Hormonal effects of prohormones Novel approaches towards effect based screening in veterinary growth promoter control Jeroen C.W. Rijk Thesis committee Thesis supervisors Prof. dr. M.W.F. Nielen Professor of Detection of Chemical Food Contaminants Wageningen University Prof. dr. ir. I.M.C.M. Rietjens Professor of Toxicology Wageningen University Thesis co-supervisors Dr. M.J. Groot Veterinary Pathologist RIKILT - Institute of Food Safety, Wageningen UR Dr. A.A.C.M. Peijnenburg Head of Toxicology and Effect analysis group RIKILT - Institute of Food Safety, Wageningen UR Other members: Prof. B. Le Bizec, LABERCA - ONIRIS, Nantes, France Dr. W.G.E.J. Schoonen, MSD, Oss Prof. dr. J. Fink-Gremmels, Utrecht University Prof. dr. M.A. Smits, Wageningen University This research was conducted under the auspices of the Graduate School VLAG. Hormonal effects of prohormones Novel approaches towards effect based screening in veterinary growth promoter control Jeroen C.W. Rijk Thesis Submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 3 December 2010 at 11 a.m. in the Aula. Jeroen C.W. Rijk Hormonal effects of prohormones Novel approaches towards effect based screening in veterinary growth promoter control 208 pages Thesis Wageningen University, Wageningen, NL (2010) ISBN 978-90-8585-819-5 Abstract 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. Table of contents Chapter 1 General introduction 9 Chapter 2 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 49 Chapter 3 Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay 69 Chapter 4 Bioassay based screening of steroid derivatives in animal feed and supplements 85 Chapter 5 Bovine liver slices: a multifunctional in vitro bioactivation model to study the prohormone dehydroepiandrosterone (DHEA) 101 Chapter 6 Metabolomics approach to anabolic steroid urine profiling of bovines treated with prohormones 121 Chapter 7 Feasibility of a liver transcriptomics approach to assess bovine treatment with the prohormones dehydroepiandrosterone (DHEA) 143 Chapter 8 General discussion and future perspectives: Towards effective screening of prohormones in livestock production 161 Summary 183 Samenvatting 191 Dankwoord 199 About the author 203 Chapter 1 General Introduction Chapter 1 1.1. History, legislation and monitoring In modern livestock production special meat producing breeds in combination with sophisticated feeding strategies are employed to assure optimal growth thereby maximizing economical benefits for the farmer. However, to further increase productivity, farmers are tempted to use illegal growth promoters like anabolic steroids, thyrostatics and ß-agonists. One of the first experiments with growth promoters in ruminants dates back from 1947 showing improved growth and feed conversion in heifers as a consequence of diethylstilbestrol (DES) administration [1]. The recognition of the growth promoting properties of estrogens, either alone or in combination with androgens led to their introduction as a tool to increase meat production. In 1955, the USA allowed DES- containing ear implants in cattle and since then, not only in the USA but also in Europe, a wide range of compounds came available for growth promoting purposes comprising synthetic as well as natural hormones. Because of the awareness that residues of growth promoters in meat may lead to disturbance of homeostatic hormone levels and might adversely affect consumers health [2,3], the use of anabolic agents for growth promoting purposes has been forbidden in The Netherlands since 1961 by a decree of the Commodity Board of Livestock and Meat (PVV) [4]. This consequently raised the need for effective methods to control and monitor abuse of growth promoters and since then various biological, histological, chemical and immunological based screening and detection methods were developed (Figure 1). One of the first screening methods to track abuse of estrogens was based on histological examinations of the prostate and Bartholin’s gland in veal calves [5,6]. When animals were found suspect the urine was checked by using more specific chemical and immunochemical methods like thin layer chromatography (TLC) and immunoassays (ELISAs and RIAs). In the beginning of the 1980s the illegal use of estrogens strongly reduced in favor of other anabolic compounds and cocktails. As a result it was harder to identify treated animals due to the fact that histological evaluations showed more variations and alterations were less pronounced [7]. Consequently, control measures shifted to the already existing detection/conformation methods like TLC and immunoassays later followed by more specific hyphenated techniques like gas chromatography (GC) and high performance liquid chromatography (HPLC) coupled to mass spectrometry [8]. These techniques allow targeted detection and quantification of a limited number of pre-selected analytes in one single run. Nowadays a typical analytical strategy for residue monitoring is in general a two-step approach. At first, a low cost screening method is applied which is optimized to prevent false negative results. Secondly, a confirmation method is used to confirm any positive screening result thereby 10 General introduction preventing false positive results. For both screening as well as confirmation procedures gas and liquid chromatography in combination with mass spectrometric detection (GC- and LC-MS/MS) are used extensively and are considered state-of-the-art in veterinary control. Apart from screening by GC- and LC-MS, traditional