carbonyl on the A-ring with the guanidinium group on the Arg234 side chain (Fig. 4). The A-ring of 17a- hydroxyprogesterone is anchored by helix G such that the C21-methyl group faces the heme iron [51], facilitating C21-hydroxylation. Clos- tebol and oxymetholone, in all predictions, showed a 180° flipped or- ientation compared to 17a-hydroxyprogesterone with the A-ring facing the heme. Clostebol and oxymetholone showed an interaction with the
heme iron. Furthermore, both steroids, like 17a-hydroxyprogesterone, are stabilized by the formation of a hydrogen bond with Arg234 (dis- tance to Arg234 for clostebol of 2.93 Å and for oxymetholone of 3.14 Å). Moreover, all three steroids formed similar hydrophobic stabilizing interactions.
4. Discussion
Evaluation of adverse cardiovascular effects associated with AAS use has mainly focused on heart structure and function by performing echocardiographic examinations. Increases in blood pressure are gen- erally assumed to be due to enhanced cardiac output, arterial stiffness and peripheral arterial resistance [52,53]. Another mechanism by which AAS might contribute to the development of hypertension and cardiovascular diseases includes the interference with adrenal ster- oidogenesis [34], leading to enhanced mineralocorticoid biosynthesis. Mineralocorticoids regulate sodium reabsorption and potassium ex- cretion by acting through mineralocorticoid receptor (MR). An ex- aggerated MR activation leads to hypernatremia and hypokalemia, re- sulting in fluid retention and edema formation and causing hypertension, thereby contributing to the development of cardiovas- cular diseases [54,55].
The androgen derivative abiraterone is a drug well known to in- terfere with steroidogenesis, causing mineralocorticoid excess, hypo- kalemia and hypertension [33,56]. Abiraterone is a drug designed to treat prostate cancer by suppressing testosterone biosynthesis through inhibition of CYP17A1, thereby blocking gonadal and adrenal androgen production. As a side effect of CYP17A1 inhibition, cortisol production is diminished. This results in an excessive activation of the hypothala- mic-pituitaryadrenal (HPA) axis and elevated ACTH release in an at- tempt to correct for the lack of cortisol along with an excessive pro- duction of the mineralocorticoid aldosterone. To avoid the mineralocorticoid excess-related side effects of abiraterone, patients are co-administered prednisone to prevent the excessive HPA axis activa- tion and ACTH release [57]. Additionally, amiloride, a non-steroidal sodium channel inhibitor, alone or in combination with hydro- chlorothiazide, can be administered to control the abiraterone-stimu- lated mineralocorticoid excess [58]. Furthermore, by blocking CYP17A1 abiraterone treatment might lead to accumulation of pro- gesterone [33], which was found to activate certain AR mutants and thereby may contribute to the development of abiraterone-resistant tumors [59].
While patients receiving AAS in a therapeutic situation can be monitored for symptoms of mineralocorticoid excess (pseudo-hyper- aldosteronism, hypertension, hypokalemia), this is not the case for re- creational users. Their uncontrolled use of supraphysiologic AAS doses exposes them to serious health risks.
Despite the well-described adverse health events of AAS, very few of them have been assessed for their impact on adrenal steroidogenesis and the mechanisms of action of such compounds is poorly in- vestigated. To our knowledge, this study is the first to compare nu- merous well-known and widely used AAS as well as three non-steroidal SARMs with respect to their potential to interfere with adrenal steroid biosynthesis.
This analysis revealed three AAS (mesterolone, mestanolone and methenolone) exhibiting effects on the steroid profile of H295R cells in the basal state and upon forskolin-stimulation that resemble those of abiraterone [47], namely increased mineralocorticoids and decreased androgens. However, abiraterone, at a high concentration of 10 uM, results only in enhanced levels of progesterone, but reduced con- centrations of corticosteroids and adrenal androgens, thus resembling an almost complete block of steroidogenesis [35,60]. The similar bioactivity of these three AAS may be explained by their related che- mical structures. They share C3-carbonyl and C17-hydroxyl groups. Mestanolone differs from mesterolone only by the lack or presence of a methyl group in C5 and C17, respectively. Moreover, methenolone has
A
mesterolone
C abiraterone
abiraterone
Ala113
Asn202
lle371
OH
Ile371
Thr306
Leu209
Ile205
Ile206
Heme
Phe114
Val482
Ala367
Asn202
Ala367
Val366
Heme
Ala302
Val483
Heme
Val483
D
mesterolone
Thr306
Heme
0
B
mestanolone
OH
abiraterone
Ala105
Ala367
Heme
Ile206
Thr306
Val483
Ala302
Leu209
Ile371
Val482
lle205
E
mestanolone
Ala105
Ile205
Ala302
Heme
OH Val482
Asn202
Thr306
Val483
Heme
Leu209
Heme
Thr306
Val483
Ala302
Ile206
a double bond between C1 and C2 that is absent in mesterolone. The observed steroid profiles indicated decreased CYP17A1 enzyme activity (Table 1 and 2, Fig. 2B), mainly affecting 17a-hydroxylase activity, in the order mesterolone > mestanolone > methenolone (Fig. 2A).
The mechanism by which these three AAS decrease CYP17A1 ac- tivity is distinct from that of abiraterone. Whereas abiraterone was found to irreversibly inhibit CYP17A1 activity by direct interaction with the substrate binding pocket due to its 16,17-double bond [61], mesterolone, mestanolone and methenolone lacking this important structural feature were unable to substantially inhibit CYP17A1 17a- hydroxylase activity in a cell-free enzyme assay (Table 3). Molecular docking indicated the lack of a stabilizing hydrogen bond between the C17-hydroxyl of mesterolone and mestanolone with residues on CYP17A1 such as Asn202 that forms a key interaction with the C3-hy- droxyl of abiraterone (Fig. 3). Thus, the predicted lack of important stabilizing interactions is in line with the results of the activity assay that do not support a direct inhibition of CYP17A1 by these AAS. Moreover, gene expression analysis did not reveal an effect of mester- olone, mestanolone and methenolone on mRNA levels of 3ß-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2, implying post-transla- tional effects. Thus, further research should focus on post-translational modifications such as phosphorylation and glycosylation and on protein stability that have been found earlier to be involved in the regulation of CYP17A1 activity [62-66].
Increased mineralocorticoid but decreased androstenedione pro- duction was also observed for stanozolol (Table 1 and 2, Fig. 2B), with a strongly decreased androstenedione/17a-hydroxyprogesterone ratio, suggesting inhibition of CYP17/20-lyase activity (Fig. 2A). Stanozolol
did not substantially inhibit CYP17A1 17a-hydroxylase activity in the cell-free assay; however, since the 17/20-lyase reaction needs the al- losteric action of cytochrome b5 to promote the interaction of CYP17A1 with P450 oxidoreductase (POR) [67,68], our cell-free CYP17A1 ac- tivity assay did not consider 17/20-lyase activity. Drostanolone, which enhanced mineralocorticoid biosynthesis but showed little effect on androgen production, did not affect CYP17A1 activity in the cell-free assay (Table 3). Both stanozolol and drostanolone did not alter the expression of steroidogenic genes. Thus, an enhanced mineralocorticoid production due to increased CYP11B2 gene expression, such as ob- served in an earlier study for the UV-filter octylmethoxycinnamate [35], seems unlikely. A limitation of the gene expression analysis in- cludes the late time point, after 48 h of treatment, and it cannot be excluded that mRNA levels were altered at an earlier time point and returned to normal after 48 h. Further research is needed to uncover the mechanism of enhanced aldosterone production by stanozolol and drostanolone.
The results of the current study show a direct effect of particular AAS on adrenal steroidogenesis to enhance aldosterone production. Aldosterone acts through mineralocorticoid receptors and regulates salt and water homeostasis and redox processes [69]. The uncontrolled and prolonged use of such AAS may cause excessive aldosterone secretion, increasing blood volume and blood pressure, decreasing potassium le- vels and promoting oxidative stress, thereby contributing to the de- velopment of cardiovascular diseases.
A different steroid pattern was observed for clostebol, turinabol and oxymetholone (Table 1 and 2, Fig. 2C). Considering that turinabol (clostebol acetate) is the non-active esterified pro-drug of clostebol and
A
clostebol
Leu364
Leu110
17aOH-Prog
C 17aOH-Prog
Leu364
Val101
HO
Arg234
O
Heme
Arg234
Trp202
Leu110
Val101
Heme
D clostebol
Leu110
Leu364
Val101
Trp202
Arg234
Heme
V
B
oxymetholone
O
OH
Leu364
Leu110
17aOH-Prog
CI
Val101
E
oxymetholone
Val101
Heme
Leu110
Arg234
Leu364
Met284
Arg234
HO
V
H
O
Heme
0
Ile231
Ile291
Val198
that such esters are rapidly hydrolyzed to the free active form, explains the almost identical steroid profiles obtained for these two compounds. Suppressed corticosteroid production along with increased dehy- droepiandrosterone levels suggest inhibition of CYP21A2 activity. Since there is no cell-free CYP21A2 activity assay available, molecular docking calculations were performed. Strong stabilizing interactions were found for clostebol and oxymetholone, supporting direct CYP21A2 inhibition (Fig. 4). Follow-on work needs to establish a cell-free CYP21A2 activity assay and address the issue of direct or indirect in- hibition. A direct effect on protein function is favored by the fact that mRNA expression levels of key steroidogenic enzymes were not affected by clostebol and oxymetholone. Furthermore, involvement of an in- hibition of sulfotransferase and/or 3ß-HSD2, which would contribute to increased DHEA formation, cannot be excluded.
In vivo, an inhibition of adrenal corticosteroid biosynthesis, as sug- gested for clostebol, turinabol and oxymetholone, is expected to result in negative feedback-mediated HPA axis activation, ultimately leading to adrenal hyperplasia. Consequently, the increased levels of steroid precursors may be mainly converted to androgens, as it is the case in patients with 21-hydroxylase deficiency suffering from congenital adrenal hyperplasia (CAH). A deficiency of gluco- and miner- alocorticoids can cause severe metabolic disturbances, ultimately leading to life-threatening adrenal crisis [70].
Not all of the tested AAS affected the adrenal steroid profile. While some of them exhibited only minor effects, one third showed no sig- nificant changes on steroid levels. Since AAS have been designed to act through the AR and only differ in their relative binding affinity, the observed effects on the adrenal steroid pattern are most likely AR-in- dependent. In line with this, none of the SARMs tested interfered with adrenal steroidogenesis. Whereas the steroidal scaffold of the AAS bears
a higher risk for interference with other steroid hormone receptors and steroid metabolizing enzymes, including steroidogenic CYPs, the non- steroidal SARMs possess an improved risk profile and should be pre- ferably considered in clinical applications.
The (adverse) effects of AAS depend on the concentrations reached in vivo at the site of a given enzyme or receptor. To our knowledge, there are no data available on intra-adrenal concentrations of AAS and exposure has to be estimated from plasma levels. For example, plasma levels of testosterone undecanoate, the active ingredient of Andriol® Testocaps®, reach about 10 to 35 nmol/L (300-1000 ng/dL) and are considered to be within accepted therapeutic range [71,72]. Bearing in mind that supraphysiologic AAS doses in non-therapeutic uses can reach up to 100 times physiologic levels, the concentration of the AAS used in the present study may be achieved in AAS abusers. Although quantitative in vitro to in vivo extrapolation of concentration-effect correlations can be problematic, the concentrations used herein, causing significant effects on adrenal steroidogenesis, might be relevant in situations of AAS misuse. An obvious limitation of the current study is the lack of negative feedback regulation by the HPA axis in the adrenal cell model applied. Another limitation, regarding the analysis of the underlying mechanisms, includes that only one time point (48 h) was considered, and follow-on work will need to address earlier time points; nevertheless, initial mechanistic information could be gathered.
In conclusion, AAS are a heterogeneous class of testosterone deri- vatives displaying a potential to interfere with different enzymes in- volved in steroidogenesis. The use of H295R cells in their basal and forskolin-stimulated state together with the simultaneous quantification of the most important adrenal steroids by UHPLC-MS/MS allowed the detection of compound-specific interferences with adrenal ster- oidogenesis and provided preliminary information on the mode-of-
action. The comparison of the tested AAS and SARMs allowed grouping compounds causing similar effects and potentially sharing the same mechanism. Mesterolone, mestanolone and methenolone were found to enhance mineralocorticoid and reduce adrenal androgen production by decreasing CYP17A1 activity, whereas clostebol, turinabol and oxy- metholone lowered corticosteroid and enhanced dehydroepian- drosterone biosynthesis, likely via inhibition of CYP21A2. The non- steroidal SARMs displayed a more favorable safety profile than most of the AAS and they should be considered as preferred treatment option for various muscle wasting conditions.
CRediT authorship contribution statement
Melanie Patt: Conceptualization, Investigation, Visualization, Writing - review & editing. Katharina R. Beck: Investigation, Visualization, Writing - review & editing. Tobias Di Marco: Investigation. Marie-Christin Jäger: Investigation. Victor González- Ruiz: Data curation, Writing - review & editing. Julien Boccard: Data curation, Writing - review & editing. Serge Rudaz: Data curation, Writing - review & editing. Rolf W. Hartmann: Writing - review & editing. Mohamed Salah: Investigation. Chris J. Koppen: Investigation, Writing - review & editing. Matthias Grill: Resources, Writing - review & editing. Alex Odermatt: Conceptualization, Writing - review & editing.
Acknowledgements
This work was supported by the Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland. We thank Dr. Denise V. Kratschmar for advice in LC-MS analytics. We are grateful to Prof. Thierry Langer, University of Vienna, Austria, and Inte:Ligand GmbH, for providing the LigandScout software.
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