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In vitro metabolism of testosterone in the horse liver and involvement of equine CYP s 3 A 89, 3 A 94 and 3 A 95
Author(s) -
Schmitz A.,
Zielinski J.,
Dick B.,
Mevissen M.
Publication year - 2014
Publication title -
journal of veterinary pharmacology and therapeutics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.527
H-Index - 60
eISSN - 1365-2885
pISSN - 0140-7783
DOI - 10.1111/jvp.12106
Subject(s) - microsome , ketoconazole , cyp3a , metabolite , cytochrome p450 , cyp3a4 , horse , metabolism , biology , hydroxylation , biochemistry , drug metabolism , testosterone (patch) , chemistry , in vitro , pharmacology , enzyme , endocrinology , microbiology and biotechnology , paleontology , antifungal
Testosterone ( TES ) 6‐ β ‐hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 ( CYP ) 3 A 4/5 specific metabolic activity. Several CYP 3 A 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 CYP 3 A 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 CYP s involved in TES biotransformation in equine microsomes. Single CYP s 3 A 89, 3 A 94, and 3 A 95, recombinantly expressed in V 79 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 CYP s. 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 CYP 3A95 catalyzed 11‐ β ‐hydroxylation of testosterone ( TES ). Metabolism of BFC was significantly inhibited by ketoconazole in CYP 3 A 95, whereas troleandomycin affected the activities of CYP 3 A 94 and CYP 3 A 95. Both inhibitors had no significant effect on CYP 3 A 89. Metabolic reactions and effects of inhibitors differed between the equine CYP 3 A isoforms investigated. This has to be considered in future in vitro studies.