Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney
Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney
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DOI：
10.1016/j.jsbmb.2019.03.027
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发表时间：
2019-07-01
影响因子：
4.1
通讯作者：
Prasad, Bhagwat
中科院分区：
文献类型：
--
作者：
Li, Cindy Yanfei;Basit, Abdul;Prasad, Bhagwat
睾酮（T）的主要代谢产物为睾酮葡萄糖醛酸苷（TG）、雄酮葡萄糖醛酸苷（AG）、胆甾烷酮葡萄糖醛酸苷（EtioG）和二氢睾酮葡萄糖醛酸苷（DHTG），经尿液和胆汁排泄。胆汁排泄后，葡萄糖醛酸苷在肠腔中可与活性雄激素解结合，进而影响雄激素的生理水平。本研究的目的是利用相对表达因子（REF）方法定量表征TG、AG、EtioG和DHTG从肝、肠和肾中消除的机制。使用重组人MRP 2、MRP 3、MRP 4、MDR 1和BCRP的囊泡转运试验，我们首先确定TG、AG、EtioG和DHTG是MRP 2和MRP 3的主要底物，尽管在MDR 1和BCRP囊泡中也观察到较低水平的转运。转运动力学分析显示，与DHTG、AG和EtioG相比，MRP 2和MRP 3对TG的固有清除率更高。MRP 3对所研究的葡萄糖醛酸苷的转运表现出比MRP 2更高的亲和力。接下来，我们定量了囊泡中这些外排转运蛋白的蛋白丰度，并通过定量LC-MS/MS蛋白质组学将其与从人体组织中分离的合并总膜组分进行比较。通过基于蛋白质组学的生理缩放因子估计单个转运蛋白的贡献分数（f（t）），即，整个组织中转运蛋白丰度与囊泡，并针对由内而外的囊泡进行校正（通过5 '-核苷酸酶测定法测定）。在肝脏中，非活性雄激素的葡萄糖醛酸苷、AG和EtioG优先由MRP 3转运，而活性雄激素的葡萄糖醛酸苷、TG和DHTG主要由MRP 2转运。通过胆小管转运的流出可能表明肠肝再循环在调节肠道中去结合后循环活性雄激素中的潜在作用。在肠道中，MRP 3可能对这些葡萄糖醛酸苷的外排贡献最大。在肾脏中，所有研究的葡萄糖醛酸苷似乎都优先被MRP 2和MDR 1（对于EtioG）排出。这些基于REF的分析需要通过体内结果进行确认。总体而言，T葡糖苷酸代谢产物外排机制的表征对于预测雄激素分布和个体间变异性（包括人体中的药物-雄激素相互作用）非常重要。通过将这些数据与定量组织蛋白质组学数据整合，可以将机制数据外推到其他雄激素相关器官（例如前列腺、睾丸和胎盘）。
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. Glucuronides can be deconjugated to active androgen in 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 glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters 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'-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.