In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95.

This research investigates how testosterone is metabolized in the horse liver and which cytochrome P450 enzymes participate in this process. The findings revealed that specific CYP3A isoforms in horses play different roles in testosterone metabolism, with differences in their response to certain inhibitors.

Research Purpose and Approach

• The objective of this study was to explore the metabolism of testosterone (TES) in the horse liver and to find out which equine CYP3A enzymes are involved.
• The researchers used in vitro methods, involving equine hepatocyte primary cultures and liver microsomes, and applied chemical inhibitors to determine the enzymes involved in TES metabolism in horses.
• The researchers also looked at three specific CYP3A isoforms (3A89, 3A94, 3A95), which were expressed in V79 hamster lung fibroblasts, then incubated with TES and a fluorescent metabolite (7-benzyloxy-4-trifluoromethylcoumarin or BFC) to understand their roles and responses to inhibitors.

Results and Discoveries

• The most prevalent metabolite in hepatocyte primary cultures was found to be androstenedione (AS), while 6-β-hydroxytestosterone was the most present in liver microsomes.
• Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was hindered by the enzymes inhibitors ketoconazole, troleandomycin, and quercetin.
• Recombinant CYP3A95 was discovered to catalyze the 11-β-hydroxylation of TES, while metabolism of BFC was significantly impeded by ketoconazole in CYP3A95.
• Troleandomycin affected the activities of CYP3A94 and CYP3A95 but not CYP3A89, showing that the metabolic reactions and effects of the inhibitors vary between the equine CYP3A isoforms.

Implications of the Research

• The data presents novel insights into horse liver metabolism of testosterone and the significant role of specific CYP3A isoforms.
• The differing responses to inhibitors amongst the isoforms demonstrate that each plays a unique function in the biotransformation process and should be considered in future in vitro studies.
• This research builds a foundation for understanding drug metabolism in horses and can be essential for the development of pharmacological treatments and management of performance in equine health and sports medicine.