Human Efflux Transport of Testosterone, Epitestosterone and Other Androgen Glucuronides

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low K-m values, between 0.4 and 4 mu M, while the K-m values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 pM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by K-m values over 100 mu M. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.Peer reviewe

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E-1-G), estradiol-3- and 17-glucuronides (E-2-3G and E-2-17G), as well as estriol-3- and 16-glucuronides (E-3-3G and E-3-16G) are found in human plasma and urine. Unlike E-2-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E-1-G, E-2-3G, E-3-3G, E-3-16G and estrone-3-sulfate (E-1-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E-1-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E-1-G and E-2-3G, were still transported by BCRP at 10-fold higher rates than E-1-S. BCRP also transported E-3-16G at higher rates than the studied MRPs, while it transported E-3-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E-1-G, E-2-3G, E-3-3G and E-3-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K-m values, between 180 and 790 mu M. MRP3 transported all the tested glucuronides at rather similar rates, at K-m values below 20 mu M, but lower V-max values than other transporters. In the case of E-3-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E-3-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.Peer reviewe

Efflux transport of nicotine, cotinine and trans-3 '-hydroxycotinine glucuronides by human hepatic transporters

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3 '-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K-m value of 895 mu mol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 mu mol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.Peer reviewe

Optical substrates for drug-metabolizing enzymes : Recent advances and future perspectives

Drug-metabolizing enzymes (DMEs), a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics, have been recognized as the critical determinants to drug safety and efficacy. Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity, as well as characterizing the interactions of central DMEs with xenobiotics require reliable, practical and highly specific tools for sensing the activities of these enzymes in biological systems. In the last few decades, the scientists have developed a variety of optical substrates for sensing human DMEs, parts of them have been successfully used for studying target enzyme(s) in tissue preparations and living systems. Herein, molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically. Furthermore, the challenges and future perspectives in this field are also highlighted. The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems, which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb-drug interactions, as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes. (C) 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.Peer reviewe

Data on biosynthesis of BPAF glucuronide, enzyme kinetics of BPAF glucuronidation, and molecular modeling

Bisphenol AF (BPAF) is in the body mainly metabolized to the corresponding bisphenol AF glucuronide (BPAF-G). While BPAF-G is not commercially available, enzyme-assisted synthesis of BPAF-G using the human recombinant enzyme UGT2A1, purification of BPAF-G by solid phase extraction and semi-preparative HPLC and chemical characterization of BPAF-G by NMR and LC-MS/MS were performed and are described here. Furthermore, BPAF glucuronidation kinetics with the UGT enzymes that showed the highest glucuronidation activity in previous studies (i.e hepatic UGTs 1A3, 2B7, and 2B17, intestinal UGT 1A10 and UGT 2A1 that is present in airways) was performed and data is presented. Hepatic enzymes exhibited high affinities toward BPAF, while extrahepatic UGTs 2A1 and 1A10 showed the high v(max), values (3.3 and 3.0 nmol/min/mg, respectively). To understand molecular interactions of BPA, BPAF and BPAF-G with ligand biding sites of several nuclear receptors, molecular modeling was performed and data on the binding modes of BPAF, BPA, and BPAF-G in the ligand-binding sites of nuclear receptors are presented. This article is related to "Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite" (Skledar et al., 2019). (C) 2018 The Authors. Published by Elsevier Inc.Peer reviewe

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p <0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues. (C) 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.Peer reviewe

A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drugiherb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived K-m values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors. (C) 2021 Elsevier B.V. All rights reserved.Peer reviewe

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.Peer reviewe

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects are Enzyme and Substrate Dependent

Peer reviewe

Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite

Bisphenol AF (BPAF) is a fluorinated analog of bisphenol A (BPA), and it is a more potent estrogen receptor (ER) agonist. BPAF is mainly metabolized to BPAF-glucuronide (BPAF-G), which has been reported to lack ER agonist activity and is believed to be biologically inactive. The main goal of the current study was to examine the influence of the metabolism of BPAF via glucuronidation on its ER activity and adipogenesis. Also, as metabolites can have different biological activities, the effects of BPAF-G on other nuclear receptors were evaluated. First, in-vitro BPAF glucuronidation was investigated using recombinant human enzymes. Specific reporter-gene assays were used to determine BPAF and BPAF-G effects on estrogen, androgen, glucocorticoid, and thyroid receptor pathways, and on PXR, FXR, and PPAR gamma pathways. Their effects on lipid accumulation and differentiation were determined in murine 3T3L1 preadipocytes using Nile Red, with mRNA expression analysis of the adipogenic markers adiponectin, Fabp4, Cebp alpha, and PPAR gamma. BPAF showed strong agonistic activity for hER alpha and moderate antagonistic activities for androgen and thyroid receptors, and for PXR. BPAF-G was antagonistic for PXR and PPAR gamma. BPAF (0.1 mu M) and BPAF-G (1.0 mu M) induced lipid accumulation and increased expression of key adipogenic markers in murine preadipocytes. BPAF-G is therefore not an inactive metabolite of BPAF. Further toxicological and epidemiological investigations of BPAF effects on human health are warranted, to provide better understanding of the metabolic end-elimination of BPAF. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe