Bioassay based screening of steroid derivatives in animal feed and supplements (original) (raw)
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Analytical Chemistry, 2011
T he over-the-counter nutritional supplement (nutraceutical) market has grown rapidly since the 1994 legislation permitting sale of steroid precursors as nutraceutical food supplements 1 with a market turnover estimated at more than US $60 billion in 2006. 2 Nutraceuticals, claiming on their labels to enhance performance legally, are marketed to athletes. However, chemical analysis has revealed undeclared compounds in these products including substances banned by the World Anti-Doping Agency (WADA) as well as novel designer androgens or proandrogens, which are not specified in the WADA Prohibited List but generically are prohibited as androgens. 2 WADA-approved doping detection tests for androgens identify banned steroids using gas or liquid chromatography with tandem mass spectrometry (MS). Although sensitive and specific, these methods can only detect specified steroid structures and not their in vivo metabolites nor whether they are androgen agonists or antagonists. Hence, MSbased methods alone cannot serve adequately to support the generic prohibition of androgens. One approach to addressing these limitations has been the development of in vitro androgen bioassays, which allow for functional characterization of steroids as androgens according to their AR-specific transcriptional activity, regardless of structural knowledge. These assays entail the use of eukaryotic cells overexpressing the androgen receptor (AR) and introduced with a reporter gene driven by an androgen ABSTRACT: Androgenic steroids marketed online as nutraceuticals are a growing concern in sport doping. The inability of conventional mass spectrometry (MS)-based techniques to detect structurally novel androgens has led to the development of in vitro androgen bioassays to identify such designer androgens by their bioactivity. The objective of this study was to determine the androgenic bioactivity of novel steroidal compounds isolated from nutraceuticals using both yeast and mammalian cell-based androgen bioassays. We developed two new in vitro androgen bioassays by stably transfecting HEK293 and HuH7 cells with the human androgen receptor (hAR) expression plasmid together with a novel reporter gene vector (enhancer/ARE/SEAP). The yeast β-galactosidase androgen bioassay was used for comparison. Our new bioassay featuring the enhancer/ARE/SEAP construct (-S) displayed simpler assay format and higher specificity with lower sensitivity compared with the commonly used mouse mammary tumour virus (MMTV)-luciferase. The relative potencies (RP), defined as [EC 50 ] of testosterone/[EC 50 ] of steroid, of nutraceutical extracts in the yeast, were 34, 333, and 80 000 for Hemapolin; 208, 250, and 80 for Furazadrol; 0.38, and 106 for Oxyguno; 2.7, 0.28, and 15 for Trena; and 4.5, 0.1, and 0.4 for Formadrol, respectively. The wide discrepancies in rank RP of these compounds was reconciled into a consistent potency ranking when the cells were treated with meclofenamic acid, a nonselective inhibitor of steroid metabolizing enzymes. These findings indicate that steroids extracted from nutraceuticals can be converted in vitro into more or less potent androgens in mammalian but not in yeast cells. We conclude that the putative androgenic bioactivity of a new compound may depend on the bioassay cellular format and that mammalian cell bioassays may have an added benefit in screening for proandrogens but sacrifice specificity for sensitivity in quantitation.
Analyst, 2000
The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, b-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17a-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 °C with the pH adjusted to 5.2 and using 12 000 units of enzyme preparation.
Analytical and Bioanalytical Chemistry, 2008
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-3,17dione (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.
Hormones and Hormonal Anabolics: Residues in Animal Source Food, Potential Public Health Impacts, and Methods of Analysis, 2020
The demand for nutritious food, especially food of animal origin, is globally increasing due to escalating population growth and a dietary shift to animal source food. In order to fulfill the requirements, producers are using veterinary drugs such as hormones and hormone-like anabolic agents. Hormones such as steroidal (estrogens, gestagens, and androgens), nonsteroidal, semisynthetic, and synthetic or designer drugs are all growth-promoting and body-partitioning agents. Hence, in food animal production practice, farm owners use these chemicals to improve body weight gain, increase feed conversion efficiency, and productivity. However, the use of these hormones and hormonal growth-promoting agents eventually ends up with the occurrence of residues in the animal-originated food. The incidence of hormone residues in such types of food and food products beyond the tolerance acts as a risk factor for the occurrence of potential public health problems. Currently, different international and national regulatory bodies have placed requirements and legislative frameworks, which enable them to implement residue monitoring test endeavors that safeguard the public and facilitate the trading activity. To make the tests on the animal-origin food matrix, there are different sample extraction techniques such as accelerated solvent extraction, supercritical fluid extraction, solid phase extraction, solid-phase microextraction, and hollow-fiber liquid-phase microextraction. After sample preparation steps, the analytes of interest can be assayed by screening and confirmatory methods of analysis. For screening, immunological tests such as ELISA and radioimmunoassay are used. Detection and determination of the specific residues will be done by chromatographic or instrumental analysis. Mainly, among high-performance liquid chromatography, liquid chromatography with mass spectrometry (LC-MS, LC-MS/MS), and gas chromatography with mass spectrometry (GC-MS and GC-MS/MS) methods, LC-MS/MS is being preferred because of easier sample preparation without a derivatization step and high detection and quantification capacity.
Evaluation of estrogenic activity in animal diets using in vitro assay
Toxicology in Vitro, 2014
A yeast estrogen bioassay (RIKILT REA) was in-house validated for feed on the 5 lg 17b-estradiol-equivalents per kg level according to EC Decision 2002/657/EC. All the performance characteristics met the criteria as defined in the Decision and the REA is able to detect 17b-estradiol in animal feed at a low level of 1.15-2 lg kg À1. Subsequently, the developed and validated procedure was applied to determine the estrogenic activity in 24 feed samples intended for food producing animals, pets and laboratory animals. Two batches of rodent diet Murigran and one dog feed have been presented as a suspect, i.e. gave responses above the determined decision limit (CCa) and detection capability (CCb). In assessing the performance of the estrogenic activity in these diets evaluated by comparison with the 17b-estradiol calibration curve, 17b-estradiol-equivalence levels of 7.07 lg EEQ kg À1 and 9.54 lg EEQ kg À1 in two batches of rodent diet and 5.3 lg EEQ kg À1 in dog feed have been established. The activities observed in the rodent feed could be explained by chemical analysis, revealing high amounts of genistein, daidzein and trace amounts of zearalenone. In addition, the estrogenic activity in one of rodent feed was above the established CCa, but below the CCb values established and all other samples showed no estrogenic activity with responses below the CCa value, which corresponds to levels below 2 lg EEQ kg À1 .
Journal of Chromatography B, 2009
We have developed a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P 5 ), progesterone (P 4 ), 17hydroxyP 5 , 17hydroxyP 4 , testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E 1 ), 2, 4 and 16 hydroxyE 1 , 2 and 4 methoxyE 1 ]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.