Is zebrafish (Danio rerio) water tank model applicable for the assessment of glucocorticoids metabolism? The budesonide assessment

Knowledge of the metabolic profile is essential for doping control analysis in sport since most drugs are excreted after an elaborate biotransformation process. Currently, Zebrafish Water Tank (ZWT) model has been applied to investigate the metabolism of different doping agents. Nevertheless, the class of glucocorticoids has not been subjected to this model for metabolism studies. In the present work, budesonide (BUD) was applied as a pilot to investigate the metabolic pathways of glucocorticoids in the ZWT model. The BUD biotransformation in ZWT model was compared to the described metabolism in humans. Samples from ZWT experiments were collected after BUD administration and analyzed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS). Following the identification and characterization of all significant metabolites described for humans, it was observed that the ZWT was able to produce in a relevant amount the main target for doping control purposes: the 6β-hydroxy BUD. In addition, prior knowledge about the lack of butyrylcholinesterase activity in the zebrafish organism was considered for the evaluation for the formation of the 16α-hydroxy prednisolone, the most intense BUD metabolite in human urine. Biotransformation of BUD by ZWT focused on metabolites with the acetal fraction preserved, including the intermediate metabolite for the 16α-hydroxy prednisolone pathway. However, an alternative metabolic pathway for the complete biotransformation of the 16α-hydroxy prednisolone intermediate was not observed, leading to the absence of the major human metabolite in the ZWT model. The findings reported in this study elucidate for the first time the application and limitations of the ZWT model to evaluate the metabolism of other glucocorticoids.