Androgenic Compounds Research Articles

Effect-directed analysis of androgenic compounds from sewage sludges in China

The safety of municipal sewage sludge has raised great concerns because of the accumulation of large-scale endocrine disrupting chemicals in the sludge during wastewater treatment. The presence of contaminants in sludge can cause secondary pollution owing to inappropriate disposal mechanisms, posing potential risks to the environment and human health. Effect-directed analysis (EDA), involving an androgen receptor (AR) reporter gene bioassay, fractionation, and suspect and nontarget chemical analysis, were applied to identify causal AR agonists in sludge; 20 of the 30 sludge extracts exhibited significant androgenic activity. Among these, the extracts from Yinchuan, Kunming, and Shijiazhuang, which held the most polluted AR agonistic activities were prepared for extensive EDA, with the dihydrotestosterone (DHT)-equivalency of 2.5 – 4.5 ng DHT/g of sludge. Seven androgens, namely boldione, androstenedione, testosterone, megestrol, progesterone, and testosterone isocaproate, were identified in these strongest sludges together, along with testosterone cypionate, first reported in sludge media. These identified androgens together accounted for 55 %, 87 %, and 52 % of the effects on the sludge from Yinchuan, Shijiazhuang, and Kunming, respectively. This study elucidates the causative androgenic compounds in sewage sludge and provides a valuable reference for monitoring and managing androgens in wastewater treatment.

A Review on Male Infertility and Herbal Medicine: Complementary and Alternative Therapies in Animal Models

Various factors contributing to male infertility include genetic determinants, hormonal/neurological imbalance, erectile/libido disorders, genital tract injuries, and toxic/ischemic testicular injuries. Herbs increase sperm count and quality parameters, as well as sexual performance in infertile men, through various mechanisms. For this purpose, efforts were made to investigate effective plants in treating infertility, focusing on those studied in animal research. In this review, we explored the latest findings from animal studies on the therapeutic applications of medicinal plants in male infertility. Based on MeSH keywords, 250 animal studies investigating the effects of herbs on male infertility were reviewed, 72 of which were selected after removing duplicated and unrelated articles. The search was conducted in the Scientific Information Database, the World Health Organization database, Web of Science, Science Direct, Iranmedex, Cochran Library, PubMed, and Google Scholar. The mechanisms underlying the therapeutic effects of herbs on male infertility caused by hormonal disorders, oxidative stress, cytokines, inflammatory pathways, as well as erectile and libido disorders were discussed. Aphrodisiac, spermatogenesis-inducing compounds, antioxidants, anti-inflammatory agents, and androgenic compounds, including anthocyanins, proanthocyanidins, ginsenosides, protodioscin, quassinoids, sesquiterpenes, diosgenin, phyto-oestrogens, thymoquinone, and bajijiasu, in these plants increase male fertility by affecting the hypothalamic-pituitary-gonadal axis and improving sperm’s functional parameters and fertility indices in relation with seminiferous tubules.

Adverse effect of environmental androgenic compounds Galaxolide and Irgacure 369 on the male reproductive system

Our recent study has found that two environmental chemicals, Galaxolide (HHCB, a raw material for synthesizing musk) and Irgacure 369 (IC-369, a photoinitiator used in packaging) are agonists for the androgen receptor in vitro and in vivo. This study aims to reveal the subchronic reproductive toxicity of these two compounds in mature male rats. The results showed that compared with the control group, HHCB and IC-369 reduced the sperm concentration and motility, increased the sperm deformity, and caused the atrophy of the seminiferous tubules in the testicles. Exposure to HHCB and IC-369 reduced testosterone level, and induced luteinizing hormone, and follicle-stimulating hormone levels in rat serum. Compared with the control group, the levels of oxidative stress markers in the serum and testicular tissue increased. HHCB and IC-369 also inhibited expression of the genes involved in androgen synthesis in testicle. The above results indicated that HHCB and IC-369 could affect the levels of sex hormones, alter gene expression profiles and induce histological damage in reproductive organs, resulting in decreased sperm quality. Therefore, HHCB and IC-369 have endocrine disruptors with prominent reproductive toxicity in males.

Where sports performance meets reproductive rights: hormonal contraception

Rational To discuss the use of hormonal contraception (HC) in elite women’s competitive sports from an anti-doping perspective because 1) it changes the natural female hormonal milieu; 2) is used to manipulate the menstrual cycle with performance enhancement intent; 3) even though lowering endogenous testosterone levels, some HCs contain testosterone-like androgenic compounds with potential performance-enhancing effects. Results A complex interaction between rapidly advancing sports-relevant biomedical scientific discoveries, societal changes concerning sex and gender, and a zero-tolerance anti-doping ideology, leads to contentious results, jeopardizing the premises defining and protecting contemporary elite sport in general and that of women in particular. Discussion and conclusions This is in part because of the two out of three criteria for inclusion on the World Anti-Doping Agency’s List of forbidden substances and methods: i) actual or potential for performance enhancement, ii) actual or potential for health risk, and iii) counter to the Spirit of Sport concept. These criteria would suffice for the inclusion of HC on the List, especially in their androgenic form. The fact that they are not is good for women’s reproductive rights but also illustrates the arbitrariness of the administration of WADA’s Prohibited List of substances and methods in elite sports.

Bioactive compounds from four Indian medicinal plants have different potency to induce sex reversal in Nile tilapia: A chromatographic, molecular docking and in silico analysis.

Herbal plants such as Basella alba (Family: Basellaceae), Tribulus terrestris (Family: Zygophyllaceae), Asparagus racemosus (Family: Asparagaceae) and Mucuna pruriens (Family: Fabaceae) are mentioned in Indian traditional system of medicine Ayurveda to possess androgenic activity and increase male virility. The plants have been reported to improve testosterone level and sperm production in experimental male rodents as well. Male Nile tilapias grow more quickly than females and hence are preferred for monosex Nile tilapia culture. Ethanol extracts of B. alba leaves (EB) and T. terrestris seeds (ET), and methanol extract of A. racemosus roots (MA) and M. pruriens seeds (MM) were found effective to induce masculinization in Nile tilapia. The present study intends to evaluate the anti-aromatase activity of EB, ET, MA and MM, to identify the androgenic bioactive compounds in the extracts, and to determine their pharmacokinetics. The study may validate the use of those plant extracts and their major bioactive phytoconstituents in the field of aquaculture and pharmaceuticals. The four crude plant extracts were first fractioned through column and thin layer chromatography (TLC). Three days old Nile tilapia juveniles (mean weight 0.025±0.009g; mean length 12.50±0.12mm; n=50 fish/replicate, 3 replicates/treatment) were then fed diets fortified with the obtained fractions for 30 days. After 30 days, fish were sacrificed and gonad aromatase mRNA expression, and 11-ketotestosterone (11-KT) and estradiol (E2) levels were measured. Fractions yielding the highest male percentage for each plant were subject to gas chromatography-mass spectrometry (GC-MS) analysis. The in silico docking and SwissADME study were conducted with the components showing higher peak percentage in chromatogram. After column chromatography and TLC analysis, EB, ET, MM and MA yielded 6 (EB1 - EB6), 8 (ET1- ET8), 14 (MM1-MM14) and 5 (MA1- MA5) fractions, respectively. Fish fed EB2, ET2, MA2 and MM13 fraction fortified diets showed significantly (p<0.05) higher male percentage (92.32%-98.39%) compared to other treatment groups. EB2, ET2, MA2 and MM13 fed fish showed significantly (p<0.05) higher 11-KT level compared to control male (+247.52 - +397.76%) and lower E2 level compared to control female (-95.92% to -90.65%). Aromatase mRNA expression was significantly (p<0.05) down-regulated by all these four fractions (-1.32 to -5.65 fold) with respect to control female. GC-MS analysis revealed the presence of 1-Octadecene (OD) in EB2, Phenol, 2,4-bis(1,1-dimethylethyl) (PD) in ET2 and MA2, 9,12-Octadecadienoic acid (Z,Z)- (ODDA) in MM13. In silico molecular docking indicated that PD is more effective than ODDA and OD to inhibit aromatase. In addition, PD showed better pharmacokinetics and more drug-likeness compared to OD and ODDA in SwissADME analysis. The present results indicate that ET and MA are more potent to produce all-male tilapia by means of aromatase inhibition. PD can be an ideal compound to achieve masculinization in Nile tilapia through dietary administration, but further investigation is required.

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone−1 for flutamide and 20.1 ± 5.1 ng zone−1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

&lt;p&gt;&lt;strong&gt;Effect of 17α-methyltestosterone (MT) on osmoregulatory responses and apoptosis in genetically improved farmed tilapia (GIFT), &lt;em&gt;Oreochromis niloticus&lt;/em&gt; (L.)&lt;/strong&gt;&lt;/p&gt;

Androgenic compounds can affect osmoregulatory response and apoptosis in fish. In the present study, we exposed genetically improved farmed tilapia (GIFT) Oreochromis niloticus (L.) to 17α-methyltestosterone (MT, 0.5 and 5 mg/L) for 7, 14 and 21 days for understanding the phenomenon. The activities of Na + /K + ATPase (NKA) and Ca 2+ /Mg 2+ ATPase (CMA) were measured in the gill, kidney and intestine to evaluate the change in osmoregulation of GIFT, and genotoxicity was also detected. Results showed that organic NKA were significantly decreased in 5 mg/L MT exposure groups. The intestine NKA was significantly increased (0.5 mg/L MT). MT exposures increased the CMA of kidney and intestine (0.5 mg/L), together with gill CMA (5 mg/L MT). The results of genotoxicity assay showed gill atp 1 a 1 a and nkcc 2 transcripts significantly increased, while intestine atp 1 a 1 a and fxyd 7 transcripts revealed significant increases for MT exposure groups. Caspases proteins demonstrated significant increases at 7 th and 21 st day, and their transcripts were enhanced in 0.5 mg/L MT exposure groups. The results have evidently demonstrated that chronic exposure of MT could result in organic osmoregulatory response and hepatic apoptosis in GIFT O . niloticus .

Comparison of metabolic effects of the progestational androgens dimethandrolone undecanoate and 11β-MNTDC in healthy men.

Dimethandrolone (DMA) and 11β-methyl-19-nortestosterone (11β-MNT) are two novel compounds with both androgenic and progestational activity that are under investigation as potential male hormonal contraceptives. Their metabolic effects have never been compared in men. Assess for changes in insulin sensitivity and adiponectin and compare the metabolic effects of these two novel androgens. In two clinical trials of DMA undecanoate (DMAU) and 11β-MNT dodecylcarbonate (11β-MNTDC), oral prodrugs of DMA and 11β-MNT, healthy men received drug, or placebo for 28days. Insulin and adiponectin assays were performed on stored samples. Mixed model analyses were performed to compare the effects of the two drugs. Student's t test, or the non-parametric Kruskal-Wallis test as appropriate, was used to evaluate for an effect of active drug versus placebo. Class effects were seen, with decrease in HDL-C and SHBG, and increase in weight and hematocrit, with no statistically significant differences between the two compounds. No changes in fasting glucose, fasting insulin, or HOMA-IR were seen with either compound. There was a slight decrease in adiponectin with DMAU that was not seen with 11β-MNTDC. An increase in LDL-C was seen with 11β-MNTDC but not with DMAU. There were no significant changes in insulin resistance after 28days of oral administration of these novel androgens despite a mild increase in weight. There may be subtle differences in their metabolic impacts that should be explored in future studies. Changes in metabolic parameters should be carefully monitored when investigating androgenic compounds.

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography.

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

Controlled release of sex steroids through osmotic pump alters brain GnRH1 and catecholaminergic system dimorphically in the catfish, Clarias gariepinus

The present study aimed to evaluate osmotic pump-mediated controlled release of estrogen in males and androgen in females to analyze the impact on gonadotropin-releasing hormone (GnRH1), catecholamines (CAs) and other associated genes in the catfish, Clarias gariepinus. During pre-spawning phase, catfish were separately implanted osmotic pumps loaded with 17β-estradiol (E2) in males and 17α-methyltestosterone (MT) in females at a dose of 10 μg/100 μl or saline (100 μl) controls into both sexes to release for 21 days and all fishes were maintained as per the duration. Further, GnRH1 expression levels were analysed in the discrete regions of brain after E2 and MT treatments in male and female catfish, respectively using qPCR which revealed that GnRH1 expression was significantly higher in E2 treated male as compared to the control. On the other hand, GnRH1 expression was lower in MT treated female when compared to the control in the discrete regions of brain. In addition, certain brain and monoaminergic system related genes showed a differential response. Catfish GnRH1 could be localized in preoptic area-hypothalamus (POA-HYP) that correlated with the expression profile in the discrete regions of catfish brain. Serum levels of sex steroids in the treated male fish indicated that the treatment of E2 could maintain and impart feminization effect even in the presence of endogenous androgen during gonadal recrudescence while such an effect was not seen in females with androgen treatment. Measurement of CAs, L-3,4-dihydroxyphenylalanine, dopamine and norepinephrine levels in the male and female brain after the controlled release of E2 and MT, respectively confirmed the modulation of neurotransmitters in the E2treated male than MT treated female fish. These results collectively suggest the severity of estrogenic over androgenic compounds to alter reproductive status even at a minimal dose by targeting CAs and GnRH1 at the level of brain of catfish. This study provides insights into the reproductive toxicity of sex steroid analogues at the level of brain GnRH1 and CA-ergic system in addition to serum T, 11-KT and E2 levels during gonadal recrudescence, which is a crucial period of gametogenesis preceding spawning.

The impact of nandrolone decanoate abuse on experimental animal model: Hormonal and biochemical assessment

Nandrolone decanoate (ND) is one of the most commonly abused anabolic androgenic steroids compounds in the world owing to its ability to improve physical performance but its abuse is associated with several adverse effects. The current study was performed to evaluate the effect of recommended and overdose of nandrolone decanoate (ND) for short and long term on the alterations of biochemical markers related to kidney, liver, adrenal, thyroid gland functions and oxidant and antioxidant activities. Sixty male rats were randomly assigned into two major groups. The first was treated with ND for 6 weeks and the second was treated with same drug for 12 weeks. Each of these groups was further subdivided into three sub groups: 1-Control (untreated rats), 2- Rats intraperitoneally injected with ND 3 mg/kg weekly, 3- Rats intraperitoneally injected with ND 15 mg/kg weekly. Administration of high ND dose for either short or long term significantly elevated kidney function biomarkers, liver enzymes both in serum, cytosol and mitochondria, insignificantly increased thyroid function, significantly increased adrenal function while, decreased ACTH. Moreover, oxidative stress biomarkers were significantly upregulated associated with depression in antioxidants activities. Administration of high ND dose for either short or long term as well as the repeated use of recommended ND dose for long term proved to have harmful effects manifested in impairing the functions of kidneys, liver, thyroid and adrenal glands as well as oxidant antioxidant balance.

A Convenient Plant-Based Detection System to Monitor Androgenic Compound in the Environment.

Environmental androgen analogues act as endocrine disruptors, which inhibit the normal function of androgen in animals. In the present work, through the expression of a chimeric gene specified for the production of the anthocyanin in response to androgen DHT (dihydrotestosterone), we generated an indicator Arabidopsis that displays a red color in leaves in the presence of androgen compounds. This construct consists of a ligand-binding domain of the human androgen receptor gene and the poplar transcription factor gene PtrMYB119, which is involved in anthocyanin biosynthesis in poplar and Arabidopsis. The transgenic Arabidopsis XVA-PtrMYB119 displayed a red color in leaves in response to 10 ppm DHT, whereas it did not react in the presence of other androgenic compounds. The transcript level of PtrMYB119 peaked at day 13 of DHT exposure on agar media and then declined to its normal level at day 15. Expressions of anthocyanin biosynthesis genes including chalcone flavanone isomerase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, UFGT (UGT78D2), and anthocyanidin synthase were similar to that of PtrMYB119. It is assumed that this transgenic plant can be used by nonscientists for the detection of androgen DHT in the environment and samples such as food solution without any experimental procedures.

The beneficial androgenic action of steroidal aromatase inactivators in estrogen-dependent breast cancer after failure of nonsteroidal drugs

Direct treatment of ER (+) breast cancer with Formestane diminishes the tumor within weeks. This is unlikely due to lack of estrogens alone. We proposed that it is the negative influence of androgens on the growth of ER(+) breast cancer. We investigated the influence of Formestane and Exemestane and of their major androgenic metabolites 4-hydroxytestosterone and 17-hydroexemestane on the proliferation of MCF-7 cells and ZR-75-1 cells. Inhibitory effects could be prevented by antiandrogens and siRNA. Activation of the AR in MCF-7 and U2-OS cells was tested by reporter gene assays. In vivo androgenicity was evaluated using the Hershberger assay. Influence on the cell cycle was demonstrated by flow-cytometry. Influence of androgens on the activity of CCND1 was demonstrated by Chip-qPCR. Antitumor activity was determined by topical treatment of DMBA tumors. We found that breast cancer cells can metabolize Formestane and Exemestane to androgenic compounds which inhibit proliferation. This can be explained by hindering the accessibility of CCND1 by histone modification. Androgenic metabolites can abolish the growth of DMBA-tumors and prevent the appearance of new tumors. The lack of cross-resistance between steroidal and nonsteroidal aromatase inhibitors is due to inhibitory effects of androgenic steroidal metabolites on the production of cyclin D1. These sterols not only inhibit proliferation of cancer cells but can also stop the growth of DMBA cancers upon direct absorption into the tumor. The quick and considerable effect on ER(+) tumors may open a new avenue for neodjuvant treatment.

Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve.

We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1–30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.

In vitro bioanalytical evaluation of removal efficiency for bioactive chemicals in Swedish wastewater treatment plants

Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge handling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50–60% for two plants) and estrogenic (>90% for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50–60% for most plants and 16% for one plant.

Role of Estrogen in Androgen-Induced Prostate Carcinogenesis in NBL Rats.

Androgens are thought to cause prostate cancer, but the underlying mechanisms are unclear. Data from animal studies suggest that for androgens to cause prostate cancer, they must be aromatized to estrogen and act in concert with estrogen metabolites. We tested the hypothesis that androgen-receptor and estrogen receptor-mediated effects of androgen and estrogen are necessary, as well as genotoxicity of estrogen metabolites. NBL rats were treated with androgenic and estrogenic compounds for 16-75weeks through slow-release silastic implants or pellets. Testosterone alone induced cancer in the prostate of 37% of rats. 5α-Dihydrotestosterone, which cannot be converted to estradiol or testosterone, did not cause a significant prostate cancer incidence (4%). Addition of estradiol to 5α-dihydrotestosterone treatment did not markedly enhance prostate cancer incidence (14%), unlike adding estradiol to testosterone treatment which induced a 100% tumor incidence. Testosterone plus estradiol treatment induced a DNA adduct detectable by 32P-postlabeling, oxidative DNA damage (8-hydroxyguanosine), and lipid peroxidation at the site within the prostate where this treatment causes cancers, preceding later cancer formation. The non-estrogenic 4-hydroxy metabolite of estradiol, when combined with testosterone, induced prostatic dysplasia within 16weeks and, after long-term treatment, a very low incidence of prostate cancer (21%). When an estrogen that cannot be hydroxylated (2-fluoroestradiol) was added to this combined treatment with testosterone and 4-hydroxyestradiol, dysplasia frequency after 16weeks was doubled. These results strongly support the hypothesis, but additional definitive studies are needed which may identify new targets to interfere with these mechanisms that are clinically feasible in humans.

Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency.In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10−6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492.In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

Enhancement of androgen transcriptional activation assay based on genome edited glucocorticoid knock out human prostate cancer cell line

Endocrine-disrupting chemicals (EDCs) interfere with the biological activity of hormones. Among EDC’s, (anti-)androgenic compounds potentially cause several androgen-related diseases. To improve the accuracy of an in vitro transactivation assay (TA) for detection of (anti-)androgenic compounds,We established the glucocorticoid receptor (GR) knockout 22Rv1/MMTV cell line by using an RNA-guided engineered nuclease (RGEN)-derived CRISPR/Cas system. The 22Rv1/MMTV GRKO cell line was characterized and validated by androgen receptor (AR)-mediated TA assay compared with the AR-TA assay using 22Rv1/MMTV. In conclusion, the AR-TA assay with the 22Rv1/MMTV GRKO cell line was more accurate, excluding the misleading signals derived from glucocorticoids or equivalent chemicals, and might be an effective method for screening potential (anti-)androgenic compounds.

Thyroid hormones and androgens differentially regulate gene expression in testes and ovaries of sexually mature Silurana tropicalis

A series of ex vivo exposures using testicular and ovarian tissues of sexually mature Western clawed frogs (Silurana tropicalis) were designed to examine molecular mechanisms of thyroid hormone (TH) and androgen crosstalk sans hypophyseal feedback as well as investigate potential sex-specific differences. Tissues were exposed ex vivo to either triiodothyronine (T3), iopanoic acid (IOP), one co-treatment of IOP + 5α-dihydrotestosterone (5α-DHT), 5α-DHT, 5β-dihydrotestosterone (5β-DHT), or testosterone (T). Direct exposure to different androgens led to androgen specific increases in thyroid receptor and deiodinase transcripts in testes (trβ and dio1) but a decrease in expression in ovaries (trβ and dio3), suggesting that male and female frogs can be differently affected by androgenic compounds. Moreover, exposure to select androgens differentially increased estrogen-related transcription (estrogen receptor alpha (erα) and aromatase (cyp19)) and production (estradiol) in ovaries and testes indicating the activation of alternate metabolic pathways yielding estrogenic metabolites. Sex-steroid-related transcription (i.e., steroid 5α-reductase type 2 (srd5α2) and erα) and production (i.e., 5α-DHT) were also differentially regulated by THs. The presence and frequency of transcription factor binding sites in the putative promoter regions of TH- and sex steroid-related genes were also examined in S. tropicalis, rodent, and fish models using in silico analysis. In summary, this study provides an improved mechanistic understanding of TH- and androgen-mediated actions and reveals differential transcriptional effects as a function of sex in frogs.

Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling — Molecular, biochemical and morphological insights

Lipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol – EE2), androgenic (testosterone – T), and hypolipidemic (clofibrate – CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n−3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERβ-1, acyl-CoA oxidase 1–3I (Acox1–3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis.