Jeroen C.W. Rijk

Metabolomics Approach to Anabolic Steroid Urine Profiling of Bovines Treated with Prohormones

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach.

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17β-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.

Identification of anabolic steroids and derivatives using bioassay-guided fractionation,UHPLC/TOFMS analysis and accurate mass database searching

Biological tests can be used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental analysis, food analysis, as well as in clinical and forensic toxicology. In this study bioassay-guided fractionation, ultra high performance liquid chromatography combined with time-of-flight mass spectrometry (UHPLC/TOFMS) and accurate mass database searching was tested to detect and identify unknown androgens. Herbal mixtures and sport supplements were tested using an androgen bioassay and modifications in sample preparations were carried out in order to activate inactive pro-androgens, androgen esters and conjugated androgens to enable their detection in the bioassay. Two of the four herbal mixtures tested positive and bioassay-guided fractionation followed by UHPLC/TOFMS of positive fractions resulted in the identification of nortestosterone phenylpropionate, testosterone cyclohexanecarboxylate and methyltestosterone. Three of the four sport supplements reacted toxic in the bioassay or gave inconclusive results and were further investigated using UHPLC/TOFMS in combination with data processing software and an accurate mass database having approximately 40,000 entries. This accurate mass database was derived from the PubChem database on the internet and coupled to the TOFMS software. This resulted in the tentative identification of several androgens, including methylboldenone, testosterone and the androgen esters methyltestosterone propionate or testosterone isobutyrate, testosterone buciclate and methylenetestosterone acetate. The study showed that bioassay-guided fractionation in combination with UHPLC/TOFMS analysis is a useful procedure to detect, isolate and identify unknown androgens in suspected samples. As an alternative, the use of data processing software in combination with an accurate mass database and coupled on-line with the TOFMS instrument software enabled the identification of androgens and androgen esters in the chromatogram even without bioassay-guided fractionation.

Detection of anabolic steroids in dietary supplements: the added value of an androgen yeast bioassay in parallel with a liquid chromatography-tandem mass spectrometry screening method.

Recently we constructed a recombinant yeast cell that expresses the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. Eighteen different dietary supplements, already analysed by a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the presence of anabolic steroids, were screened for androgenic activity. Eleven samples containing at least one anabolic steroid, with a concentration that was around or above 0.01 mgunit(-1) according to LC-MS/MS, were also positive in the bioassay. Seven samples did not contain any of the 49 compounds screened for in LC-MS/MS. In contrast two of them were positive in the bioassay. Bioassay-directed identification, using the bioassay as an off-line LC-detector and LC-time of flight-MS with accurate mass measurement was carried out in these two samples and revealed the presence of 4-androstene-3beta,17beta-diol and 5alpha-androstane-3beta,17beta-diol in the first and 1-testosterone in the second supplement, showing the added value of the bioassay in comparison with a LC-MS/MS screening method alone.

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay.

Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17β-testosterone (17β-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.

Phytoestrogens in menopausal supplements induce ER-dependent cell proliferation and overcome breast cancer treatment in an in vitro breast cancer model

Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor Modulator Tamoxifen (TAM) can result in the onset of menopausal symptoms. Women often try to relieve these symptoms by taking menopausal supplements containing high levels of phytoestrogens. However, little is known about the potential interaction between these supplements and breast cancer treatment, especially aromatase inhibitors. In this study, interaction of phytoestrogens with the estrogen receptor alpha and TAM action was determined in an ER-reporter gene assay (BG1Luc4E2 cells) and human breast epithelial tumor cells (MCF-7). Potential interactions with aromatase activity and LET were determined in human adrenocorticocarcinoma H295R cells. We also used the previously described H295R/MCF-7 co-culture model to study interactions with steroidogenesis and tumor cell proliferation. In this model, genistein (GEN), 8-prenylnaringenin (8PN) and four commercially available menopausal supplements all induced ER-dependent tumor cell proliferation, which could not be prevented by physiologically relevant LET and 4OH-TAM concentrations. Differences in relative effect potencies between the H295R/MCF-7 co-culture model and ER-activation in BG1Luc4E2 cells, were due to the effects of the phytoestrogens on steroidogenesis. All tested supplements and GEN induced aromatase activity, while 8PN was a strong aromatase inhibitor. Steroidogenic profiles upon GEN and 8PN exposure indicated a strong inhibitory effect on steroidogenesis in H295R cells and H295R/MCF-7 co-cultures. Based on our in vitro data we suggest that menopausal supplement intake during breast cancer treatment should better be avoided, at least until more certainty regarding the safety of supplemental use in breast cancer patients can be provided.

Endocrine-Disrupting Effects of Thioxanthone Photoinitiators

Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. Recently, several studies indicated that 2-ITX might be an endocrine-disrupting chemical. In this work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthio xanthone (2,4-diethyl-TX), 2-chlorothioxanthone (2-chloro-TX), and 1-chloro-4-propoxythioxanthone (1-chloro-4-propoxy-TX) on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. None of the compounds showed androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities were observed for 2-ITX, 4-ITX, and 2,4-diethyl-TX, whereas 2-chloro-TX showed only antiandrogenic activity. In an adapted version of the H295R steroidogenesis assay, using gas chromatography-tandem mass spectrometry analysis of H295R media, all five compounds increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX, 4-ITX, and 2,4-diethyl-TX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.

Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids.

AbstractPreviously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T. FigureBovine liver slices for exposure studies in a 6-well format.

Extending an In Vitro Panel for Estrogenicity Testing: The Added Value of Bioassays for Measuring Antiandrogenic Activities and Effects on Steroidogenesis

In the present study, a previously established integrated testing strategy (ITS) for in vitro estrogenicity testing was extended with additional in vitro assays in order to broaden its sensitivity to different modes of action resulting in apparent estrogenicity, i.e., other than estrogen receptor (ER) binding. To this end, an extra set of 10 estrogenic compounds with modes of action in part different from ER binding, were tested in the previously defined ITS, consisting of a yeast estrogen reporter gene assay, an U2OS ERα CALUX reporter gene assay and a cell-free coregulator binding assay. Two androgen reporter gene assays and the enhanced H295R steroidogenesis assay were added to that previous defined ITS. These assays had added value, as several estrogenic model compounds also elicited clear and potent antiandrogenic properties and in addition also showed effects on steroidogenesis that might potentiate their apparent estrogenic effects in vivo. Adding these assays, examining mechanisms of action for estrogenicity apart from ERα binding, gives a more complete and comprehensive assessment of the ability of test compounds to interfere with endocrine signaling. It was concluded that the extended ITS will go beyond in vivo estrogenicity testing by the uterotrophic assay, thereby contributing to the 3R-principles.

Bovine liver slices: A multifunctional in vitro model to study the prohormone dehydroepiandrosterone (DHEA).

Biotransformation of inactive prohormones like dehydroepiandrosterone (DHEA) can lead to the formation of potent androgens and subsequent androgenic responses in target tissues. In the present study, precision-cut bovine liver slices were used to study the effects of DHEA on the metabolite, transcript and androgenic activity level. Bovine liver slices were exposed for 6h to various concentrations of DHEA. Changes in androgenic activity of the DHEA containing cell culture media were measured using a yeast androgen bioassay and metabolites were identified using ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS), while gene expression in the DHEA-treated liver slices was examined using bovine microarrays and compared with the profile as obtained with 17ß-testosterone (17ß-T). An increase in androgenic activity was observed in the bioassay upon testing of samples from incubations of DHEA with liver slices and the formation of 4-androstenedione (4-AD), 5-androstene-3ß,17ß-diol, 17ß-T, 7α-hydroxy-DHEA, 7-keto-DHEA and 17α-T could be confirmed by UPLC-TOFMS analysis. Exposure of liver slices to DHEA and the strong androgen 17ß-T resulted in the identification of significantly up- and down-regulated genes and revealed similar gene expression profiles for both compounds. The results indicate that DHEA itself is biologically not very active, but is rapidly converted by the liver slices into the more androgen active compounds 4-AD and 17ß-T. Moreover, the present data highlight the multi-functionality of bovine liver slices as an in vitro bioactivation model, allowing the assessment of androgen activity or gene expression as effect-based endpoints for prohormone exposure.