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

Abstract

Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.

Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences

“…The stock solutions for testosterone and the expected metabolites, 6b-hydroxy-testosterone (Lipomed, Arlesheim, Switzerland), 11b-hydroxy-testosterone [kind gift from Dr. B. Dick (Inselspital, Bern, Switzerland)] and androstenedione (Sigma, Buchs, Switzerland) were prepared in methanol (HPLC grade, Sigma, Buchs, Switzerland) with the final methanol concentration not exceeding 0.1% (v/v) as described previously (Schmitz et al, 2014). Solvent controls were performed in parallel.…”

“…Inhibitor concentrations were chosen based on literature and previous work done in our laboratory (Mossner et al, 2011;Schmitz et al, 2014). The compound, 17-spiro-d-lactone-E2 (EM-919), [a generous gift from Dr. D. Poirier (CHU de Québec and Université Laval, Canada)], an enzyme inhibitor that has been demonstrated to inhibit formation of testosterone from androstenedione in human embryonic kidney cells (HEK293) was used (Bydal et al, 2004); 5 different EM-919 concentrations (5 lM; 10 lM; 25 lM; 50 lM and 100 lM) were investigated.…”

“…Testosterone metabolites were analyzed as described previously (Schmitz et al, 2014). Briefly, testosterone and metabolites were analyzed by HPLC at 254 nm UV detection on an EC 125/4 Lichrospher 100-5 RP-8 column (Machery-Nagel; # 720742.40).…”

“…The 6b-hydroxylation has also been used to investigate potential CYP3A-mediated activity in liver microsomes of other species including dogs and horses (Chauret et al, 1997;Schmitz et al, 2014;Shou et al, 2003). CYP3A96 has been reported to be involved in testosterone 6b-hydroxylation in horses (Knych et al, 2010).…”

“…Some inhibition studies using canine microsomes or single CYPs confirmed that some of these inhibitors can be used in canine species while inhibition studies using equine liver microsomes are still rare (Chauret et al, 1997;Mossner et al, 2011;Peters et al, 2013;Schmitz et al, 2014;van Beusekom et al, 2010).…”