Author's personal copy Analytica Chimica Acta 638 (2009) 106–113 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Identification of potential gene expression biomarkers for the surveillance of anabolic agents in bovine blood cells Irmgard Riedmaier ∗ , Ales Tichopad, Martina Reiter, Michael W. Pfaffl, Heinrich H.D. Meyer Physiology Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising, Germany article info Article history: Received 21 November 2008 Received in revised form 9 February 2009 Accepted 9 February 2009 Available online 20 February 2009 Keywords: Anabolic agents Trenbolone acetate Estradiol Biomarker Gene expression Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) Principal components analysis abstract In the EU, the use of anabolic steroids in food producing animals has been forbidden since 1988. The routine methods used in practice are based on the detection of hormonal residues. To overcome these routine methods, growth-promoting agents are sometimes administered at concentrations below the detection limit and new anabolic substances are designed. Therefore, new monitoring systems are needed to overcome the misuse of anabolic agents in meat production. In this study, a new monitoring system was applied: the quantification of mRNA gene expression changes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Blood was selected as ideal tissue for biomarker screening. From the literature, it is known that steroid hormones affect mRNA gene expression of the different blood cells, which can easily be taken from the living animal. In an animal trial, 18 Nguni heifers were separated to two groups of nine animals. One group served as untreated control and the other group was treated with a combination of trenbolone acetate plus estradiol for 39 days in order to allow the detection of the effect on mRNA expression in blood at three time points. Candidate genes used for developing a biomarker pattern were chosen by screening the actual literature for anabolic effects on blood cells. It could be demonstrated that the combination of trenbolone acetate plus estradiol significantly influences mRNA expression of the steroid receptors (ER- and GR-), the apotosis regulator Fas, the proinflammatory interleukins IL-1, IL-1 and IL-6 and of MHCII, CK, MTPN, RBM5 and Actin-. Advanced statistical analysis by Principal Components Analysis (PCA) indicated that these genes represent potential biomarkers for this hormone combination in whole blood. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Growth-promoting agents like anabolic steroids or -agonists are used in meat producing animals to improve weight gain and feed efficiency in order to increase the productivity and to reduce production costs [13,20]. Due to adverse effects of hormone residues for the consumer [4,27] the use of anabolic hormones for growth promotion is forbidden in the European Union since 1988 under Directive 88/146/EEC. Routine methods like immuno assays, either radio immuno assay (RIA) or enzyme immuno assay (EIA), and chromatographical methods combined with mass spectrom- etry are used to detect hormone residues [18,19,26,29]. To avoid detection of residues during routine control, growth-promoting agents are often administered in cocktails with such low amounts per agent that residues are below the detection limit [1]. Alterna- tively, new compounds, not yet included in testing programs, are ∗ Corresponding author. Tel.: +49 8161 715552; fax: +49 8161 714204. E-mail address: irmgard.riedmaier@wzw.tum.de (I. Riedmaier). used. Therefore, it is necessary to develop new monitoring systems to detect a broad range of agents at the lowest concentration that is used to get a growth-promoting effect. A potential way to develop a new monitoring system is to find gene expression biomarkers for the illegal use of anabolic steroids [23,24,28]. It is well known that steroid hormones influence biochemical pathways of different organs and tissues. mRNA expression of hor- mone dependent genes can be activated or suppressed. Using appropriate specific and sensitive quantification methods, like quantitative real time reverse transcription polymerase chain reaction (qRT-PCR), such mRNA expression changes are measurable at very low levels. From the literature it is known that sex steroid hormones show physiological effects on the different blood cells [3,9,16]. The aim of this pilot study was to monitor the effects of a commercially available combination of trenbolone acetate plus estradiol on mRNA expression of selected target genes in bovine whole blood and to perform a bioinformatic evaluation in order to find potential biomarkers for the effective surveillance of this hormone combination. 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.02.014