Estradiol Glucuronide Research Articles

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis

The endogenous metabolite of estradiol, estradiol 17β-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.

Kinetic Characterization of Estradiol Glucuronidation by Liver Microsomes and Expressed UGT Enzymes: The Effects of Organic Solvents.

In vitro glucuronidation of 17β-estradiol (estradiol) is often performed to assess the role of uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) in xenobiotic/drug metabolism. The objective of this study was to determine the effects of four commonly used organic solvents [i.e., dimethyl sulfoxide (DMSO), methanol, ethanol, and acetonitrile] on the glucuronidation kinetics of estradiol, which can be glucuronidated at C3 and C17 positions. The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. In human liver microsomes (HLM), methanol, ethanol, and acetonitrile significantly altered estradiol glucuronidation kinetics with increased Vmax (up to 2.6-fold) and CLmax (up to 2.8-fold) values. Altered estradiol glucuronidation in HLM was deduced to be attributed to the enhanced metabolic activities of UGT1A1 and UGT2B7, whose activities differ at the two glucuronidation positions. The effects of organic solvents on estradiol glucuronidation were glucuronidation position-, isozyme-, and solvent-specific. Furthermore, both ethanol and acetonitrile have a greater tendency to modify the glucuronidation activity of estradiol in animal liver microsomes. Organic solvents such as methanol, ethanol, and acetonitrile showed great potential in adjusting the glucuronidation of estradiol. DMSO is the most suitable solvent due to its minimal influence on estradiol glucuronidation. Researchers should be cautious in selecting appropriate solvents to get accurate results when assessing the metabolism of a new chemical entity.

Can Cannabinoids Precipitate UGT‐mediated Drug Interactions?

Background: Cannabidiol (CBD) and tetrahydrocannabinol (THC) are cannabinoids used predominately for medicinal and recreational purposes, respectively. Because these cannabinoids are often co-consumed with conventional drugs, there is concern for deleterious cannabinoid-drug interactions. Both cannabinoids and their major circulating metabolites (7-OH CBD or 11-OH THC) are potent inhibitors of CYPs, but their potency to inhibit UGTs has not been investigated. Objective The objective of the study was to predict the potential for CBD and THC to precipitate UGT-mediated drug interactions using an IVIVE approach. The aims were to determine the following for CBD, THC, and their metabolites: 1) fraction unbound in incubations (fu,inc) containing recombinant UGT (rUGTs) and 0.2% BSA and 2) unbound UGT inhibitory potency (IC50,u). These data were used to predict the potential for interactions between each cannabinoid and UGT-metabolized drugs after typical doses used orally or by inhalation. Methods Fraction unbound (fu,inc) of CBD, THC, or metabolite in incubations containing rUGTs and 0.2% BSA was determined by ultracentrifugation. The inhibition extent (at 10 μM) or potency (IC50,u) of CBD, 7-OH CBD, 7-COOH CBD, THC, 11-OH THC, or 11-COOH THC towards the glucuronidation of estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-OH indole (UGT1A6), propofol (UGT1A9), gemfibrozil (UGT2B4), naloxone (UGT2B7), amitriptyline (UGT2B10), oxazepam (UGT2B15), or testosterone (UGT2B17) was determined using rUGTs. Estimated IC50,u and unbound hepatic inlet (oral dose) or systemic plasma (inhaled dose) concentrations were used to predict the potential for interactions between CBD (700 mg) or THC (20 mg) and drugs metabolized by UGT with fm values of 0.25, 0.5, or 0.75. Results fu,inc of CBD, 7-OH CBD, and THC was 0.05, 0.20, and 0.04, respectively. CBD, 7-OH CBD, THC, and 11-OH THC at 10 μM inhibited all UGTs tested (by 30-98%), whereas 7-COOH CBD inhibited only UGT2B7 (by 25%) and 11-COOH THC inhibited only UGT1A1, 1A4, and 1A9 (by 20-35%). IC50,u of CBD, 7-OH CBD, and THC against UGT1A9, 2B4, and 2B7 ranged from 0.002-0.02 μM, 0.6-1.6 μM, and 0.07-0.13 μM, respectively. IC50,u of CBD against UGT1A4, 1A6, 2B10, and 2B15 was < 1.3 μM. A mechanistic static model predicted strong in vivo interactions (AUC ratio > 3.3) between CBD and drugs extensively metabolized (fm = 0.75) by UGT1A9, 2B4, or 2B7. CBD was also predicted to result in moderate or weak interactions (1.3 ≥ AUC ratio ≤ 2.0) with drugs not extensively metabolized by these UGTs (fm = 0.25 or 0.5). In contrast, oral or inhaled THC was not predicted to precipitate UGT-mediated drug interactions (AUC ratio < 1.2). UGT inhibition by 7-OH CBD did not change the predicted potential of CBD-drug interactions. Conclusion This study is the first to determine the UGT inhibition potencies of cannabinoids. CBD was a potent inhibitor of UGT1A9, 2B4, and 2B7 and was predicted to interact with drugs metabolized by these UGTs. Further investigation via dynamic PBPK modeling and simulation and clinical evaluation is warranted to verify these predictions.

Conserved amino acids in the region connecting membrane spanning domain 1 to nucleotide binding domain 1 are essential for expression of the MRP1 (ABCC1) transporter.

Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C4. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser612, Arg615, His622, Glu624) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg615 and Glu624 might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe619 and distal Lys406 in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.

An emerging powerful technique for distinguishing isomers: Trapped ion mobility spectrometry time-of-flight mass spectrometry for rapid characterization of estrogen isomers.

Isomer metabolites are involved in metabolic pathways, and their characterization is essential but remains challenging even using high-performance analytical platforms. The addition of ion mobility prior to mass analysis can help to separate isomers. Here, the ability of a recently developed trapped ion mobility spectrometry system to separate metabolite isomers was examined. Three pairs of estrogen isomers were studied as a model of isomeric metabolites under both negative and positive electrospray ionization (ESI) modes using a commercial trapped ion mobility spectrometry-TOF mass spectrometer. The standard metabolites were also spiked into human urine to evaluate the efficiency of trapped ion mobility spectrometry to separate isomers in complex mixtures. The estradiol glucuronide isomers (E2 β-3G and E2 β-17G) could be distinguished as deprotonated species, while the estradiol epimers (E2 β and E2 α) and the methoxyestradiol isomers (2-MeO-E2 β and 4-MeO-E2 β) were separated as lithiated adducts in positive ionization mode. When performing analyses in the urine matrix, no alteration in the ion mobility resolving power was observed and the measured collision cross section (CCS) values varied by less than 1.0%. The trapped ion mobility spectrometry-TOF mass spectrometer enabled the separation of the metabolite isomers with very small differences in CCS values (ΔCCS% = 2%). It is shown to be an effective tool for the rapid characterization of isomers in complex matrices.

Hepatic bile formation: bile acid transport and water flow into the canalicular conduit.

Advances in molecular biology identifying the many carrier-mediated organic anion transporters and advances in microscopy that have provided a more detailed anatomy of the canalicular conduit make updating the concept of osmotically determined canalicular flow possible. For the most part water flow is not transmembrane but via specific pore proteins in both the hepatocyte and the tight junction. These pores independently regulate the rate at which water flows in response to an osmotic gradient and therefore are determinants of canalicular bile acid concentration. Review of the literature indicates that the initial effect on hepatic bile flow of cholestatic agents such as Thorazine and estradiol 17β-glucuronide are on water flow and not bile salt export pump-mediated bile acid transport and thus provides new approaches to the pathogenesis of drug-induced liver injury. Attaining a micellar concentration of bile acids in the canaliculus is essential to the formation of cholesterol-lecithin vesicles, which mostly occur in the periportal region of the canalicular conduit. The other regions, midcentral and pericentral, may transport lesser amounts of bile acid but augment water flow. Broadening the concept of how hepatic bile flow is initiated, provides new insights into the pathogenesis of canalicular cholestasis.

Human Liver Microsomes Immobilized on Magnetizable Beads: A Novel Approach to Study In Vitro Drug Metabolism.

Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CLint) of CYP450 substrates was comparable; however, CLint of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.

Simultaneous UHPLC-MS/MS method of estradiol metabolites to support the evaluation of Phase-2 metabolic activity of induced pluripotent stem cell derived hepatocytes

The goal of this study was to develop and validate a high-throughput UHPLC-MS/MS method for simultaneous quantitation of three estradiol metabolites namely estradiol 3-β-D-glucuronide (E3G), estradiol 17-β-D-glucuronide (E17G) and estradiol 3-sulfate (E3S) in cell culture medium to support the characterization of metabolic function of induced pluripotent stem cell (iPSC) derived hepatocytes. To achieve this goal, a simple protein precipitation method was used for sample cleanup. All the metabolites were separated chromatographically using a C-18 column where 10 mM ammonium acetate and acetonitrile was used in gradient flow for 4 min. The analytes were quantitated by triple quadrupole mass spectrometer with the use of isotopically labeled internal standard (IS). This method was validated as per the U.S Food and Drug Administration's Bioanalytical Method Validation, Guidance for Industry. Linearity for E3G and E17G was in the range of 2–1500 ng/mL whereas for E3S it was 0.3–500 ng/mL. Inter-day and intra-day accuracy and precision of this method were in the acceptable limits. In addition, multiple stability tests (freeze thaw, autosampler, water bath (37 °C), bench top and long term) were performed for all the metabolites in cell culture medium. All the metabolites were stable up to 3 freeze thaw cycles at −20 °C and − 80 °C, 48 h in autosampler, 24 h at 37 °C, 48 h at room temperature and 173 days at −20 °C. Extraction recoveries for the metabolites were reproducible and were in the range of 94–108%. This method was used to quantitate estradiol metabolites generated by iPSC hepatocytes in-vitro studies.

Ion Mobility Spectrometry and Tandem Mass Spectrometry Analysis of Estradiol Glucuronide Isomers.

Estradiol is an estrogenic steroid that can undergo glucuronidation at two different sites, which results in two estradiol glucuronide regioisomers. These isomers can be produced by different enzymes and can have different biological activities before being eliminated from the body. Although there have been previous methods that can distinguish the two isomers, these methods often have long acquisition times or high cost per analysis. In this study, traveling wave ion mobility spectrometry (TWIMS) coupled with mass spectrometry (MS) was employed to separate estradiol glucuronides using alkali metal adduction in positive ion mode, where the sodiated dimer adduct provided adequate separation both in single-component standards and in two-component mixtures. Additionally, in negative ion mode, tandem mass spectrometry (MS/MS) was used to quantitatively determine the relative composition of the two isomers. This was possible due to differences in the energetic requirements for loss of the glucuronic acid, which was characterized by energy-resolved collision-induced dissociation (CID). This work demonstrated that the intensity of the glucuronic acid neutral loss product as compared with the intensity of the intact precursor ion can be used to determine the percentage of each isomer present in a mixture. Overall, TWIMS successfully separated estradiol glucuronide isomers in positive ion mode and MS/MS via CID enables relative quantitation of each isomer in negative ion mode.

Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

The human multidrug resistance protein 1 (hMRP1) transporter is implicated in cancer multidrug resistance as well as immune responses involving its physiologic substrate, glutathione (GSH)-conjugated leukotriene C4 (LTC4). LTC4 binds a bipartite site on hMRP1, which a recent cryoelectron microscopy structure of LTC4-bound bovine Mrp1 depicts as composed of a positively charged pocket and a hydrophobic (H) pocket that binds the GSH moiety and surrounds the fatty acid moiety, respectively, of LTC4. Here, we show that single Ala and Leu substitutions of H-pocket hMRP1-Met1093 have no effect on LTC4 binding or transport. Estrone 3-sulfate transport is also unaffected, but both hMRP1-Met1093 mutations eliminate estradiol glucuronide transport, demonstrating that these steroid conjugates have binding sites distinct from each other and from LTC4. To eliminate LTC4 transport by hMRP1, mutation of 3 H-pocket residues was required (W553/M1093/W1246A), indicating that H-pocket amino acids are key to the vastly different affinities of hMRP1 for LTC4vs. GSH alone. Unlike organic anion transport, hMRP1-mediated drug resistance was more diminished by Ala than Leu substitution of Met1093. Although our findings generally support a structure in which H-pocket residues bind the lipid tail of LTC4, their critical and differential role in the transport of conjugated estrogens and anticancer drugs remains unexplained.-Conseil, G., Arama-Chayoth, M., Tsfadia, Y., Cole, S. P. C. Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin.Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells.Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CLint = Vmax/Km) value of 66.5 μl/min/nmol, followed by UGT1A1 (CLint = 48.6 μl/min/nmol), UGT1A9 (CLint = 21.0 μl/min/nmol), UGT2B15 (CLint = 16.7 μl/min/nmol) and UGT1A10 (CLint = 1.60 μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin.Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.

LST-3TM12 is a member of the OATP1B family and a functional transporter

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2 pmol mg−1 min−1; Km 34.2 µm) and estradiol 17β-glucuronide (Vmax 29.9 mol mg−1 min−1 and Km 32.8 µM).In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver.

Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-D-glucuronide-induced cholestasis in rats.

Estradiol-17β-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

Development of a sensitive and robust online dual column liquid chromatography-tandem mass spectrometry method for the analysis of natural and synthetic estrogens and their conjugates in river water and wastewater.

An online ultra-high-performance-liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method for detection and quantification of natural and synthetic estrogens and their conjugates in aqueous matrices was developed. Target compounds include the natural estrogen estradiol (E2) and its main metabolites estrone (E1) and estriol (E3), the synthetic estrogens ethinylestradiol (EE2) and diethylstilbestrol (DES) and their conjugates estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), estradiol 17-glucuronide (E2-17G), estrone 3-glucuronide (E1-3G), and estriol 16-glucuronide (E3-16G). After pH adjustment, sample filtration and addition of internal standards (IS), water samples (5mL) were preconcentrated on a Hypersil GOLD aQ column after which chromatographic separation was achieved on a Kinetex C18 column using methanol and water as a mobile phase. The experimental parameters, such as sample loading flow rate, elution time, the percentage of organic solvent in the aqueous-organic eluent mixture, pH, and volume of analyzed samples, were optimized in detail. The benefits of the method compared to previously published methods include minimum sample manipulation, lower detection limits, reduced total analysis time, and overall increased method accuracy and precision. Method detection limits (MDLs) are in subnanogram per liter, complying with the requirements of the EC Decision 2015/495 (Watch list) for hormones listed therein. Applicability of the developed method was confirmed by analysis of river and raw wastewater samples taken directly from urban sewerage systems before being discharged into the river. Graphical abstract Sheme of online SPE-UHPLC-MS/MS system.

Uridine Diphosphate-Glucuronosyltransferase (UGT) 2B Subfamily Interspecies Differences in Carnivores

UDP-glucuronosyltransferases (UGTs) are among the most important xenobiotic metabolizing enzymes that conjugate a wide range of chemicals. Previous studies showed that Felidae and Pinnipedia species have very low UGT activities toward some phenolic compounds because of the UGT1A6 pseudogene and small numbers of UGT1A isozymes. In addition to the UGT1As, UGT2Bs isozymes also conjugate various endogenous (eg, estrogens, androgens, and bile acids) and exogenous compounds (opioids, non-steroidal anti-inflammatory drugs, and environmental pollutants). However UGT2B activity and genetic background are unknown in carnivore species. Therefore, this study was performed to elucidate the species differences of UGT2Bs. Using typical substrates for UGT2Bs, UGT activity was measured in vitro. In addition, UGT2B genetic features are analyzed in silico. Results of UGT activity measurement indicate marked species differences between dogs and other carnivores (cats, Northern fur seals, Steller sea lions, Harbor seals, and Caspian seals). Dogs have very high Vmax/Km toward estradiol (17-glucuronide), estrone, lorazepam, oxazepam, and temazepam. Conversely, cats and pinniped species (especially Caspian seals and Harbor seals) have very low activities toward these substrates. The results of genetic synteny analysis indicate that Felidae and pinniped species have very small numbers of UGT2B isozymes (one or none) compared with dogs, rodents, and humans. Furthermore, Felidae species have the same nonsense mutation in UGT2B, which suggests that Felidae UGT2B31-like is also a pseudogene in addition to UGT1A6. These findings of lower activity of UGT2B suggest that Felidae and some pinniped species have very low UGT activity toward a wide range of chemicals. These results are important for Felidae and Pinnipedia species that are frequently exposed to drugs and environmental pollutants.

High-content screening of clinically tested anticancer drugs identifies novel inhibitors of human MRP1 (ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2–6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.

N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity

Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity.

Solitary Inhibition of the Breast Cancer Resistance Protein Efflux Transporter Results in a Clinically Significant Drug-Drug Interaction with Rosuvastatin by Causing up to a 2-Fold Increase in Statin Exposure.

The intestinal efflux transporter breast cancer resistance protein (BCRP) restricts the absorption of rosuvastatin. Of the transporters important to rosuvastatin disposition, fostamatinib inhibited BCRP (IC50 = 50 nM) and organic anion-transporting polypeptide 1B1 (OATP1B1; IC50 > 10 μM), but not organic anion transporter 3, in vitro, predicting a drug-drug interaction (DDI) in vivo through inhibition of BCRP only. Consequently, a clinical interaction study between fostamatinib and rosuvastatin was performed (and reported elsewhere). This confirmed the critical role BCRP plays in statin absorption, as inhibition by fostamatinib resulted in a significant 1.96-fold and 1.88-fold increase in rosuvastatin area under the plasma concentration-time curve (AUC) and Cmax, respectively. An in vitro BCRP inhibition assay, using polarized Caco-2 cells and rosuvastatin as probe substrate, was subsequently validated with literature inhibitors and used to determine BCRP inhibitory potencies (IC50) of the perpetrator drugs eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe, fenofibrate, and fluconazole. OATP1B1 inhibition was also determined using human embryonic kidney 293-OATP1B1 cells versus estradiol 17β-glucuronide. Calculated parameters of maximum enterocyte concentration [Igut max], maximum unbound hepatic inlet concentration, transporter fraction excreted value, and determined IC50 value were incorporated into mechanistic static equations to compute theoretical increases in rosuvastatin AUC due to inhibition of BCRP and/or OATP1B1. Calculated theoretical increases in exposure correctly predicted the clinically observed changes in rosuvastatin exposure and suggested intestinal BCRP inhibition (not OATP1B1) to be the mechanism underlying the DDIs with these drugs. In conclusion, solitary inhibition of the intestinal BCRP transporter can result in clinically significant DDIs with rosuvastatin, causing up to a maximum 2-fold increase in exposure, which may warrant statin dose adjustment in clinical practice.

EGFR participates downstream of ERα in estradiol-17β-D-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets.

Estradiol-17β-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.