Organic Anion Transporting Polypeptide-Mediated Hepatic Uptake of Glucuronide Metabolites of Androgens

We previously established that androgen glucuronides are effluxed by multidrug resistance-associated proteins 2 and 3. However, no data exist on the mechanism of hepatic uptake of these metabolites. The first goal of this study was to explore the role of hepatic uptake transporters and characterize transport kinetics of glucuronides of testosterone (TG), dihydrotestosterone (DHTG), androsterone (AG), and etiocholanolone (EtioG) using cell lines overexpressing organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1). Using a quantitative proteomics-guided approach, we then estimated the fractional contribution of individual OATPs in hepatic uptake of these glucuronides. The transport screening assays revealed that the glucuronides were primarily taken up by OATP1B1 and OATP1B3. The K _m values for OATP1B1-mediated uptake were low for EtioG (6.2 µM) as compared with AG, TG, and DHTG (46.2, 56.7, and 71.3 µM, respectively), whereas the K _m value for OATP1B3-mediated uptake for EtioG, AG, DHTG, and TG were 19.8, 29.3, 69.6, and 110.4 µM, respectively. Both OATP1B1 and OATP1B3 exhibited the highest transport rate toward AG as compared with other glucuronides. When adjusted for the transporter abundance in human livers, EtioG and DHTG were predicted to be transported by both OATP1B1 and OATP1B3, whereas TG and AG were preferentially (>68%) transported by OATP1B3. Collectively, this report elucidates the mechanisms of hepatic uptake of androgen glucuronides. Perturbation of these processes by genetic polymorphisms, disease conditions, or drug interactions can lead to changes in enterohepatic recycling of androgens. TG and AG can be further investigated as potential biomarkers of OATP1B3 inhibition. SIGNIFICANCE STATEMENT: This is the first study to elucidate the mechanism of hepatic uptake of androgen glucuronides and estimate the fractional contribution of individual OATPs using quantitative proteomics. Our results show that both OATP1B1 and OATP1B3 are responsible for the hepatic uptake of major circulating testosterone glucuronides. The apparent higher selectivity of OATP1B3 toward testosterone glucuronide and androsterone glucuronide can be leveraged for establishing these metabolites as clinical biomarkers of OATP1B3 activity.