Development of a dual luciferase reporter screening assay for the detection of synthetic glucocorticoids in animal tissues

Abstract

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 dual luciferase reporter assay that detects residues or contaminants with glucocorticoid activity. This screening assay is performed by transfection of human cell lines with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for assay variabilities and unspecific matrix effects. The limit of detection (1.25 microg for dexamethasone in liver) depends on the biological potency of each synthetic glucocorticoid but, with all drugs tested, the maximal response reaches a 20 to 30 fold induction of luciferase activity. In combination with an appropriate sample clean-up method (recovery of 82%), this luciferase assay has been applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual luciferase reporter assay provides a new screening tool to detect unwanted glucocorticoid activities in animal tissues or other crude biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.

Effect-based detection of synthetic glucocorticoids in bovine urine

“…The endogenous hormone cortisol showed a low and comparable activity in U2OS, T47D and HeLa cells. Among synthetic molecules, flumethasone proved to be one of the most active molecules in all the RGAs evaluated, methylprednisolone and prednisolone exhibited a weak glucocorticoid activity in U2OS, T47D and HeLa cells, even if GR-CALUX demonstrated to be the most suitable assay for the detection of these feeble GR agonists (Willemsen et al 2002;Schumacher et al 2003;Connolly et al 2009). …”

“…A wide variety of RGAs for glucocorticoid detection have been described (Willemsen et al 2002(Willemsen et al , 2005Schumacher et al 2003;Connolly et al 2009;Bovee et al 2011), but only two assays are commercially available to date: the yeast glucocorticoid biosensor and the chemical-activated luciferase gene expression (CALUX) in vitro bioassay based on human bone cells (U2OS). Of the two, only GR-CALUX is suited for sample analysis requiring a low sensitivity (Bovee et al 2011(Bovee et al , 2012.…”

“…Among these innovative methods are assays employing indirect biomarkers, like receptors (Odore et al 2006), metabolites (Courant et al 2009), proteins (Gardini et al 2006) or gene expression (Toffolatti et al 2006;Reiter et al 2007), and in vitro system for effect-based detection of the analytes, like histopathology-based methods Cannizzo et al 2011;Vascellari et al 2012) and reporter gene assays (RGAs) (Willemsen et al 2002(Willemsen et al , 2005Schumacher et al 2003;Connolly et al 2009;Bovee et al 2011). RGAs based on stably transfected cell lines may provide one of the most specific, responsive and biologically relevant means to screen biological samples for hormone activity, with a high throughput potential (Bovee et al 2011(Bovee et al , 2012.…”

Simultaneous Determination of Three Synthetic Glucocorticoids by Differential Pulse Stripping Voltammetry with the Aid of Chemometrics

“…The therapeutic indications of glucocorticoid compounds include inflammatory diseases, disorders of the musculoskeletal system, and skin disease (Schimmer and Parker 1996;Schumacher et al 2003). Synthetic glucocorticoids are frequently administered to animals in livestock production because of their inhibition of inflammatory, allergic, and immunological responses.…”

Microwave-assisted enzymatic hydrolysis followed by extraction with restricted access nanocomposites for rapid analysis of glucocorticoids residues in liver tissue

“…Glucocorticoids ( Table 1) are medications that have been used in livestock production due to their pharmacological activities such as inhibition of inflammatory, allergic and immunological responses [1][2][3]. However, it has been observed that overdosed glucocorticoids are illegally added in animal fodder, aiming to improve feed intake and weight-gain [4].…”