Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Abstract Testosterone glucuronide(TG), androsterone glucuronide(AG), etiocholanolone glucuronide(EtioG) and dihydrotestosterone glucuronide(DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronidescan be deconjugated to active androgenin gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronidesthan MRP2. We next quantified the protein abundances of these effluxtransporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (f t ) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′- nucleotidaseassay). The glucuronidesof inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronidesof active androgens, TG and DHTG were mainly transported by MRP2 in liver. Effluxby bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgensafter deconjugation in the gut. In intestine, MRP3 possibly contributes most to the effluxof these glucuronides. In kidney, all studied glucuronidesseemed to be preferentially effluxedby MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the effluxmechanisms of T glucuronidemetabolites is important for predicting the androgendisposition and interindividual variability, including drug- androgeninteraction in humans. The mechanistic data can be extrapolated to other androgenrelevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.