Relevance of Human Aldoketoreductases and Microbial /3-Glucuronidases in Testosterone DispositionS

Testosterone exhibits high variability in pharmacokinetics and glucuro-nidation after oral administration. Although testosterone metabolism has been studied for decades, the impact of UGT2B17 gene deletion and the role of gut bacterial /3-glucuronidases on its disposition are not well characterized. We first performed an exploratory study to investi-gate the effect of UGT2B17 gene deletion on the global liver proteome, which revealed significant increases in proteins from multiple biological pathways. The most upregulated liver proteins were aldoketoreduc-tases [AKR1D1, AKR1C4, AKR7A3, AKR1A1, and 7-dehydrocholesterol reductase (DHCR7)] and alcohol or aldehyde dehydrogenases (ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A). In vitro assays revealed that AKR1D1 and AKR1C4 inactivate testosterone to 5/3-dihydrotestos-terone (5/3-DHT) and 3a,5/3-tetrahydrotestosterone (3a,5/3-THT), re-spectively. These metabolites also appeared in human hepatocytes treated with testosterone and in human serum collected after oral tes-tosterone dosing in men. Our data also suggest that 5/3-DHT and 3a, 5/3-THT are then eliminated through glucuronidation by UGT2B7 in UGT2B17 deletion individuals. Second, we evaluated the potential reactivation of testosterone glucuronide (TG) after its secretion into the intestinal lumen. Incubation of TG with purified gut microbial /3-glucuronidase enzymes and with human fecal extracts confirmed tes-tosterone reactivation into testosterone by gut bacterial enzymes. Both testosterone metabolic switching and variable testosterone activation by gut microbial enzymes are important mechanisms for explaining the disposition of orally administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-associated pathophysiological conditions. SIGNIFICANCE STATEMENT This study investigated the association of UGT2B17 gene deletion and gut bacterial /3-glucuronidases with testosterone disposition in vitro. The experiments revealed upregulation of AKR1D1 and AKR1C4 in UGT2B17 deletion individuals, and the role of these en-zymes to inactivate testosterone to 5/3-dihydrotestosterone and 3a, 5/3-tetrahydrotestosterone, respectively. Key gut bacterial spe-cies responsible for testosterone glucuronide activation were iden-tified. These data are important for explaining the disposition of exogenously administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-asso-ciated pathophysiological conditions.