Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells
Dungen, M.W. van den; Rijk, J.C.W. ; Kampman, E. ; Steegenga, W.T. ; Murk, A.J. - \ 2015
Toxicology in Vitro 29 (2015)4. - ISSN 0887-2333 - p. 769 - 778.
Persistent organic pollutants (POPs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg) can be accumulated in seafood and then form a main source for human exposure. Some POPs have been associated with changes in steroid hormone levels in both humans and animals. This study describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analyzed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17ß-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17ß-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded.
Transcription profiling by array of Bos taurus liver from control and either orally or intramuscularly DHEA treated animals
Rijk, J.C.W. - \ 2014
E-MEXP-2611 - A-MEXP-1810 - Bos taurus
Comparing gene expression profiles of bovines treated orally or intramuscularly with DHEA versus control animals.
Extending an in vitro panel for estrogenicity testing: the added value of bioassays for measuring antiandrogenic activities and effects on steroidogenesis
Wang, S. ; Rijk, J.C.W. ; Besselink, H.T. ; Houtman, J. ; Peijnenburg, A.A.C.M. ; Brouwer, A. ; Rietjens, I. ; Bovee, T.F.H. - \ 2014
Toxicological sciences 141 (2014)1. - ISSN 1096-6080 - p. 78 - 89.
coregulator binding assay - bisphenol-a - androgen receptor - transcriptional activities - environmental chemicals - pubertal development - thyroid-function - calux method - wistar rats - cell-line
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 ERa 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 ERa 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.
Phytoestrogens in menopausal supplements induce ER-dependent cell proliferation and overcome breast cancer treatment in an in vitro breast cancer model
Duursen, M.B.M. van; Smeets, E.E.J.W. ; Rijk, J.C.W. ; Nijmeijer, S.M. ; Berg, M. - \ 2013
Toxicology and Applied Pharmacology 269 (2013)2. - ISSN 0041-008X - p. 132 - 140.
expression patterns - plasma estrogen - gene-expression - mcf-7 cells - aromatase - tamoxifen - letrozole - women - bioavailability - metabolism
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. (C) 2013 Elsevier Inc. All rights reserved.
|2-Isopropylthioxantone (ITX) induces endocrine disrupting effects and increases liver weight during puberty in male Wistar rats.
Hendriksen, P.J.M. ; Kramer, E.H.M. ; Groot, M.J. ; Rijk, J.C.W. ; Hoogenboom, L.A.P. ; Peijnenburg, A.A.C.M. - \ 2013
In: 52nd Annual Meeting and ToxExpo, San Antonio, Texas, USA, 10 - 14 March, 2013. - Reston : Society of Toxicology (The Toxicologist ) - p. 413 - 413.
Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis
Rijke, E. de; Essers, M.L. ; Rijk, J.C.W. ; Thevis, M. ; Bovee, T.F.H. ; Ginkel, L.A. van; Sterk, S.S. - \ 2013
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 30 (2013)9. - ISSN 1944-0049 - p. 1517 - 1526.
doping control purposes - mass-spectrometric characterization - urinary metabolites - sarms
For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l–1 and a detection capability of 0.025 µg l–1 in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.
Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices
Szalowska, E. ; Stoopen, G.M. ; Rijk, J.C.W. ; Wang, S. ; Hendriksen, P.J.M. ; Groot, M.J. ; Ossenkoppele, J.S. ; Peijnenburg, A.A.C.M. - \ 2013
Toxicology in Vitro 27 (2013)5. - ISSN 0887-2333 - p. 1513 - 1524.
precision-cut liver - toxicity - metabolism - culture - glucose - tension - systems - cells - toxicogenomics - activation
Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24 h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.
A low-density DNA microchip for the detection of (anti-)estrogenic compounds and their relative potencies
Wang, S. ; Rijk, J.C.W. ; Pen, M.J. ; Aarts, J.M. ; Peijnenburg, A.A.C.M. ; Rietjens, I. ; Bovee, T.F.H. - \ 2013
Analytical Biochemistry 435 (2013)1. - ISSN 0003-2697 - p. 83 - 92.
i gene-expression - disrupting chemicals - breast-cancer - growth-factor - estrogen - receptors - protein - assays - alpha - identification
In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17ß-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography–tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen)
Endocrine disrupting effects of thioxanthone photoinitiators
Reitsma, M. ; Bovee, T.F.H. ; Peijnenburg, A.A.C.M. ; Hendriksen, P.J.M. ; Hoogenboom, L.A.P. ; Rijk, J.C.W. - \ 2013
Toxicological sciences 132 (2013)1. - ISSN 1096-6080 - p. 64 - 74.
tandem mass-spectrometry - cell-line - liquid-chromatography - gas-chromatography - in-vitro - expression - bioassay - itx - steroidogenesis - protein
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 the present work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthioxanthone (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 anti-androgenic as well as anti-estrogenic activities were observed for 2-ITX, 4-ITX and 2,4-diethyl-TX, while 2-chloro-TX showed only anti-androgenic 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), were significantly up-regulated 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 signalling and steroid hormone production.
|Metabolic profiling of steroidogenesis in the human H295R adrenocortaical cell line for detection of endocrine disruptors
Rijk, J.C.W. ; Bovee, T.F.H. ; Lommen, A. ; Hoogenboom, L.A.P. ; Peijnenburg, A.A.C.M. - \ 2012
Effect of oxygen on liver slices viability
Szalowska, E. ; Stoopen, G.M. ; Wang, S. ; Rijk, J.C.W. ; Ossenkoppele, J.S. ; Peijnenburg, A.A.C.M. - \ 2012
In: 51st Annual Meeting of the Society of Toxicology and ToxExpo, March 11-15, 2012, San Fransisco, California. - Oxford University Press - p. 394 - 395.
Screening for Modulatory Effects on Steroidogenesis Using the Human H295R Adrenocortical Cell Line: A Metabolomics Approach
Rijk, J.C.W. ; Peijnenburg, A.A.C.M. ; Blokland, M.H. ; Lommen, A. ; Hoogenboom, L.A.P. ; Bovee, T.F.H. - \ 2012
Chemical Research in Toxicology 25 (2012)8. - ISSN 0893-228X - p. 1720 - 1731.
mass-spectrometry - in-vitro - expression - receptor - assay - disruption - inhibitors - chemicals - bioassays - model
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 17ß-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.
Bovine liverslices: A multifunctional in vitro model to study the prohormone dehydroepiandrosterone (DHEA)
Rijk, J.C.W. ; Bovee, T.F.H. ; Peijnenburg, A.A.C.M. ; Groot, M.J. ; Rietjens, I.M.C.M. ; Nielen, M.W.F. - \ 2012
Toxicology in Vitro 26 (2012)6. - ISSN 0887-2333 - p. 1014 - 1021.
prepubertal children - exposure assessment - androgen bioassay - metabolism - expression - rat - estrogens - urine - hepatocytes - microsomes
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, 7a-hydroxy-DHEA, 7-keto-DHEA and 17a-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.
Bioassay based screening of steroid derivatives in animal feed and supplements
Rijk, J.C.W. ; Ashwin, H.M. ; Kuijk, S.J.A. van; Groot, M.J. ; Heskamp, H.H. ; Bovee, T.F.H. ; Nielen, M.W.F. - \ 2011
Analytica Chimica Acta 700 (2011)1-2. - ISSN 0003-2670 - p. 183 - 188.
testosterone-undecanoate - liquid-chromatography - androgen bioassay - yeast bioassay - validation - urine
Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90–100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 µg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by ß-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.
Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA
Becue, I. ; Poucke, C. ; Rijk, J.C.W. ; Bovee, T.F.H. ; Nielen, M.W.F. ; Peteghem, C. van - \ 2010
Analytical and Bioanalytical Chemistry 396 (2010)2. - ISSN 1618-2642 - p. 799 - 808.
mass-spectrometry - androgen profile - dehydroepiandrosterone - gas - consequences - prohormones - supplements - bioassay - men
DHEA (3ß-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17a- and 17ß-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17a-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the ¿5-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible.
Identification of anabolic steroids and derivatives using bioassay-guided fractionation,UHPLC/TOFMS analysis and accurate mass database searching
Peters, R.J.B. ; Rijk, J.C.W. ; Bovee, T.F.H. ; Nijrolder, A.W.J.M. ; Lommen, A. ; Nielen, M.W.F. - \ 2010
Analytica Chimica Acta 664 (2010)1. - ISSN 0003-2670 - p. 77 - 88.
liquid-chromatography - androgen bioassay - directed identification - veterinary drugs - screening method - human urine - hplc-uv - lc-ms - spectrometry - testosterone
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.
Feasibility of a liver transcriptomics approach to assess bovine treatment with the prohormone dehydroepiandrosterone (DHEA)
Rijk, J.C.W. ; Peijnenburg, A.A.C.M. ; Hendriksen, P.J.M. ; Hende, J. van; Groot, M.J. ; Nielen, M.W.F. - \ 2010
BMC Veterinary Research 6 (2010). - ISSN 1746-6148
gene-expression-biomarkers - messenger-rna expression - anabolic agents - androgen - tissues - muscle - abuse - sport
Background Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet. Results As determined by principal component analysis (PCA), large differences in liver gene expression profiles were observed between treated and control animals as well as between the two control groups. When comparing the gene expression profiles of PO and IM treated animals to that of all control animals, the number of significantly regulated genes (p-value 1.5) was 23 and 37 respectively. For IM and PO treated calves, gene sets were generated of genes that were significantly regulated compared to one control group and validated versus the other control group using Gene Set Enrichment Analysis (GSEA). This cross validation, showed that 6 out of the 8 gene sets were significantly enriched in DHEA treated animals when compared to an 'independent' control group. ConclusionThis study showed that identification and application of genomic biomarkers for screening of (pro)hormone abuse in livestock production is substantially hampered by biological variation. On the other hand, it is demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation. Therefore, DNA-microarray expression profiling together with statistical tools like GSEA represent a promising approach to screen for (pro)hormone abuse in livestock production. However, a better insight in the genomic variability of the control population is a prerequisite in order to define growth promoter specific gene sets that can be used as robust biomarkers in daily practice.
Hormonal effects of prohormones : novel approaches towards effect based screening in veterinary growth promoter control
Rijk, J.C.W. - \ 2010
Wageningen University. Promotor(en): Michel Nielen; Ivonne Rietjens, co-promotor(en): Maria Groot; Ad Peijnenburg. - [S.l. : S.n. - ISBN 9789085858195 - 207
groeibevorderaars - hormonen - biotesten - metabolisme - metabolomica - growth promoters - hormones - bioassays - metabolism - metabolomics
Within the European Union the use of growth promoting agents in cattle fattening is prohibited according to Council Directive 96/22/EC. Interestingly, there is not a black list of substances, but 96/22/EC states that all substances having thyrostatic, estrogenic, androgenic or gestagenic activity are prohibited. Besides abuse of the “classical” synthetic steroids there is a tendency towards misuse of natural steroids and prohormones. Prohormones are compounds that exhibit limited or no hormonal activity but are direct precursors of bioactive hormones and are intended to be converted to full active hormones via enzymatic processes in the body. However, knowledge about metabolism, the mode of action and excretion profiles in cattle is often unclear, and methods to detect abuse of prohormones in livestock production are lacking. Therefore, the aim of this thesis was to get insight into the hormonal action of prohormones and to develop novel in vitro and in vivo screening methods allowing effective surveillance on the illegal use of prohormones in livestock production. Hereby the emphasis was on developing effect based approaches to better meet Council Directive 96/22/EC.
The bioactivity of a wide variety of supplements which contained prohormones were tested using a yeast androgen bioassay. For supplements containing solely prohormones the value of this bioactivity based screening appeared to be limited as they require metabolism to become active. Therefore, screening methods for animal feed, supplements and preparations were set-up by using the same yeast androgen bioassay in combination with bovine liver models as well as enzymatic and chemical deconjugation procedures to mimic in vivo metabolic bioactivation. The use of either bovine liver S9, liver slices, pure enzymes or alkaline hydrolysis showed that prohormones could be activated, resulting in a significant increase in bioactivity as determined by the androgen yeast bioassay.
For the detection of prohormone abuse at the farm and/or slaughterhouse the usefulness of ‘omics’ based profiling techniques was investigated. Within this scope a comprehensive metabolomics based screening strategy for steroid urine profiling was developed. Comparison of urinary profiles revealed large differences between the profiles of controls and dehydroepiandrosterone (DHEA) as well as pregnenolone treated animals. Moreover this steroid urine profiling approach allowed identification of biomarkers for treatment by specific prohormones. This resulted in respectively 7 and 12 specific mass peak loadings which could potentially be used as biomarkers for pregnenolone and DHEA treatment.
In addition, the feasibility of a liver gene expression profiling approach was investigated to monitor the effects of DHEA treatment at the transciptome level. It was shown that identification and application of genomic biomarkers for screening of DHEA abuse in cattle is substantially hampered by biological variation. On the other hand, it was demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation.
Altogether the results of this thesis increase the knowledge about the metabolism and bioactivation of prohormones in vitro as well as in vivo. Based on this knowledge, a panel of new effect based concepts and screening methods was developed that complement and improve the current testing programs. These new concepts will facilitate better implementation of the European ban on growth promoters in livestock production as described in Council Directive 96/22/EC.
Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids
Wang, S. ; Rijk, J.C.W. ; Poortman, J.H. ; Kuijk, S. ; Peijnenburg, A.A.C.M. ; Bovee, T.F.H. - \ 2010
Analytical and Bioanalytical Chemistry 397 (2010)2. - ISSN 1618-2642 - p. 631 - 641.
human hepatocytes - drug-metabolism - gene-expression - small-intestine - rat - bioassay - system - colon
Previously 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 17ß-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 6a, 6ß, 15ß, and 16a-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
Identification of unknown residues : using bioassay directed fractionation, UPLC/TOFMS analysis and database searching
Peters, R.J.B. ; Rijk, J.C.W. ; Oosterink, J.E. ; Nijrolder, A.W.J.M. ; Nielen, M.W.F. - \ 2009
Wageningen : Rikilt - Institute of Food Safety (Report / RIKILT 2009.013) - 47
residuen - analytische methoden - biotesten - vloeistofchromatografie - massaspectrometrie - residues - analytical methods - bioassays - liquid chromatography - mass spectrometry
Nowadays a large number of compounds are determined in environmental and food samples. Biological tests are 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. This study reports on the development of a procedure for the identification of unknown residues in samples suspected of containing illegal substances and samples showing bioactivity in bioassay - or microbiological screening assays. For testing purposes several samples were selected; a number of so-called "cold cases", historical samples that were suspected of containing illegal growth promoting substances, herbal mixtures and sport supplements.