Structurally different anabolic androgenic steroids reduce neurite outgrowth and neuronal viability in primary rat cortical cell cultures.

The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.

Simple Synthesis of 17-ß-O-hemisuccinate of Stanozolol for Immunoanalytical Methods.

The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17ß-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

Design, synthesis and characterization of a PEGylated stanozolol for potential therapeutic applications.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signaling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH2)10kDa, (MeO-PEG-NH)10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, 1H NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.

Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics.

Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.

Is zebrafish (Danio rerio) a tool for human-like metabolism study?

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd.

Stanozolol-soaked grafts enhance new bone formation in rat calvarial critical-size defects.

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 µm/day ± 0.37 versus 6.50 µm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm-2 ± 2.01) to three months (28.26 n mm-2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm-2 ± 0.32 versus 1.01 n mm-2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm-2 ± 6.29 versus 6.29 n mm-2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Multimodal plasmonic biosensing nanostructures prepared by DNA-directed immobilization of multifunctional DNA-gold nanoparticles.

Biofunctional multimodal plasmonic nanostructures suitable for multiplexed localized surface plasmon resonance (LSPR) biosensing have been created by DNA-directed immobilization (DDI) of two distinct multifunctional biohybrid gold nanoparticles. Gold nanoparticles (AuNP) of distinct sizes, and therefore showing distinct plasmon resonant peaks (RP), have been biofunctionalized and codified with two different single stranded-DNA (ssDNA) chains. One of these oligonucleotide chains has been specifically designed to direct each AuNP to a distinct location of the surface of a DNA microarray chip through specific hybridization with complementary oligonucleotide strands. Scanning Electron Microscopy (SEM) has been used to demonstrate selective immobilization of each AuNP on distinct spots. The second ssDNA chain of the AuNPs provides the possibility to introduce by hybridization distinct types of bioactive molecules or bioreceptors, on a reversible manner. In this work, hapten-oligonucleotide bioconjugate probes, with sequences complementary to the second ssDNA linked to the AuNP, have been synthesized and used to create multiplexed hapten-biofuncionalized plasmonic nanostructures. The oligonucleotide probes consist on anabolic androgenic steroid haptens (AAS) covalently linked to specifically designed oligonucleotide sequences. The biofunctionality of these plasmonic nanostructures has been demonstrated by fluorescent microarray immunoassay and LSPR measurements, recording the shift of the RP produced after the antibody binding to the corresponding hapten-oligonucleotide probes immobilized on the nanostructured surface. Preliminary data show that this approach could allow manufacturing multifunctional multimodal LSPR chips for multiplexed analysis of different substances reaching very good detectability. Thus, small molecular weigh, analytes such as stanozolol (ST,) could be detected at concentrations in the low nM range. The results here presented open the door for an easy way to construct site-encoded multiplexed multimodal LSPR sensor transducers, combining the DDI strategies with multimodal biohybrid nanoparticles showing distinct optical properties.

Stanozolol regulates proliferation of growth plate chondrocytes via activation of ERalpha in GnRHa-treated adolescent rats.

Improving the final adult height is one of the most important aims for treatment of central precocious puberty. Stanozolol (ST) is a synthetic derivative of androgen. In this study, we investigated the effects and the mechanisms of ST on the proliferation of growth plate chondrocytes isolated from adolescent rats treated with gonadotropin-releasing hormone analogue (GnRHa). Treatment with ST resulted in time- and concentration-dependent effects on proliferation as determined by MTT and proliferating cell nuclear antigen (PCNA) assays. Western blotting showed that ST increased the phosphorylation level of the estrogen receptor alpha (ERalpha), but not the androgen receptor (AR). Pharmacological inhibition of ERalpha and mitogen-activated protein kinase (MAPK) attenuated the effects of ST on the proliferation of growth plate chondrocytes. A molecular dynamics simulation showed hydrophobic interactions between ST and ERalpha. These results suggested that ERalpha, but not AR, partially mediates the ST-driven proliferation of growth plate chondrocytes, and that multiple pathways may be involved in the mechanism of action of ST.

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry.

The applicability of LC-MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4xi,16xi-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3'-hydroxy-stanozolol, 16beta-hydroxy-stanozolol and 4beta-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC-QTOF MS.

Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-orbitrap hybrid mass analyzers.

Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of stanozolol, its metabolites 17-epistanozolol, 3'-OH-stanozolol, 4alpha-OH-stanozolol, 4beta-OH-stanozolol, 17-epi-16alpha-OH-stanozolol, 16alpha-OH-stanozolol, 16beta-OH-stanozolol, as well as the synthetic analogues 4-dehydrostanozolol, 17-ketostanozolol, and N-methyl-3'-OH-stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.

Studies on anabolic steroids. 9. Tertiary sulfates of anabolic 17 alpha-methyl steroids: synthesis and rearrangement.

A simple and convenient method has been developed to prepare sulfates of anabolic 17 beta-hydroxy-17 alpha-methyl steroids. The sulfates of methandienone, 17 alpha-methyltestosterone, mestanolone, oxandrolone, and stanozolol were prepared. Different A-ring functions were not affected under the sulfation condition. The buffered hydrolyses of these sulfates provided the 17-epimers of the original steroids and 17,17-dimethyl-18-nor-13(14)-ene steroids, presumably via the 17-carbocations.