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A reporter gene-based bioassay to replace animal testing for measuring the pharmacological potency of glucocorticoids and their metabolites or derivatives

Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed.

To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a luciferase reporter assay that detects a wide range of residues or contaminants with glucocorticoid activity.

This new screening assay is performed by transfection of human cells with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for unspecific matrix effects. The limit of detection in the assay medium ranges from 0.2nM for flumethasone to 3nM for methylprednisolone, while the maximal response reaches a 20- to 30-fold induction of luciferase activity. In combination with an appropriate sample clean-up method, this new assay has been successfully applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual reporter assay provides a sensitive, specific, highly responsive and robust screening tool to detect unwanted glucocorticoid activities in biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.

The methodology has been presented to representatives of the National Reference Laboratories at a practical workshop. The method is being presented at various International Congresses. Part of the methodology has already been published (van den Hauwe et al., J. Agric. Food Chem., Vol. 51, pp. 326-330, 2003). A second manuscript focussed on the reporter assay will be submitted to an International refereed Journal. All vectors and cell lines will be made available to interested laboratories.

The glucocorticoid reporter assay provides an alternative screening tool for residue analysis and, therefore, the initial objectives were met.

Reported by

University of Zurich
August Forel-Strasse
8008 Zürich
Switzerland
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